WO1992013559A1 - CD4-GAMMA1 AND CD4-IgG1 CHIMERAS - Google Patents

Abstract

This invention provides an expression vector encoding a CD4-gamma1 chimeric heavy chain homodimer. This invention also provides an expression vector encoding the heavy chains of a CD4-IgG1 chimeric heterotetramer. Finally, this invention provides an expression vector encoding the light chains of a CD4-IgG1 chimeric heterotetramer.

Description

CD4-GAMMA1 AND CD4-IσGl CHIMERAS

Background of the Invention

Throughout this application, various publications are referenced by Arabic numerals within parentheses. Full citations for these publications may be found at the end of the specification immediately preceding the claims. The disclosures of these publications in their entireties are hereby incorporated by reference into this application in order to more fully describe the state of the art as known to those skilled therein as of the date of the invention described and claimed herein.

The life cycle of animal viruses is characterized by a series of events that are required for the productive infection of the host cell. The initial step in the replicative cycle is the attachment of the virus to the cell surface which is mediated by the specific interaction of the viral attachment protein (VAP) to receptors on the surface of the target cell. The pattern of expression of these receptors is largely responsible for the host range and tropic properties of viruses. The interaction of the VAP with cellular receptors therefore plays a critical role in infection and pathogenesis of viral diseases and represents an important area to target the development of anti-viral therapeutics.

Cellular receptors may be comprised of all the components of membranes, including proteins, carbohydrates, and lipids.

Identification of the molecules mediating the attachment of viruses to the target cell surface has been made in a few instances. The most extensively characterized viral receptor protein is CD4 (T4) (1). CD4 is a nonpolymorphic cell surface glycoprotein that is expressed primarily on the surface of helper T lymphocytes and cells of the monocyte/macrophage lineage. CD4 associates with major histocompatibility complex (MHC) class II molecules on the surface of antigen-presenting cells to mediate efficient cellular immune response interactions. In man, CD4 is also the target of interaction with the human immunodeficiency virus (HIV) .

HIV infects primarily helper T lymphocytes and monocytes/macrophages, cells that express surface CD4, leading to a gradual loss of immune function which results in the development of the human acquired immune deficiency syndrome (AIDS) . The initial phase of the HIV replicative cycle involves the high affinity interaction between the HIV exterior envelope glycoprotein gpl20 and surface CD4 (Kd approximately 4 x IO^"9 M) (2) . Several lines of evidence demonstrate the. requirement of this interaction for viral infectivity. In vitro, the introduction of a functional cDNA encoding CD4 into human cells which do not express CD4 is sufficient to render otherwise resistant cells susceptible to HIV infection (3) . In vivo, viral infection appears to be restricted to cells expressing CD4. Following the binding of HIV gpl20 to cell surface CD4, viral and target cell membranes fuse, resulting in the introduction of the viral capsid into the target cell cytoplasm.

Characterization of the interaction between HIV gpl20 and CD4 has been facilitated by the isolation of cDNA clones encoding both molecules (4, 5). CD4 is a nonpolymorphic, lineage-restricted cell surface glycoprotein that is a member of the immunoglobulin gene superfamily. High-level expression of both full-length CD4 and truncated, soluble versions of CD4 (εCD4) have been described in stable expression systems. The availability of large quantities of purified sCD4 has permitted a detailed understanding of the structure of this complex glycoprotein. Mature CD4 has a relative molecular mass (Mr) of 55 kilodaltons and consists of an amino-terminal 372 amino acid extracellular domain containing four tandem immunoglobulin-like regions denoted

V1-V4, followed by a 23 amino acid transmembrane domain and a 38 amino acid cytoplasmic segment. The amino-terminal immunoglobulin-like domain VI bears 32% homology with kappa light chain variable domains. Three of the four immunoglobulin-like domains contain a disulphide bond (VI,

V2 and V4) , and both N-linked glycosylation sites in the carboxy-terminal portion of the molecule are utilized (4,

6).

Experiments using truncated sCD4 proteins demonstrate that the determinants of high-affinity binding to HIV gpl20 lie within the amino-terminal immunoglobulin-like domain VI (7- 9) . Mutational analysis of VI has defined a discrete gpl20 binding site (residues 38-52 of the mature CD4 protein) that comprises a region structurally homologous to the second complementarity-determining region (CDR2) of immunoglobulins (9) . The production of large quantities of V1V2 has permitted a structural analysis of the two amino-terminal immunoglobulin-like domains. The structure determined at 2.3 angstrom resolution reveals that the molecule has two tightly associated domains containing the immunoglobulin¬ fold connected by a continuous beta strand. The putative binding sites for monoclonal antibodies, class II MHC molecules and HIV gpl20 (as determined by mutational analysis) map on the molecular surface (10, 11) .

A soluble version of the entire extracellular segment of CD4 (V1-V4, termed sCD4) has been described and appears to be a potential therapeutic approach to the treatment of HIV infection (12) . In vitro experiments demonstrate that: 1) SCD4 acts as a "molecular decoy" by binding to HIV gpl20 and inhibiting viral attachment to and subsequent infection of human cells; 2) sCD4 "strips" the viral envelope glycoprotein gpl20 from the viral surface; and 3) cCD4 blocks the intercellular spread of virus from HIV-infected cells to uninfected cells by inhibiting virus-mediated cell fusion (1, 13) .

In addition to in vitro results, experiments with sCD4 in simian immunodeficiency virus (SIV)-infected rhesus monkeys have been described. These studies demonstrated that administration of 2 milligrams (intramuscular) of sCD4 for 28 days to SIV-infeσted rhesus monkeys led to a decreased ability to isolate virus from peripheral blood lymphocytes and bone marrow. In addition, the growth of granulocyte- macrophage and erythrocyte progenitor colonies in the bone marrow returned to normal levels. These data suggest that administration of sCD4 to SIV-infected rhesus monkeys leads to a diminution of the viral reservoir.

Phase I human clinical trials demonstrated that there is no significant toxicity or immunogenicity associated with administration of sCD4 at doses as high as 30 mg/day. Pharmacokinetic studies revealed the serum half-life of sCD4 to be 45 minutes following intravenous administration, 9.4 hours after intramuscular dosing, and 10.3 hours after the drug was given subcutaneously (14, 15) . Preliminary antiviral studies were inconclusive with respect to CD4 cell count and levels of HIV antigen. Because the maximum tolerated dose was not reached, the antiviral effect of sCD4 may have been underestimated, especially in light of recent data concerning differences in sCD4 concentrations required to inhibit laboratory strains of HIV-1 compared to primary viral isolates (16) .

Although these in vitro, primate, and human clinical studies with sCD4 have produced encouraging results, they have also defined several limitations. First, the measured serum half-life of sCD4 iε relatively short. Second, sCD4 is monovalent with respect to gpl20 binding in contrast with cell surface CD4 and viral surface gpl20 which are multivalent. Third, sCD4 is not cytotoxic for HIV-infected cells. Fourth, sCD4 may not cross the placenta to a significant degree. Therefore, chimeric CD4 molecules have been described which take advantage of the immunoglobulin- like nature of CD4 and several beneficial properties of immunoglobulins themselves (i.e. CD4-immunoglobulin fusions) .

Immunoglobulins, or antibodies, are the antigen-binding molecules produced by B lymphocytes which comprise the humoral immune response. The basic unit of an immunoglobulin molecule consists of two identical heavy chains and two identical light chains. The amino-terminus of each chain contains a region of variable amino acid sequence (variable region) . The variable regions of the heavy and light chains interact to form two antigen binding sites. The carboxy-terminus of each chain contains a region of constant amino acid sequence (constant region) . The light chain contains a single constant domain, whereas the heavy chain constant domain is subdivided into four separate domains (CHI, hinge, CH2, and CH3) . The heavy chains of immunoglobulin molecules are of several types, including mu (M) , delta (D) , gamma (G) , alpha (A) and epsilon (E) . The light chains of immunoglobulin molecules are of two types, either kappa or lambda. Within the individual types of heavy and light chains exist subtypes which may differ in effector function. An assembled immunoglobulin molecule derives its name from the type of heavy chain that it possesses.

The development of monoclonal antibodies has circumvented the inherent heterogeneity of antibodieε obtained from serum of animals or humans. However, most monoclonal antibodies are derived from cellε of mouεe origin and therefore are immunogenic when administered to humans. More recent developments combining the techniques of molecular genetics with monoclonal antibody technology has lead to the production of "humanized" chimeric antibodies in vitro. In these chimeric antibodies, the variable domains of human immunoglobulin heavy and light chains are replaced with specific heavy and light chain variable domains from a murine monoclonal antibody (17-19) . The result of thiε genetic manipulation is a molecule with specificity for a particular antigen and the characteristics of human immunoglobulins.

Sequence and structural analyses of CD4 indicate that the four extracellular domains are immunoglobulin-like. Since the Fc portion of immunoglobulins controls the rate of catabolism of the molecules (serum half-life ranging from 14 to 21 days) and provides various effector functions, several reports describe the replacement of variable and constant domains of immunoglobulins with the immunoglobulin-like domains of CD4 (21-24) .

CD4-IgGl heavy chain fusion proteins resulting in chimeric gammal heavy chain dimers have been described (21) . These molecules contain the gammal heavy chain CHI domain in addition to the hinge, CH2 and CH3 domains. However, heavy chain assembly and secretion from mammalian cells is less efficient if the CHI domain is expressed in the absence of light chains (25) . Subsequently, a CD4-IgGl heavy chain fuεion protein lacking the CHI domain and the firεt five amino acidε of the hinge region waε described which was secreted to high levels (22) . These fusion proteins retain various effector functions of immunoglobulin molecules, such as Fc receptor binding, antibody-dependent cell-mediated cytotoxicity (ADCC) toward HIV-l-infected cells, and placental transfer via an Fc receptor-dependent mechanism (22) . CD4-IgM heavy chain fusion proteins have also been described (26) . In addition, CD4-IgGl fuεion proteinε have been described wherein the V1V2 domains of CD4 are fused to the CHI, hinge, CH2 and CH3 domains of a gammal heavy chain, and wherein the V1V2 domains of CD4 are fused to the constant domain of a kappa light chain (29) .

Fusion proteins linking CD4 to toxins have also been constructed and tested for their ability to kill HIV- infected cells. In one study, sCD4 was coupled to the deglycosylated A chain of ricin which inactivates ribosomes, therefore inhibiting protein synthesis and killing the cell (27) . This fusion protein was reported to specifically lyse cells infected with five different isolates of HIV, but was nontoxic to uninfected cells. In another study, the V1V2 domains of CD4 were coupled to domains II and III of Pseudomonas exotoxin A (28) . This fusion protein was reported to specifically bind and inhibit protein synthesis in cells expressing the HIV envelope glycoprotein gpl20 (25).

We have now discovered that a specific CD4-gammal chimeric heavy chain homodimer provides advantages relative to those

CD4-IgGl heavy chain homodimers which have been described more than one year ago. Specifically, we have constructed a CD4-gammal chimeric heavy chain homodimer which contains the V1V2 domains of CD4 and which is efficiently assembled intracellularly and efficiently secreted from mammalian cells as a homodimer, enabling high recovery and purification from the medium of cells expressing this chimeric heavy chain homodimer. To construct this homodimer, we have used the entire hinge, CH2. and CH3 domains from a human gammal heavy chain, which results in a chimeric molecule containing the constant domains of a human

IgGl molecule responsible for dimerization and efficient secretion. This iε in contraεt to the heavy chain dimerε deεcribed by Capon and Gregory (20) which include the CHI domain in the CD4-IgGl heavy chain dimer, resulting in poor secretion and recovery from cell culture medium of the recombinant molecule. We have also included the entire hinge domain of gammal heavy chain in the CD4-gammal chimeric heavy chain homodimer of this invention to provide efficient dimerization, since the cysteine residues contained in this domain are responsible for forming the disulphide links to the second chain of the homodimer, poεitioning the two chains in the correct spatial alignment and facilitating formation of the antigen combining site.

Furthermore, by including the entire hinge domain, we have maintained the segmental flexibility of the heavy chain dimers, thus enabling modulation of biological function such as complement activation and Fc receptor binding (29) .

In addition to the CD4-gammal chimeric heavy chain homodimers, we have also constructed CD4-IgGl heavy chains, which contain the V1V2 domains of CD4 fused to the CHI, hinge, CH2 and CH3 domains of human gammal heavy chain. These molecules encode a CD4-IgGl chimeric heterotetramer and, when co-expressed in the presence of CD4-kappa chimeric light chains containing the VI and V2 domains of CD4 fused to the entire conεtant domain of human kappa light chainε (or lambda light chainε) , enable the production of said heterotetramer. This heterotetramer compriseε two CD4-IgGl chimeric heavy chainε and two CD4-kappa chimeric light chainε. Producing heavy chainε which contain the CHI domain enables efficient association with the CD4-kappa chimeric light chains, resulting in efficient secretion of a CD4-IgGl chimeric heterotetramer. These CD4-IgGl chimeric heterotetramers posseεs increased serum half-lives and increased avidity for HIV as compared with heavy chain dimers. finm-ma-rγ of the Invention

This invention provides an expression vector encoding a CD4- gammal chimeric heavy chain homodimer. This invention also provides an expression vector encoding the heavy chains of a CD4-IgGl chimeric heterotetramer. Finally, this invention provides an expression vector encoding the light chains of a CD4-IgGl chimeric heterotetramer.

Brief Description of the Figures

Figure 1: A) Domain structure of CD4-gammal chimeric heavy chain gene; B) Protein structure of CD4-gammal chimeric heavy chain homodimer. The sequence shown below is the single letter amino acid code of the junction between CD4 (phel79) and the hinge region of human gammal heavy chain. Note that the hinge region of a gammal heavy chain contains three cysteines (see text for discussion) . Abbreviations: L, leader (signal) sequence of human CD4; V1V2, amino- terminal variable-like domains of human CD4; H, hinge region of human gammal heavy chain; CH2 and CH3, second and third constant regions of human gammal heavy chain.

Figure 2: A) Domain structure of chimeric genes used to express CD4-IgGl chimeric heterotetramer. Top, CD4-gammal chimeric heavy chain gene; Bottom, CD4-kappa chimeric light chain gene. B) Protein structure of CD4-IgGl chimeric heterotetramer. Abbreviations: CH1-CH2-CH3, first, second and third constant regions of human gammal heavy chain; C- kappa, constant region of human kappa light chain.

Figure 3: DNA and predicted protein sequence of a CD4- gam al chimeric heavy chain homodimer (one chain) . The numbers at the end of each line indicate the nucleotide positions. The numbers above each line indicate the amino acid positionε (given in εingle letter code) . The protein domainε are indicated above the sequences by arrows.

Figure 4: DNA and predicted protein sequence of a CD4-IgGl chimeric heavy chain of the CD4-IgGl chimeric heterotetramer. The numbers at the end of each line indicate the nucleotide positions. The numbers above each line indicate the amino acid positions (given in single letter code) . The protein domains are indicated above the sequenceε by arrowε.

Figure 5: DNA and predicted protein sequence of a CD4-kappa chimeric light chain of the CD4-IgGl chimeric heterotetramer. The numbers at the end of each line indicate the nucleotide positions. The numbers above each line indicate the amino acid positions (given in single letter code) . The protein domains are indicated above the sequences by arrows.

Figure 6: Secretion of CD4-gammal chimeric heavy chain homodimer from transfected cells. Cos-M5 cells were mock transfected, transfected with CD4-IgGl-pcDNAl DNA, or transfected with CD4-gamma2 chimeric heavy chain mammalian expression vector DNA. At 48-72 hours post-tranεfection, the cellε were radiolabelled with -"S-methionine. Radiolabelled medium waε precipitated with Protein-A sepharose beads. The precipitated proteins were analyzed by SDS-PAGE under reducing or non-reducing conditions and were visualized by fluorography. Lane M, medium from mock tranεfected cellε; Lane l, medium from cells transfected with CD4-IgGl-pcDNAl DNA; Lane 2 , medium from cells transfected with CD4-gamma2 chimeric heavy chain mammalian expression vector DNA.

Figure 7: Precipitation of HIV-1 gpl20 with CD4-gammal chimeric heavy chain homodimer. Cos-M5 cells were mock transfected, transfected with the CD4-IgGl-pcDNAl, or transfected with CD4-gamma2 chimeric heavy chain mammalian expression vector DNA. At 48-72 hours post transfection, unlabelled aliquots of medium were incubated with an aliquot of ³⁵S-methionine-labelled gpl20. The complexes were precipitated with Protein A-sepharose beads. The precipitateε were then analyzed by SDS-PAGE followed by fluorography. Lane M, medium from mock transfected cells; Lane 1, medium from cells transfected with CD4-IgGl-pcDNAl DNA; Lane 2, medium from cells transfected with CD4-gamma2 chimeric heavy chain mammalian expression vector DNA.

Figure 8: Purification of CD4-gammal chimeric heavy chain homodimer from CHO cell-conditioned medium. Stable CHO cells constitutively secreting CD4-gammal chimeric heavy chain homodimer, or CD4-gamma2 chimeric heavy chain homodimer, were grown in roller bottles. Conditioned medium was passed over a Protein A-sepharose column and bound material was eluted from the column. The peak fractions were identified by SDS-PAGE followed by silver staining and pooled. The purified proteins were then analyzed by SDS- PAGE under reducing conditions followed by silver staining. Lane 1, CD4-gammal chimeric heavy chain homodimer; Lane 2, CD4-gamma2 chimeric heavy chain homodimer.

Figure 9: Inhibition of HIV binding to CEM cells by CD4- based molecules. Soluble CD4 (sCD4) , partially purified CD4-gammal, or partially purified CD4-gamma2 were tested for inhibition of virus binding to CD4 positive cells. Bound virus was detected by indirect immunofluorescence and cytofluorography. Resultε are expressed as percent inhibition versus concentration of inhibiting agent.

Figure 10: Inhibition of HIV infection of CD4-positive cellε by CD4-baεed molecules. sCD4, partially purified CD4- gammal, or partially purified CD4-gamma2 were incubated with an HIV-1 inoculum (100 TCID₅₀) , and mixtures were added to PHA-stimulated lymphocytes and incubated at 37^βC overnight. The cells were washed and plated in microculture (1 x 10⁵ cells/culture; 10 cultures per dilution) and monitored for reproductive viral replication by detection of HIV antigen in culture supernates 8 and 12 dayε later. Results are expressed as percent positive cultures at a given concentration of inhibiting agent. Figure 11: Purification of CD4-gammal chimeric heavy chain homodimer. Stable CHO cells constitutively secreting CD4- gammal chimeric heavy chain homodimer were grown in roller bottles. Conditioned medium was passed over a Protein A- sepharose column and bound material was eluted from the column (see Figure 8) . The peak fractions were then pooled and passed over an S-sepharose column. After extensive washes, the CD4-gammal chimeric heavy chain homodimer was eluted with 50mM BES pH 7.0, 500mM NaCl. The peak fractions were identified by SDS-PAGE followed by silver staining and pooled. The purified protein was then analyzed by SDS-PAGE under non-reducing and reducing conditions followed by silver staining. Lane 1: approximately 1.5μg protein run under non-reducing conditions. Lane 2: approximately 1.5μg protein run under reducing conditions.

Figure 12: Secretion of CD4-IgGl chimeric heterotetramer from stably transfected cells. CHO cells stably expressing both CD4-IgGl chimeric heavy chains and CD4-kappa chimeric light chains were radiolabelled with ³⁵S-methionine and cysteine. Radiolabelled medium waε precipitated with Protein-A sepharose beads. (A) The precipitated proteins were analyzed by SDS-PAGE under non-reducing conditions, and were visualized by fluorography. Lane 1: medium from untransfected CHO cells, Lane 2: medium from cells stably expressing both the CD4-IgGl chimeric heavy chains, and CD4- kappa chimeric light chainε. (B) An identical sample to that run in lane 2 from (A) was run on SDS-PAGE under non- reducing conditions. The lane from this SDS-PAGE gel was excised and the proteinε reduced by incubation of the gel slice for 45 minutes at 4"C in equilibration buffer (62.5 mM TrisHCl pH 6.8, 2.3% SDS, 5% β-mercaptoethanol , 10% glycerol) . After incubation of the gel εlice under reducing conditionε, the proteins contained within the gel were analyzed by SDS-PAGE and viεualized by fluorography. Detailed Description of the Invention

Five expression vectors and two plasmids designated CD4- IgG2-Rf, CD4-IgGl-Rf, CD4-IgGlHC-pRcCMV, CD4-IgG2HC-pRcCMV, CD4-kLC-pRcCMV, CD4-IgGl-pcDNAl, and CD4-IgG2-pcDNA, respectively have been deposited with the American Type Culture Collection, Rockville, Maryland, U.S.A. 20852, under ATCC Accession No. 40949, 40950, 75192, 75193, 75194, 40951, and 40952, respectively. These deposits were made pursuant to the provisions of the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the Purposeε of Patent Procedure (Budapest Treaty) .

Specifically, the invention provides an expression vector designated CD4-IgGl-pcDNAl (ATCC No. 40951) encoding a CD4- gammal chimeric heavy chain homodimer. The invention additionally provides a CD4-gammal chimeric heavy chain homodimer encoded by this expression vector or any other expression vector having the same DNA coding region inserted therein. Specifically, the invention also provides expresεion vectorε designated CD4-IgGlHC-pRcCMV and CD4-kLC- pRcCMV (ATCC Nos. 75192 and 75194), encoding a CD4-IgGl chimeric heavy chain and a CD4-kappa chimeric light chain. The invention additionally provides a CD4-IgGl chimeric heterotetramer encoded by theεe expression vectors or any other expresεion vector having the same DNA encoding region inserted therein.

In accordance with the invention, numerous vector syεtemε for expression may be employed. For example, one clasε of vectorε utilizes DNA elementε which are derived from animal viruses such as bovine papilloma virus, polyoma virus, adenoviruε, vaccinia viruε, baculoviruε, retroviruεeε (RSV, MMTV or MOMLV) or SV40 viruε. Additionally, cells which have stably integrated the DNA into their chromosomeε may be selected by introducing one or more markers which allow selection of transfected host cells. The marker may provide for prototrophy to an auxotrophic host, biocide resistance, e.g., antibiotics, or resistence to heavy metals such as copper or the like. The selectable marker gene can be either directly linked to the DNA sequenceε to be expressed, or introduced into the same cell by cotransformation. Additional elements may also be needed for optimal synthesiε of mRNA. These elements may include splice signals, as well as transcriptional promoters, enhancers, and termination signals. The cDNA expression vectors incorporating such elements include those described by Okayama. (32)

Thus, the invention further provides a method of producing a CD4-gammal chimeric heavy chain homodimer. This method compriseε

a) transfecting a mammalian cell with an expression vector for producing the CD4-gammal chimeric heavy chain homodimer; b) culturing the resulting transfected mammalian cell under conditionε such that CD4-gammal chimeric heavy chain homodimer is produced; and c) recovering the CD4-gammal chimeric heavy chain homodimer so produced.

Once the vector or DNA sequence containing the constructs has been prepared for expresεion, the expression vectors may be transfected or introduced into an appropriate mammalian cell host. Various techniqueε may be employed εuch as protoplast fusion, calcium phosphate precipitation, electroporation or other conventional techniqueε. In the caεe of protoplaεt fuεion, the cellε are grown in media and εcreened for the appropriate activity. Expression of the gene(s) resultε in production of the fusion protein which correspondε to one chain of the CD4-gammal chimeric heavy chain homodimer. This fusion protein may then be treated to form the chimeric heavy chain homodimer.

Further, methods and conditions for culturing the resulting transfected cells and for recovering the chimeric heavy chain homodimer so produced are well known to those skilled in the art and may be varied or optimized depending upon the specific expression vector and mammalian host cell employed.

In accordance with the claimed invention, the preferred host cells for expresεing the chimeric heavy chain homodimers of this invention are mammalian cell lines, including, for example, monkey kidney CV1 line transformed by SV40 (COS-7) ; human embryonic kidney line 293; baby hamster kidney cells (BHK) ; Chinese hamster ovary-cells-DHFR (CHO) ; monkey kidney cells (CV1) ; African green monkey kidney cells (VERO-76) ; human cervical carcinoma cells (HELA) ; canine kidney cells (MDCK) ; human lung cellε (W138) ; human liver cellε (Hep G2) ; mouse mammary tumor (MMT 060562) ; mouse cell line (C127) and myeloma cell lines.

The invention further provides a method of inhibiting the HIV infection of a CD4+ cell which compriεes treating the CD4+ cell with the CD4-gammal chimeric heavy chain homodimer in an amount which is effective to inhibit infection of the cell.

Additionally, the invention provides a method of preventing a subject from being infected with HIV which comprises administering to the subject the CD4-gammal chimeric heavy chain homodimer in an amount which iε effective to prevent the εubject from being infected with HIV.

Although the invention encompaεses the adminiεtration of the chimeric heavy chain homodimer to various subjectε, AIDS patientε are of particular intereεt. Further, methodε of administering the homodimer are well known in the art and include, merely by way of example, subcutaneous, intramuscular and intravascular injection, alone or in combination with other agentε εuch as AZT or DDL

Further provided is a method of treating a subject infected ,_ . ,_ with HIV so as to block the spread of HIV infection which comprises administering to the subject an amount of the CD4- gammal chimeric heavy chain homodimer in an amount which is effective to block the spread of HIV infection.

For example, the homodimer may be administered to patients having HIV infection at a dosage capable of maintaining a concentration of greater than about 100 ng of CD4-gammal chimeric heavy chain homodimer/ml plasma. For CD4-gammal chimeric heavy chain homodimer variants having different molecular weights, about 2 picomoles of soluble receptor per ml of plasma, an amount for example, sufficient to establish a stoichiometric equivalence with native (membrane bound) and soluble receptor is administered. Typically, the dosage of εoluble CD4 iε about 100 μg/kg of patient weight/day.

The foregoing method may be used to help prevent the spread of the HIV virus within the body of a HIV infected patient. Additionally, CD4-gammal chimeric heavy chain homodimer may be administered as a prophylactic measure to render a εubject's blood less susceptible to the spread of the HIV virus. Such prophylactic administration includes administration both prior to HIV contact or shortly thereafter, or both.

A pharmaceutical composition which compriseε the CD4-gammal chimeric heavy chain homodimer of thiε invention in an amount effective to inhibit HIV infection of a CD4+ cell and a pharmaceutically acceptable carrier iε further provided. Pharmaceutically acceptable carriers are well known in the art to which the present invention pertains and include, but are not limited to, 0.01-O.lM and preferably 0.05 M phosphate buffer or 0.8% saline. Additionally, such pharmaceutically acceptable carriers may be aqueous or non¬ aqueous solutions, suspensions, and emulsions. Examples of non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate. Aqueous carriers include water, alcoholic/aqueous solutions, emulsions or suspensions, including saline and buffered media. Parenteral vehicles include sodium chloride solution. Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's or fixed oils. Intravenous vehicles include fluid and nutrient replenishers, electrolyte replenishers such as those based on Ringer's dextrose, and the like. Preservatives and other additives may also be present, such as, for example, antimicrobials, antioxidantε, chelating agents, inert gases and the like. (33)

The invention further provides a composition of matter comprising a CD4-gammal chimeric heavy chain homodimer and a toxin linked thereto.

Some example of toxins are the deglycosylated A chain of ricin, domainε II or III of Pseudomonas exotoxin A, Diphtheria toxin, or a non-peptidyl cytotoxin. These toxinε may be linked uεing conventional in vitro protein cross- linking agents (34-36) . Additionally the toxins may be linked by recombinant syntheεis as a fusion protein (see for example U.S. Patent 4,765,382).

The invention also provides a diagnostic reagent comprising a CD4-IgGl chimeric heavy chain homodimer and a detectable marker linked thereto. By employing a molecule which bindε to the HIV viruε and additionally has attached to it a detectable marker, one may identify cells which are infected with HIV. Examples of conventional detectable markers includes radioisotopes such as 1125, chromophores , and fluorophores.

Thus, the chimeric heavy chain homodimer of the invention may be used in an assay for HIV or SIV viral infection in a biological sample by contacting a sample derived from an animal suspected of having an HIV or SIV infection, with the homodimer of the invention, and detecting whether a complex forms with gpl20, either alone or on the surface of an HIV- infected cell. For this purpose the homodimer may be labeled with a detectable marker or may be unlabeled and then be detected with another reagent which is detectably labeled and is specifically directed to the homodimer or to a complex between it and gpl20.

For example, a biological sample may be treated with nitro¬ cellulose, or another solid support which is capable of immobilizing cells, cell particles or soluble protein. The support may then be washed with suitable buffers followed by treatment with the chimeric heavy chain homodimer which may be detectably labeled. The solid phase support may then be washed with buffer a second time to remove unbound fusion protein and the labeled homodimer detected.

In carrying out the assay the following steps may be employed.

a) contacting a sample suspected of containing gpl20 with a solid εupport to effect immobilization of gpl20, or cellε which expreεs gpl20 on their surface; . b) contacting said εolid εupport with the detectably labeled chimeric heavy chain homodimer of the invention; c) incubating said detectably labeled homodimer with said support for a sufficient amount of time to allow the homodimer to bind to the immobilized gpl20 or cell which expresses gpl20 on its surface; d) separating the solid phase support from the incubation mixture obtained in step c) ; and e) detecting bound labeled homodimer and thereby detecting gpl20.

Such a method may be formatted either as a qualitative or as a quantitative test using methods well known in the art.

Alternatively, labeled homodimer-gpl20 complex may be separated from a reaction mixture by contacting the complex with an immobilized antibody or protein which is specific for an immunoglobulin or, e.g., protein A, protein G, or anti-IgG antibodies. such anti-immunoglobulin antibodies may be monoclonal or polyclonal. The solid support may then be washed with suitable buffers to obtain an immobilized gpl20-labeled homodimer-antibody complex. The label on the homodimer may then be detected so aε to measure endogenous gpl20, and thereby detect the presence of HIV.

In one embodiment of the invention, a method for detecting HIV or SIV viral infection in a sample is provided comprising:

a) contacting a sample suspected of containing gpl20 with a CD4-gammal chimeric heavy chain homodimer in accordance with this invention, and the Fc portion of an immunoglobulin chain; and b) detecting whether a complex is formed.

The invention also provides a method of detecting gpl20 in a sample comprising: a) contacting a mixture obtained by contacting a sample suspected of containing gpl20 with a homodimer of this invention, and the Fc portion of an immunoglobulin chain, with an Fc binding molecule, such as an antibody, protein A, or protein G, which is immobilized on a solid phase support and is specific for the homodimer, to obtain a gpl20-homodimer immobilized antibody complex, b) washing the solid phase support obtained in step (a) to remove unbound homodimer; and c) detecting the homodimer.

Of course, the specific concentrations of unlabeled or detectably labeled homodimer and gpl20, the temperature and time of incubation, as well as other assay conditions, may be varied depending on various factors including the concentration of gpl20 in the sample, the nature of the sample, and the like. Those skilled in the art are readily able to determine operative and optimal assay conditions for each determination.

Also provided is an enzyme-linked immunoadsorbent assay (ELISA) to detect and quantify soluble CD4 (sCD4) or CD4 chimeric proteins. In carrying out the assay, the process comprises: a) contacting a sample containing sCD4 with a solid support to immobilize soluble sCD4; b) contacting said εolid εupport with the detectably labeled monoclonal antibody OKT4a alone, or with a sample containing sCD4 or CD4 chimeric proteinε and OKT4a; c) incubating εaid detectably labeled OKT4a containing media for εufficient time to allow for binding to immobilized SCD4 ; d) separating the solid phase support from the incubation mixture in step (c) ; e) detecting the bound 0KT4a and thereby quantifying the amount of CD4 contained in the sample.

The invention further provides an expression vector encoding the heavy chains of a CD4-IgGl chimeric heterotetramer, designated CD4-IgGlHC-pRcCMV (ATCC No. 75192). The invention also provides a CD4-IgGl chimeric heterotetramer, the heavy chains of which are encoded by this expression vector or another vector containing the same coding sequence.

Additionally, the invention provides an expression vector encoding the light chains of a CD4-IgGl chimeric heterotetramer, deεignated CD4-kLC-pRcCMV (ATCC No. 75194) . Finally, the invention provideε a CD4-IgGl chimeric heterotetramer, the light chainε of which are encoded by the CD4-kLC-pRcCMV expression vector or another vector containing the same coding sequence.

Further, the invention provides a CD4-IgGl chimeric heterotetramer both the heavy and light chains of which are encoded by the aforementioned expression vectors.

The invention further provides a method of producing εuch a CD4-IgGl chimeric heterotetramer. Thiε method comprises: a) cotransfecting a mammalian cell with the expression vector for producing the light chains of a CD4-IgGl chimeric heterotetramer and an expression vector encoding a light chain; b) culturing the resulting cotransfected mammalian cell under conditions such that CD4-IgGl chimeric heterotetramer iε produced; and c) recovering the CD4-IgGl chimeric heterotetramer εo produced. Methods of cotransfecting mammalian cells are well known in the art and include those discussed hereinabove. Similarly, expression vectors encoding light chains are well known in the art.

The invention additionally provides a method of producing a CD4-IgGl chimeric heterotetramer which comprises: a) cotransfecting a mammalian cell with the expression vector for producing the light chains of a CD4-IgGl chimeric heterotetramer and with an expression vector encoding an IgGl heavy chain; b) culturing the resulting cotransfected mammalian cell under conditions such that a CD4-IgGl chimeric hetero-tetramer is produced; and c) recovering the CD4-IgGl chimeric heterotetramer so produced.

Further the invention provides a method of producing an CD4- IgGl chimeric heterotetramer which comprises: a) cotransfecting a mammalian cell with the expression vector for producing the heavy chains of a CD4-IgGl chimeric heterotetramer and an expression vector for producing the light chains of a CD4-IgGl chimeric heterotetramer; b) culturing the resulting cotransfected mammalian cell under conditions such that the CD4-IgGl chimeric heterotetramer is produced; and c) recovering the CD4-IgGl chimeric heterotetramer so produced.

The invention also includes a method of inhibiting HIV infection of a CD4+ cell which comprises treating the CD4+ cell with either a CD4-IgGl chimeric heterotetramer, the heavy chains of which are encoded by the expression vector designated CD4-IgGlHC-pRcCMV; a CD4-IgGl chimeric heterotetramer, the light chainε of which are encoded by the expression vector designated CD4-kLC-pRcCMV; or a CD4-IgGl chimeric heterotetramer, both the heavy and the light chains of which are encoded by both of the above expression vectors, in an amount effective to inhibit infection of the cell.

The invention further provides a method of preventing a subject from being infected with HIV. This method comprises administering to the subject either a CD4-IgGl chimeric heterotetramer, the heavy chains of which are encoded by the expression vector designated CD4-IgGlHC-pRcCMV; a CD4-IgGl chimeric heterotetramer, the light chains of which are encoded by the expreεεion vector designated CD4-kLC-pRcCMV; or a CD4-IgGl chimeric heterotetramer, both the heavy and the light chains of which are encoded by the above expression vectors, in an amount which is effective to prevent the subject from being infected with HIV.

The invention also provides a method of treating a subject infected with HIV so aε to block the spread of HIV infection. This method comprises administering to the subject either a CD4-IgGl chimeric heterotetramer, the heavy chains of which are encoded by the expression vector designated CD4-IgGlHC-pRcCMV; a CD4-IgGl chimeric heterotetramer, the light chains of which are encoded by the expression vector designated CD4-kLC-pRcCMV; or a CD4-IgGl chimeric heterotetramer, both the heavy and the light chains of which are encoded by the above-described expreεεion vectorε, in an amount effective to block εpread of HIV infection, for example, within the subject or an AIDS patient's body.

The invention also provides a pharmaceutical composition which compriεeε either a CD4-IgGl chimeric heterotetramer, the heavy chains of which are encoded by the expression vector designated CD4-IgGlHC-pRcCMV; a CD4-IgGl chimeric heterotetramer, the light chains of which are encoded by the expression vector designated CD4-kLC-pRcCMV, or a CD4-IgGl chimeric heterotetramer, both the heavy and the light chains of which are encoded by the above described expression vectors, in an amount effective to inhibit HIV infection of a CD4+ cell, and a pharmaceutically acceptable carrier.

Further provided by the invention is a composition of matter comprising either a CD4-IgGl chimeric heterotetramer, the heavy chains of which are encoded by the expression vector designated CD4-IgGlHC-pRσCMV; a CD4-IgGl chimeric heterotetramer, the light chains of which are encoded by the expression vector designated CD4-kLC-pRcCMV, or a CD4-IgGl chimeric heterotetramer, both the heavy and the light chains of which are encoded by the above described expression vectors, and a toxin linked thereto.

In one embodiment of the invention, the toxin is the deglycosylated A chain of ricin, domains II or III of Pseudomonas exotoxin A, Diphtheria toxin, or a non-peptidyl cytotoxin.

The invention further provides a diagnostic reagent either comprising a CD4-IgGl chimeric heterotetramer, the heavy chains of which are encoded by the expression vector designated CD4-IgGlHC-pRcCMV; a CD4-IgGl chimeric heterotetramer the light chains of which are encoded by the expression vector designated CD4-kLC-pRcCMV; or a CD4-IgGl chimeric heterotetramer both the heavy and the light chains of which are encoded by both of those expresεion vectorε, and a detectable marker linked thereto. Exampleε of εuitable detectable markerε are radioisotopes, chromophores or fluorophoreε. In order to facilitate understanding of the following examples, certain frequently occurring methods and/or terms are best described in Maniatis et al. (37)

This invention is illustrated in the Experimental Details section which follows. These sections are set forth to aid in an understanding of the invention but are not intended to, and should not be construed to, limit in any way the invention as set forth in the claims which follow thereafter.

Bxperimental Details

A. Materials and Methods

1.

encoding CD4-gammal chimeric heavy chain homodimer:

The human CD4 cDNA was excised from the plasmid pSP6T4 (4) as an EcoRl/Stul restriction fragment. The 0.70 kilobase fragment was isolated and cloned into EcoRl/Smal digested

M13mpl8. This intermediate vector (M13mpl8(CD4) ) was then isolated, linearized with Pstl, purified, and treated with

Bacterial Alkaline Phosphatase (BAP). The 2.0 Kb Pstl/Pstl fragment from the plasmid pBr gammal containing the human gammal heavy chain gene (30), (containing the hinge, CH2, and CH3 exons) was isolated and cloned into the BAP-treated

M13mpl8/CD4 vector. Resulting recombinants were then screened for the correct orientation of the Pstl fragment

(with respect to the CD4 sequence) to obtain a vector which contains in tandem CD4(EcoRl/Stul) - gammal(Pstl/Pstl) . To obtain a CD4-gammal chimeric heavy chain gene, oligonucleotide-mediated site-directed mutagenesis was performed to juxtapose the CD4 and gammal heavy chain DNA sequences, ligating the CD4 sequence in frame to the hinge exon. The resulting chimeric DNA molecule encodes a protein containing the V1V2 domains of CD4 followed by the hinge,

CH2, and CH3 domains of gammal heavy chain (Figure IA) .

Mutagenesis was performed on single-εtranded DNA isolated from recombinant phage from transformed TGI cells

(Amersham) . Briefly, template DNA was annealed with a 34- mer oligonucleotide (5'-GTCACAAGATTTGGGCTCGAAAGCTAGCACCACG-

3') , containing sequenceε which join the laεt codon encoding

Phe(179) from V1V2 of CD4 to the firεt codon of the hinge for IgGl (encoding Glu) (Figures IA and 3) . After second strand synthesis, double stranded DNA was transformed into competent TGI cells. Isolated plaques were then grown in fresh TGI cells and single stranded DNA was purified for DNA sequencing. All mutations were verified and confirmed by dideoxy sequencing using the Sequenase system (USB) .

Plaques containing the chimeric gene with the correct sequence were then grown in TGI cells, and Rf DNA

(designated CD4-IgGl-Rf) was isolated from the cells.

2. Construction of Mammalian Expression Vector Encoding CD4- gammal chimeric heavy chain homodimer:

The CD4-gammal chimeric heavy chain gene was isolated from the recombinant Rf DNA following Rf linearization with EcoRl. The EcoRl sites in the linearized DNA were filled in with the Klenow fragment of DNA polymerase I. The flush ended DNA was then ligated overnight at 15 degrees Celsius with T4 DNA ligase to a 100-fold molar excess of Hindlll linkers. After heat inactivation of T4 DNA ligase for 15 minutes at 70 degrees Celsius, the Hindlll-linkered DNA was extensively digested with Hindlll to liberate a fragment containing the CD4-gammal chimeric heavy chain gene. This Hindlll fragment waε then purified and ligated to the expression vector pcDNA-1 (Invitrogen) , which was previously digested with Hindlll and BAP treated. The resulting plasmid was then transformed into MC1061/P3 cells. Plasmid DNA was isolated from recombinant clones, and verification of the presence of the Hindlll insert and orientation of the insert with respect to the cytomegalovirus (CMV) promoter in the plaεmid was made by reεtriction enzyme analyεis. The resulting mammalian expression plasmid which encodes a CD4- gammal chimeric heavy chain homodimer iε deεignated CD4IgGl- pcDNAl.

3. Expression of CD4-IgGl-pcDNAl in mammalian cellε:

a. Tranεient expression. CosM5 cells grown in DMEM containing 10% fetal calf serum were split to 75% confluence. On the following day, the cells were transfected for 16-20 hours with 10 micrograms of

CsCl-purified plasmid CD4IgGl-pcDNAl DNA by the standard

CaPO(4) precipitation technique. After transfection, fresh medium was added to the cells. Analysis of the products synthesized 48-72 hours post-transfection was performed by radiolabelling of transfectants with ³⁵S-methionine for 12-18 hours followed by precipitation of media and cell lysates using anti-CD4 antibodies or by incubation with Protein A- sepharose beadε alone followed by SDS-PAGE under reducing or non-reducing conditions (Figure 6) . In addition, analysis of media and cell lysates was performed 48-72 hours post- transfection by standard Western blotting procedures.

b. Stable expression.

Dhfr-Chinese hamster ovary cells (CHO) were transfected with 20 micrograms of CsCl purified DNA in a 1000:1 molar ratio of CD4IgGl-pcDNAl:p410 (p410 iε an expreεsion plasmid containing the dhfr gene) , although other ratios may also be used. Approximately 3-5 days post-transfection, cells were placed in selective medium (nucleoside-free alpha MEM containing 10% dialyzed fetal calf serum) . Approximately 10-15 days post-εelection, individual cell cloneε were picked and analyzed for stable expression of CD4-gammal chimeric heavy chain homodimer by several screening techniques, such as ELISA and precipitation with Protein A- εepharoεe beadε followed by SDS-PAGE under reducing and non- reducing conditionε. Cloneε expreεεing the higheεt levelε were εubjected to succesεive roundε of amplification of the newly introduced DNA sequences in increasing concentrations of methotrexate. Stable CHO cell lines were thus generated which secrete between 10-100 micrograms/milliliter of CD4- gammal chimeric heavy chain homodimer. 4. Purification of CD4-gammal chimeric heavy chain homodimer from CHO conditioned media:

CD4-gammal chimeric heavy chain homodimer was purified in a single step using Protein A-Sepharose column chromatography. CHO cells secreting CD4-gammal chimeric heavy chain homodimer were grown to high density in roller bottles in medium containing alpha MEM with 10% IgG-free fetal calf serum. Conditioned media was collected, clarified by centrifugation, and diluted 1:1 with PBS with/or without detergent (i.e. Tween) in this and subsequent buffers. The diluted media was then applied to a 5ml column of Protein A- Sepharose fast flow previously equilibrated with PBS, at a flow rate of 60ml/hour. After extensive washing, the specifically bound material was eluted with lOOmM glycine/HCl, pH 3.5, directly into an aliquot of 1M Tris.HCl pH 8.0 to immediately neutralize the eluted fractions. The fractions were then analyzed by SDS-PAGE under reducing and non-reducing conditions followed by silver staining and pooled (Figure 8) .

The pooled fractions were then applied to a 10 ml column of S-sepharose fast flow previously equilibrated with 50mM BES pH 7.0 at a flow rate of 120ml/hr. After application of the εample, a step elution gradient (consisting of the following 4 stepε: 5 column volumes of 50mM BES pH 7.0, 4 column volumes of 50mM BES pH 70, lOOmM NaCl, 6 column volumes of 50mM BES pH 7.0225mM NaCl, followed by 8 column volumes of 50mM BES pH 7.0, 500mM NaCl) was employed for specific elution of the CD4-gammal chimeric heavy chain homodimer. The CD4-gammal chimeric heavy chain homodimer was eluted from the column in 50mM BES pH 7.0, 500mM NaCl. The peak fractions were then pooled and concentrated to yield a final protein concentration of at least lmg/ml. 5. Demonstration of binding of CD4-gammal chimeric heavy chain homodimer to the HIV envelope glycoprotein gpl20:

CosM5 transfectants expressing CD4-gammal chimeric heavy chain homodimer were incubated for 72 hours in DMEM containing 10% IgG-free fetal calf serum. Unlabelled medium « was then collected and used to precipitate ""S-methionine- radiolabelled HIV gpl20. After incubation of CD4-gammal chimeric heavy chain homodimer containing medium with ³⁵S- methionine-labelled gpl20, the complexes were adsorbed to

Protein A-sepharose. Protein A-sepharose complexes were recovered by centrifugation, and the precipitates were analyzed by SDS-PAGE under reducing conditions followed by fluorography (Figure 7) . Alternatively, aliquots of purified CD4-gammal chimeric heavy chain homodimer from CHO cellε were alεo used to precipitate ³⁵S-radiolabelled gpl20 using the same procedure.

6. Determination of plasma half-life and placental transfer of CD4-gammal chimeric heavy chain homodimer:

Determination of the plasma half-life and placental transfer are performed by well established techniques. Briefly, rabbits or monkeys are injected intravenously or intramuscularly with purified CD4-gammal chimeric heavy chain homodimer. At various time points post-injection, plasma samples are taken, and the quantity of the CD4-gammal chimeric heavy chain homodimer present in the serum is measured by ELISA. In addition, pregnant monkeys are also injected either IV or IM with CD4-gammal chimeric heavy chain homodimer and the concentration determined in the cord blood and the serum of the newborn monkey. Determination and compariεon of the quantity of the CD4-gammal chimeric heavy chain homodimer in the mother's serum aε well aε in the cord blood and εerum of the newborn indicateε the relative rate of transport across the placenta of these molecules.

7. Determination of FcR binding and macrophage infectivitv of C A-qap al chimeric heavy chain homodimer. ^"~

Determination of FcR binding and macrophage infectivity of

CD4-gammal chimeric heavy chain homodimer are performed by well established techniques. For these studies, U937 cells (a human monocytic cell line expressing FcRl and FcRII) , purified monocyte/macrophage populations from human peripheral blood, and Hela cells constitutively expressing recombinant human FcRs are utilized. In addition, monoclonal antibodies specific for FcRl and FcRII are commercially available. Briefly, radiolabelled monomeric or aggregated CD4-gammal chimeric heavy chain homodimer is incubated with the above cells and appropriate control cells at 4 degrees Celsius over various time points. At the end of each incubation, the cells are solubized and the cell- associated radioactivity is determined to establish the amount of CD4-gammal chimeric heavy chain homodimer specifically bound to each cell type. As controls, radiolabelled normal monomeric or aggregated human IgGl are used to determine the levels of specific antibody binding. Furthermore, competing the radiolabelled component with unlabelled monomeric or aggregated normal human IgGl, or monoclonal antibodies to FcRl or FcRII, will establish the binding efficiency and specificity of CD4-gammal chimeric heavy chain homodimer to each cell type.

To ascertain whether the CD4-gammal chimeric heavy chain homodimer mediates enhancement of HIV infection of monocyteε/macrophages, HIV-1 is incubated with media alone or either monomeric or aggregated CD4-gammal chimeric heavy chain homodimer at several dilutions. As controls, sera from normal individuals and HIV-infected individuals are used (31) . After incubation for one hour at 4 degrees Celsius, the 'opsonized' virus is added to the cell types described in the paragraph above. At various time points after infection, the media is harvested and assayed for viral reverse transcriptase activity to determine the degree of viral infection. As controls, sCD4, 0KT4a or eu3a are included during the infection of the cells. In addition, various dilutions of the CD4-gammal chimeric heavy chain homodimer and appropriate controls are first incubated with the cells at 4 degress Celsius to allow binding. HIV is then added and infection assayed by viral reverse transcriptase activity.

8. HIV binding assay:

Binding of HIV was performed as previously described (38, 39) . Briefly, concentrated HIV-1 preparations were incubated with various dilutionε of sCD4, CD4-gammal, or CD4-gamma2, for 30 minutes and then added to 5 x 10⁵ CEM cells. Bound virus was detected by indirect immunofluoreεcence and cytofluorography as previously described (39) .

9. Neutralization assay:

The microculture asεay for productive viral replication waε aε previouεly deεcribed (38, 40). Briefly dilutionε of εCD4, CD4-gammal, or CD4-gamma2 were incubated for 30 minuteε with 100 TCID₅₀ HIV-1 at room temperature. The mixtureε were added to PHA-stimulated lymphocytes and incubated at 37"C overnight. The cells were then washed and plated in microculture at 1 x 10⁵ eelIs/culture; and 10 cultureε per dilution and monitored for reproductive viral replication by detection of HIV antigen in culture εupernates 8 and 12 days later. B. Construction of CD4-IOG1 chimeric heavy chain and CD4- kappa chimeric light chain for expression of CD4-IgGl chimeric heterotetramer:

1. Introduction

This invention describes a CD4-gammal chimeric heavy gene encoding a CD4-gammal chimeric heavy chain homodimer which is efficiently secreted from transformed mammalian cells. This chimeric molecule was designed to contain sequences from the human IgGl heavy chain which allow for efficient homodimer assembly and secretion. The CHI region of the IgG heavy chains is responsible for retaining heavy chain molecules intracellularly and for formation of heterotetramers with light chains (25) . In order to efficiently produce CD4-gammal chimeric heavy chain homodimers, the CD4-gammal chimeric heavy chain gene described above specifically lacks the CHI domain. The resulting homodimer contains two CD4 V1V2 moieties and therefore has the potential of being bivalent with respect to gpl20 binding and having enhanced avidity for HIV compared to sCD4.

In addition, this invention describeε the construction of CD4-IgGl chimeric heterotetramers which contain two heavy chains and two light chains. The resulting heterotetramer, containing two or four CD4 V1V2 moieties, and has the potential of being tetravalent with respect to gpl20 binding and having enhanced avidity for HIV compared to sCD4. The CD4-IgGl chimeric heavy chain gene used to produce CD4-IgGl chimeric heterotetramer contains the entire heavy chain constant region, including the CHI domain. The inclusion of the CHI domain facilitates efficient intracellular asεociation with light chainε, affording the potential for secreted, disulfide-bonded heterotetramerε. Both the CD4- IgGl chimeric heavy chain gene and the CD4-kappa chimeric light chain gene contain the V1V2 domains of CD4. Efforts to express CD4-IgGl chimeric heavy chains or CD4-kappa chimeric light chains (either alone or in combination) containing only the VI domain of CD4 were unsuccessful.

2. Construction of CD4-IgGl chimeric heavy chain expression vector and CD4-kappa chimeric liσht chain expression vector for production of CD4-IgGl chimeric heterotetramers.

a. Construction of CD4-IgGl chimeric heavy chain mammalian expression vector.

The human CD4 cDNA sequence is excised from the plasmid pSP6T4 (4) as an EcoRl/Stul restriction fragment. The 0.70 kilobase fragment is isolated and cloned into EcoRl/Smal- digeεted M13mpl8. The resulting vector (M13mpl8(CD4) ) is then isolated and digested with BamHl. The BamHl sites of the M13mpl8(CD4) are made flush ended with the Klenow fragment of DNA polymerase 1. After heat inactivation of the polymerase for 15 minutes at 65 degrees Celsius, the linearized M13mpl8(CD4) vector is then digested with Pstl and purified.

In order to excise a fragment containing the CHI exon of the human gammal heavy chain gene, the plasmid pBr gammal (30) iε digested with SacII, and the SacII sites are then made flush using T4 DNA polymerase. After heat inactivation of the polymerase, the fragment is then digested with Pstl. The resulting SacII(flush)-Pstl fragment containing the CHI exon is then purified and ligated to the M13mpl8(CD4) vector described in the above paragraph. After tranεformation of competent TGI cellε, the reεulting recombinantε are εcreened by reεtriction analyεis for the presence of both CD4 and CHI sequences which contain in tandem CD4 (EcoRl/Stul) - CHI (SacII(flush)/Pstl) . Oligonucleotide-mediatedsite-directed mutagenesis is then performed to juxtapose the CD4 and CHI sequences in frame. The resulting chimeric DNA molecule contains the V1V2 domains of CD4 fused to the CHI domain of gammal heavy chain. Mutagenesis is performed on single- stranded DNA isolated from recombinant phage from transformed TGI cells (Amersham) . Template DNA is annealed with a 33-mer oligonucleotide (S'-GGGCCCTTGGTGGA GGCGAAAGCTAGCACCACG-3') containing sequences which join the last codon encoding Phe (179) from V1V2 of CD4 to the first codon of the CHI domain for gammal heavy chain (encoding Ala) . After second strand synthesis, double stranded DNA is transformed into competent TGI cells. Isolated plaques are then grown in fresh TGI cells and single-stranded DNA is purified for DNA sequencing. All mutations are confirmed by dideoxy sequencing using the Sequenase system (USB) . Plaques containing the chimeric genes with the correct sequence as determined by restriction analysis are then grown in TGI cells, and the Rf DNA is isolated from the cells.

Rf DNA from the CD4-CH1 chimeric gene is then linearized by digestion with Pstl. The Pstl linearized vector is then BAP treated and ligated to the Pstl-Pstl DNA fragment of the plaεmid pBr gammal containing the hinge, CH2, and CH3 exons of the human gammal heavy chain gene. The correct orientation of the Pstl-Pstl fragment with respect to the chimeric CD4-CH1 fragment is then verified by restriction analysiε. The resulting chimeric gene encodes a protein containing the VIV2 domains of CD4 followed by the CHI, hinge, CH2, and CH3 regions of gammal heavy chain (Figures 2A, 2B, and 4) .

The CD4-IgGl chimeric heavy chain DNA molecule is isolated from the recombinant Rf DNA following Rf linearization with EcoRl. The EcoRl sites in the linearized DNA are filled in with the Klenow fragment of DNA polymerase I. The fluεh ended DNA is then ligated overnight at 15 degrees Celsius with T4 DNA ligase to a 100-fold molar excess of Hindlll linkers. After heat inactivation of T4 DNA ligase for 15 minutes at 70 degrees Celsius, the Hindlll-linkered DNA is extensively digested with Hindlll to liberate a fragment containing the CD4-IgGl chimeric heavy chain gene. This Hindlll fragment is then purified and ligated to the expression vector pcDNA-1 (Invitrogen) , which was previously digested with Hindlll and BAP treated. The resulting plasmid is then transformed into MC1061/P3 cells. Plasmid DNA is isolated from recombinant clones, and verification of the presence of the Hindlll insert and orientation of the insert with respect to the cytomegalovirus (CMV) promoter in the plasmid is made by restriction analysis. The resulting mammalian expression plasmid which encodes a CD4-IgGl chimeric heavy chain is designated CD4-IgGlHC-pRcCMV.

b. Construction of a CD4-kappa chimeric light chain mammalian expresεion vector:

The human kappa light chain constant region is excised from the plasmid pCNkappa light as an Mεel fragment. The purified Mεel fragment iε then made flush ended using the Klenow fragment of DNA polymerase 1. M13mpl8 Rf is then linearized with Hindi, and the flush ended Msel kappa light chain fragment is ligated to M13mpl8 at the flush ended Hindi site in the vector. After transformation of TGI cells, the recombinants are confirmed for the presence of the insert and the correct orientation within the vector by reεtriction analyεis. Rf is purified from infected TGI cellε and digeεted with EcoRl and Smal. The purified vector containing the kappa light chain constant region is then ligated to the EcoRl/Stul fragment of the human CD4 cDNA described above. The reεulting recombinants are then verified for the presence and orientation of both insertε containing in tandem CD4 (EcoRl/Stul) - Ckappa (Msel(flush)/Msel(flush)) , and single-stranded DNA is purified for oligonucleotide-mediated site directed mutagenesis. Template DNA is annealed to a 33-mer oligonucleotide (5'-GATGGTGCAGCCACAGTGAAAGCTAGCACCACG-3 ' ) containing sequences which join the last codon encoding Phe(179) from V1V2 of CD4 to the first codon of the kappa light chain constant domain (encoding thr) . After second strand synthesis, double-stranded DNA is transformed into competent TGI cells, and isolated plaques are grown in fresh TGI cells for DNA sequencing. The presence of the mutation is confirmed by dideoxy sequencing. Plaques containing chimeric genes with the correct sequence are then grown in TGI cells, and Rf DNA is isolated from the cells. The resulting DNA molecule encodes a protein containing the V1V2 domains of CD4 followed by the constant region of kappa light chains (Figures 2A, 2B and 5) .

The CD4-kappa chimeric light chain DNA molecule is isolated from the recombinant Rf DNA following Rf linearization with EcoRl. The EcoRl sites in the linearized DNA are filled in with the Klenow fragment of DNA polymerase I. The flush ended DNA iε then ligated overnight at 15 degrees Celsius with T4 DNA ligase to a 100-fold molar excess of Hindlll linkers. After heat inactivation of T4 DNA ligase for 15 minutes at 70 degrees Celsius, the Hindlll linkered DNA is extensively digested with Hindlll to liberate a fragment containing the CD4-kappa chimeric light chain gene. This Hindlll fragment is then purified and ligated to the expression vector pcDNA-1 (Invitrogen) , which was previously digested with Hindlll and BAP treated. The reεulting plasmid is then transformed into MC1061/P3 cells. Plasmid DNA iε isolated from recombinant clones, and verification of the presence of the Hindlll insert and orientation of the insert with reεpect to the cytomegaloviruε (CMV) promoter in the plaεmid iε made by restriction enzyme analysiε. The resulting mammalian expression plasmid which encodes a CD4- kappa chimeric light chain is designated CD4-kLC-pRcCMV.

3. Co-expression of CD4-IOG1HC-PRCCMV and CD4-kLC-pRcCMV in mammalian cells to produce CD4-IoGl chimeric heterotetramer.

a. Transient expression.

CosM5 cells grown in DMEM containing 10% fetal calf serum are split to 75% confluence. On the following day, the cells are transfected for 16-20 hours with 5 micrograms of CsCl purified CD4-IgGlHC-pRcCMV DNA and 5 micrograms of CsCl-purified CD4-kLC-pRcCMV plasmid DNA by the standard CaPO(4) precipitation technique. After transfection, fresh medium is added to the cells. Analysis of the products synthesized 48-72 hours post-transfection is performed by radiolabelling of transfectants with ³⁵S-methionine for 12-18 hours followed by precipitation of media and cell lysates using anti-CD4 antibodies or by incubation with Protein A- sepharose beads alone followed by SDS-PAGE under reducing or non-reducing conditions. In addition, analysis of media and cell lysates is performed 48-72 hourε post-transfection by standard Western blotting procedures.

b. Stable expression.

Dhfr-Chinese hamεter ovary cells (CHO) are transfected with 20 micrograms of CsCl purified DNA in a ratio of 1000:1000:1 CD4-IgGlHC-pRcCMV:CD4-kLC-pRcCMV:p410 (p410 is an expression plasmid containing the dhfr gene) , although other ratios may also be used. At approximately 3-5 days post-transfection, cells are placed in selective medium (nucleoside-free alpha MEM containing 10% dialyzed fetal calf serum) . At approximately 10-15 days post-εelection, individual cell clones are picked. The clones are then analyzed for stable expression of CD4-IgGl chimeric heterotetramers by several screening techniques, such as ELISA and precipitation with Protein A-sepharose beads followed by SDS-PAGE under reducing or non-reducing conditions. Clones expressing the highest levels are subjected to successive rounds of amplification of the newly introduced DNA sequences in increasing concentrations of methotrexate. Stable CHO cell lines are thus generated which secrete high levels of CD4-

IgGl chimeric heterotetramer.

4. Purification of CD4-IgGl chimeric heterotetramers from CHO conditioned media:

CD4-IgGl chimeric heterotetramers are purified using Protein A-Sepharose column chromatography. CHO cells secreting CD4- IgGl chimeric heterotetramers are grown to high density in roller bottles in medium containing alpha MEM with 10% IgG- free fetal calf serum. Conditioned media is collected, clarified by centrifugation, and diluted 1:1 with PBS with/or without detergent (i.e. Tween) in this and subsequent buffers. The diluted media is then applied to a 5ml column of Protein A-Sepharoεe fast flow previously equilibrated with PBS, at a flow rate of 60ml/hour. After extensive waεhing, the bound material is eluted with lOOmM glycine/HCl, pH 3.5, directly into an aliquot of 1M Tris.HCl pH 8.0 to immediately neutralize the eluted fractions. Fractions are then analyzed by SDS-PAGE under reducing and non-reducing conditions followed by silver staining and pooled.

5. Demonstration of binding of CD4-IgGl chimeric heterotetramer to the envelope glycoprotein gp!20: CosM5 transfectants expressing CD4-IgGl chimeric heterotetramers are incubated for 72 hours in DMEM containing 10% IgG-free fetal calf serum. Unlabelled medium is then collected and used to precipitate ³⁵S-methionine- radiolabelled HIV gpl20. After incubation of CD4-IgGl chimeric heterotetramer containing medium with ³⁵S- methionine-labelled gpl20, the complexes are adsorbed to

Protein A-sepharose. Protein A-sepharose complexes are recovered by centrifugation, and the precipitates are analyzed by SDS-PAGE followed by fluorography.

Alternatively, aliquots of purified CD4-IgGl chimeric heterotetramers from CHO cells are also used to precipitate

³⁵S-radiolabelled gpl20 using the same procedure.

6. Determination of plasma half-life and placental transfer of CD4-IgGl chimeric heterotetramer:

Determination of the plasma half-life and placental transfer are performed by well established techniques. Briefly, rabbits or monkeys are injected intravenously or intramuscularly with purified CD4-IgGl chimeric heterotetramer. At various time points post-injection, plasma sampleε are taken, and the quantity of the CD4-IgGl chimeric heterotetramer present in the serum is measured by ELISA. In addition, pregnant monkeys are also injected either IV or IM with CD4-IgGl chimeric heterotetramer and the concentration determined in the cord blood and the serum of the newborn monkey. Determination and comparison of the quantity of the CD4-IgGl chimeric heterotetramer in the mother's serum as well as in the cord blood and εerum of the newborn indicateε the relative rate of transport acrosε the placenta of theεe moleculeε.

7. Determination of FcR binding and macrophage infectivity of CD4-IgGl chimeric heterotetramer: Determination of FcR binding and macrophage infectivity of CD4-IgGl chimeric heterotetramer are performed by well established techniques. For these studies, U937 cells (a human monocytic cell line expressing FcRl and FcRII) , purified monocyte/macrophage populations from human peripheral blood, and Hela cells constitutively expressing recombinant human FcRs are utilized. In addition, monoclonal antibodies specific for FcRl and FcRII are commercially available. Briefly, radiolabelled monomeric or aggregated CD4-IgGl chimeric heterotetramer is incubated with the above cells and appropriate control cells at 4 degrees Celsius over various time points. At the end of each incubation, the cells are solubized and the cell- associated radioactivity is determined to establish the amount of CD4-IgGl chimeric heterotetramer specifically bound to each cell type. As controls, radiolabelled normal monomeric or aggregated human IgGl are used to determine the levelε of εpecific antibody binding. Furthermore, competition of the radiolabelled component with unlabelled monomeric or aggregated normal human IgGl, or monoclonal antibodies to FcRl or FcRII, will establish the binding efficiency and specificity of CD4-IgGl chimeric heterotetramer to each cell type.

To ascertain whether the CD4-IgGl chimeric heterotetramer mediates enhancement of HIV infection of monocyteε/macrophages, HIV-1 iε incubated with media alone or either monomeric or aggregated CD4-IgGl chimeric heterotetramer at εeveral dilutionε. Aε controlε, sera from normal individuals and HIV-infected individuals are used (31) . After incubation for one hour at 4 degrees Celsiuε, the 'opεonized' viruε is added to the cell typeε described in the paragraph above. At various time points after infection, the media is harvested and assayed for viral reverse transcriptaεe activity to determine the degree of viral infection. As controlε, sCD4, OKT4a or Leu3a are included during the infection of the cells. In addition, various dilutions of the CD4-IgGl chimeric heterotetramer and appropriate controls are incubated first with the cells at 4 degress Celsius to allow binding. HIV is then added and infection assayed by viral reverse transcriptase activity.

B. Results:

A CD4-gammal chimeric heavy chain gene encoding a CD4-gammal chimeric heavy chain homodimer was generated by ligating the leader-Vl-V2 segment of the human CD4 cDNA (4) to the hinge exon of the human gammal heavy chain gene (30) (Figure IA) . The resulting recombinant DNA molecule (designated CD4-IgGl- Rf) encodes the signal sequence and two amino-terminal immunoglobulin-like domains of the CD4 protein (the first 179 amino acids of mature CD4) followed by the hinge (15 amino acids) , CH2 (110 amino acids) , and CH3 (107 amino acids) regions of the gammal heavy chain protein (Figure 3) . This recombinant DNA molecule also contains two introns present within the gammal heavy chain gene: between the H and CH2 domains, and between the CH2 and CH3 domains. This CD4-gammal chimeric gene was deεigned to encode a CD4-gammal chimeric heavy chain homodimer which specifically lacks the CHI domain of the gammal heavy chain. Expression of the CHI domain without accompanying light chains prevents efficient heavy chain secretion from mammalian cells (25) .

In the CD4-gammal chimeric heavy chain homodimer, the hinge region of one chain contains three cyεteine residues, affording the potential of three interchain disulfide bonds

(Figure IB) . In contrast, naturally-occurring human IgGl contains two interchain disulfide bondε between the gammal heavy chainε; the amino-terminal cyεteine in the gammal hinge region iε disulfide bonded to the final cysteine in the light chain constant region, while the two remaining cysteines in the hinge region form two interchain disulfide bonds between the heavy chains.

The CD4-gammal chimeric heavy chain gene was subcloned into the mammalian expression vector pcDNAl. This vector contains the following DNA elements: the cytomegalovirus

(CMV) immediate early promoter and enhancer driving transcription of the CD4-gammal chimeric heavy chain gene; an SV40 polyadenylation sequence; and an SV40 origin of replication which allows replication of the plasmid to high copy number in CosM5 cells. The resulting CD4-gammal heavy chain mammalian expression vector (designated CD4-IgGl- pcDNAl) was transfected into CosM5 cells which were then radiolabelled with ³⁵S-methionine 48-72 hours post- transfection. The radiolabelled medium was analyzed by precipitation with Protein A-sepharose beads and SDS-PAGE followed by fluorography (Figure 6) . Under reducing conditions, a protein migrating at a relative molecular mass (Mr) of approximately 47 kilodaltons is precipitated. When the precipitated material was run on SDS-PAGE under nonreducing conditions, a protein migrating at an Mr of approximately 94 kilodaltons is observed, indicating that the CD4-gammal chimeric heavy chains assemble and are secreted as homodimers. In addition, these results demonstrate that the secreted CD4-gammal chimeric heavy chain homodimers contain an intact immunoglobulin Fc domain since they bind Protein A. Further characterization by Western blot analysis of the proteins secreted into the medium 48-72 hours post-transfection was performed using a rabbit polyclonal antiserum raised against purified soluble human CD4. Similar to the results obtained by precipitation, when the medium was run on SDS-PAGE under reducing conditions, followed by Western transfer to nitrocellulose, the major immunoreactive protein migrates at an Mr of approximately 47 kilodaltons. Under nonreducing conditions, the major immunoreactive protein migrates at an Mr of approximately 94 kilodaltons. Taken together, these results demonstrate that the CD4-gammal chimeric heavy chain is produced and secreted as a homodimer of the predicted molecular weight.

The above results demonstrate that the Fc portion of CD4- gammal chimeric heavy chain homodimer, encoded by the constant regions of the gammal heavy chain gene, binds Protein A and is therefore functionally active. In order to determine if the CD4 portion is functionally intact, CD4- gammal chimeric heavy chain homodimers were assayed for their ability to bind to the HIV exterior envelope glycoprotein, gpl20 (Figure 7) . Unlabelled medium from CosM5 cells transfected with CD4-IgGl-pcDNAl DNA was incubated with ^*?⁵S-methionine-labelled gpl20. CD4-gammal chimeric heavy chain homodimer/gpl20 complexes were precipitated by incubation with Protein A-sepharose beads, and the precipitates were analyzed by SDS-PAGE under reducing conditions followed by fluorography. These results demonstrate that the CD4-gammal chimeric heavy chain homodimer efficiently recognizes HIV gpl20 and binds with high affinity. These observations, taken together with the results described in the above paragraph, demonstrate that CD4-gammal chimeric heavy chain homodimer contains functionally active regions of both CD4 and gammal heavy chain.

In order to stably produce large quantities of the CD4- gammal chimeric heavy chain homodimerε, the CD4-IgGl-pcDNAl vector waε cotransfected with the plasmid p410 (encoding the enzyme dihydrofolate reductase (dhfr) ) into dhfr-Chineεe Hamεter Ovary (CHO) cellε. Approximately two weekε post- transfection, individual clones growing in nucleoside free alpha MEM and 10% dialyzed fetal calf serum (and therefore dhfr+) were iεolated and analyzed for co-expreεεion of CD4- gammal chimeric heavy chain homodimers by precipitation and ELISA. The highest producing cell lines were identified and subjected to stepwise increasing concentrations of methotrexate which selects for amplification of the newly introduced DNA sequences. A CHO cell line expressing 10 micrograms/milliliter of CD4-gammal chimeric heavy chain homodimer was used for stable, constitutive production in roller bottles. The cells were grown to confluence in alpha MEM containing 10% IgG-free fetal calf serum. The cells were then fed every other day and two day old conditioned medium was used for purification of the CD4-gammal chimeric heavy chain homodimer. Conditioned medium was diluted 1:1 with phosphate-buffered saline (PBS) and applied to a 5ml column of Protein A-sepharose fast flow (Pharmacia) at a flow rate of 60 milliliters/hour. The column was then washed with 10 column volumes of PBS and the bound material was eluted with 100 mM glycine pH 3.5. The eluted material was collected directly into 50μl of 1M Tris. HCl pH 8.0 to neutralize the eluant. Fractions having an 0D(280) of greater than 0.1 were analyzed by SDS-PAGE followed by εilver εtaining or Weεtern blot analysis, and the peak fractions were pooled. A single band was specifically eluted from the Protein A-sepharose column with an Mr corresponding to the CD4-gammal chimeric heavy chain homodimer (Figure 8) . Western blot analysis confirms that the eluted protein is immunoreactive with polyclonal antiεerum raiεed against soluble human CD4. In addition, the purified protein retains the ability to bind with high affinity to ³⁵S-methionine-labelled gpl20. These results demonstrate the εtable, high-level production of CD4-gammal chimeric heavy chain homodimerε in mammalian cellε, and the purification of CD4-gammal chimeric heavy chain homodimer which retainε biological function.

The partially purified CD4-gammal heavy chain homodimer purified as described in Figure 8 was effective at preventing HIV binding to CD4 cells (Figure 9) and neutralization of infectivity of a fixed HIV inoculum (Figure 10) . In this later assay, approximately 10-25 μg/ml of CD4-gammal as well as sCD4 were required to prevent 50% of the cultures from becoming infected by HIV.

Further purification of CD4-gammal heavy chain homodimer was achieved using ion-exchange chromatography. The peak fraction from the protein A-sepharose column was applied to a 10ml S-sepharose fast flow column preequilibrated with 50mM BES pH 7.0, at a flow rate of 120ml/hr. After application of the sample, the column was extensively washed with 50mM BES pH 7.0 with increasing salt concentration (see materials and methods) . A single band of CD4-gammal heavy chain homodimer was specifically eluted from the column in 50mM BES pH 7.0 containing 500mM NaCl. These peak fractions were pooled and analyzed by SDS-PAGE and silver staining under non-reducing conditions (Figure 11, lane 1), and reducing conditions (Figure 11, lane 2) . When the purified CD4-gammal chimeric heavy chain homodimer was run on SDS- PAGE under reducing conditions, a doublet was observed which appeared to be due to differences in glycosylation of the CD4-gammal chimeric heavy chain homodimer (data not shown) .

A CD4-IgGlHC chimeric heavy chain gene encoding a CD4-IgGl chimeric heavy chain was generated by ligating the leader- V1-V2 segment of the human CD4 cDNA to the CHI exon of the human IgGl heavy chain gene (Figure 2A) . In addition, a CD4-kappa chimeric light chain gene encoding a CD4-kappa light chain was generated by ligating the leader-Vl-V2 segment of the human CD4 cDNA to the conεtant domain of the kappa light chain gene (Figure 2A) . Theεe CD4-IgGl chimeric heavy chain geneε and CD4-kappa chimeric light chain geneε were deεigned to encode a CD4-IgGl chimeric heterotetramer, in which the CD4-IgGl heavy chain contains a CHI domain for efficient association with kappa light chains. Both the CD4-IgGl chimeric heavy chain and the CD4-kappa chimeric light chain genes were subcloned into the mammalian vectors pRcCMV or pPPI-2. Both vectors contain the cytomegalovirus immediate early promoter and enhancer driving transcription of the chimeric genes. In the vector pRcCMV, a second transcriptional cassette which contains the RSV promoter and enhancer is used to direct the transcription of the neomycin resistance gene. In pPPI-2, a second transcriptional cassette which contains the β- globin promoter directs the tranεcription of the dhfr gene (see supra) . In order to stably produce large quantities of the CD4-IgGl chimeric heterotetramer, the CD4-IgGl chimeric heavy chain expression vector and the CD4-kappa chimeric light chain expression vector were transfected simultaneously (typically the CD4-IgGl chimeric heavy chain gene cloned in pRcCMV was used, and CD4-kappa chimeric light chain gene cloned in pPPI-2 was used in a ratio of 1:1). Approximately two weeks post-tranεfection, individual clones growing in nucleoside-free alpha MEM containing 1 mg/ml G418 and 10% dialyzed fetal calf serum were iεolated and analyzed for co-expression of CD4-IgGl chimeric heavy chains and CD4- kappa chimeric light chainε by immunopreσipitation and ELISA. Figure 12 demonstrates one clone selected and analyzed for expression of both CD4-IgGl chimeric heavy chains and CD4-kappa chimeric light chains. The CHO cell line or the untransfected parental CHO cell line were radiolabelled with ³⁵S-methionine and ³⁵S-cysteine for 16 hours. The radiolabelled medium was analyzed by precipitation with Protein A-εepharose beads and SDS-PAGE under non-reducing conditions followed by fluorography (Figure 12A) . Under non-reducing conditions 2 proteins migrating at relative molecular masεes of approximately 140 kilodaltons and 210 kilodaltons are precipitated. When the precipitated material was run on SDS-PAGE under non-reducing conditions, 2 proteins migrating at relative molecular masses of 69 kilodaltons and 35 kilodaltons were observed. which are consistent with the relative predicted molecular masses of the CD4-IgGl chimeric heavy chains, and CD4-kappa chimeric light chains respectively (data not shown) . Further characterization has shown that the protein migrating at 210 kilodaltons on SDS-PAGE under non-reducing conditions contains both CD4-IgGl chimeric heavy chains and CD4-kappa chimeric light chains which are covalently asεociated, while the protein migrating at 140 kilodaltons on SDS-PAGE under non-reducing conditions contains only CD4- IgGl chimeric heavy chains (Figure 12B) . These data are consistent with the predicted molecular weight for the 210 kilodalton protein being comprised of 2 CD4-IgGl chimeric heavy chains and 2 CD4-kappa chimeric light chains, covalently associated to form a molecule with the structure H_jl^ (H=heavy chain, L=light chain) . Furthermore, the 140 kilodalton protein seen on SDS-PAGE under non-reducing conditions is conεiεtent with the predicted molecular weight of a CD4-IgGl chimeric homodimer with the structure H₂. Taken together, these results indicate that a CHO cell line which expresses both CD4-IgGl chimeric heavy chains and CD4- kappa chimeric light chains iε able to efficiently assemble and secrete CD4-IgGl chimeric heterotetramers.

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Claims

What is claimed is :

1. An expression vector encoding a CD4-gammal chimeric heavy chain homodimer designated CD4-IgGl-pcDNAl (ATCC No. 40951) .

2. A CD4-gammal chimeric heavy chain homodimer encoded by the expression vector of claim 1.

3. A method of producing a CD4-gammal chimeric heavy chain homodimer which comprises:

a) transfecting a mammalian cell with the expresεion vector of claim 1;

b) culturing the reεulting transfected mammalian cell under conditions such that chimeric heavy chain homodimer is produced; and

c) recovering the chimeric heavy chain homodimer so produced.

4. A method of claim 3, wherein the mammalian cell is a COS cell, CHO cell or myeloma cell.

5. A method of inhibiting HIV infection of a CD4+ cell which comprises treating the CD4+ cell with an amount of the CD4-gammal chimeric heavy chain homodimer of claim 2 effective to inhibit infection of the cell.

6. A method of preventing a subject from being infected with HIV which compriseε adminiεtering to the εubject an amount of the CD4-gammal chimeric heavy chain homodimer of claim 2 effective to prevent the εubject from being infected with HIV.

7. A method of treating a subject infected with HIV so as to block the spread of HIV infection which comprises administering to the subject an amount of the CD4-gammal chimeric heavy chain homodimer of claim 2 effective to block the spread of HIV infection.

8. A pharmaceutical composition which comprises the CD4-gammal chimeric heavy chain homodimer of claim 2 in an amount effective to inhibit HIV infection of a CD4+ cell and a pharmaceutically acceptable carrier.

9. A composition of matter comprising a CD4-gammal chimeric heavy chain homodimer of claim 2 and a toxin linked thereto.

10. A composition of claim 9, wherein the toxin is the deglycosylated A chain of ricin, domains II or III of Pseudomonas exotoxin A, or Diphtheria toxin.

11. A diagnostic reagent comprising a CD4-gammal chimeric heavy chain homodimer of claim 2 and a detectable marker linked thereto.

12. A diagnostic reagent of claim 11 wherein the detectable marker is a radioisotope, chromophore, or fluorophore.

13. An expression vector encoding the heavy chains of a CD4-IgGl chimeric heterotetramer designated CD4- IgGlHC-pRcCMV (ATCC No. 75192).

14. An expression vector encoding the light chainε of a CD4-IgGl chimeric heterotetramer designated CD4- kLC-pRcCMV (ATCC No. 75194) .

15. A CD4-IgGl chimeric heterotetramer, the heavy chains of which are encoded by the expression vector of claim 13.

16. A CD4-IgGl chimeric heterotetramer, the light chains of which are encoded by the expression vector of claim 14.

17. A CD4-IgGl chimeric heterotetramer the heavy and the light chains of which are encoded by the expression vectors of claims 13 and 14, respectively.

18. A method of producing a CD4-IgGl chimeric heterotetramer which comprises:

a) cotransfecting a mammalian cell with the expression vector of claim 13 and an expression vector encoding a light chain;

b) culturing the resulting cotransfected mammalian cell under conditions such that the CD4-IgGl chimeric heterotetramer is produced; and

c) recovering the CD4 -IgGl chimeric heterotetramer so produced.

19 , A method of producing an CD4-IgGl chimeric heterotetramer which comprises :

a) cotransfecting a mammalian cell with the expresεion vector of claim 14 and an expression vector encoding an IgGl heavy chain and; b) culturing the resulting cotransfected mammalian cell under conditions such that the chimeric heterotetramer is produced; and

c) recovering the chimeric heterotetramer εo produced. 5

20. A method of producing a CD4-IgGl chimeric heterotetramer which comprises:

a) cotransfecting a mammalian cell with the

10 expression vectors of claim 13 and 14;

b) culturing the resulting cotransfected mammalian cell under conditions such that the chimeric heterotetramer is produced; and

15 c) recovering the chimeric heterotetramer so produced.

21. A method of claim 18, 19 or 20, wherein the on mammalian cell is a COS cell, CHO cell or myeloma cell.

22. A method of inhibiting HIV infection of a CD4+ cell which comprises treating the CD4+ cell with an

25 amount of the CD4-IgGl chimeric heterotetramer of claim 15, 16 or 17 effective to inhibit infection of the cell.

23. A method of preventing a subject from being 0 infected with HIV which comprises administering to the subject an amount of the CD4-IgGl chimeric heterotetramer of claim 15, 16 or 17 effective to prevent the subject from being infected with HIV.

5

24. A method of treating a subject infected with HIV so as to block the spread of HIV infection which comprises administering to the subject an amount of CD4-IgGl chimeric heterotetramer of claim 15, 16 or 17 effective to block spread of HIV infection.

25. A pharmaceutical composition which comprises the

CD4-IgGl chimeric heterotetramer of claim 15, 16 or 17 in an amount effective to inhibit HIV infection of a CD4+ cell and a pharmaceutically acceptable carrier.

26. A composition of matter comprising a CD4-IgGl chimeric heterotetramer of claim 15, 16 or 17 and a toxin linked thereto.

27. A composition of claim 26, wherein the toxin is the deglycosylated A chain of ricin, domains II or III of Pseudomonas exotoxin A, and Diphtheria toxin.

28. A diagnostic reagent comprising a CD4-IgGl chimeric heterotetramer of claim 15, 16 or 17 and a detectable marker linked thereto.

29. A diagnostic reagent of claim 28 wherein the detectable marker is a radioisotope, chromophore or fluorophore.