IE912416A1 - Streptomyces vectors for production of heterologous proteins - Google Patents

Abstract

Nucleic acid sequences and DNA vectors useful for the production of CD4 chimeric proteins, as well as other heterologous proteins, in Streptomyces are disclosed. The nucleic acid sequences of the invention comprise the coding sequence for the signal peptide of the Streptomyces longisporus tyrosine inhibitor (LTI) gene operatively linked with a propeptide sequence consisting essentially of an amino acid sequence coding for from one to about 6 amino acids, the sequence of said amino acids selected to result in the formation in Streptomyces of a protein product having a homogeneous amino terminus after processing to remove said signal peptide formed on the protein product during synthesis of the protein product. In an alternative embodiment of the invention, the propeptide is omitted and the LTI signal peptide is operatively linked with a nucleic acid sequence coding for a heterologous protein which has been modified to code for the sequence lys-ala- at the 3' end. The invention also provides cells transformed with the nucleic acid sequences or vectors of the invention, and methods of using the nucleic acid sequences and vectors of the invention to produce heterologous proteins in Streptomyces.

Description

The present invention relates to production of 5 heterologous proteins in microorganisms. More particularly, the present invention relates to nucleic acid sequences and expression vectors for expression of heterologous proteins, particularly soluble CD-4 proteins, in Streptomyces.

Background of the Invention The principal route of infection of HIV viruses is mediated through attachment of the virus to the CD4 protein. CD4 (also referred to as T4) is a T cell surface glycoprotein that associates with the major histocompatibility complex class II molecules. However, CD4 is also the target for the HIV-l envelope glycoprotein, gpl20. Once the HIV-l virus binds to CD4 on the target cell surface, the viral RHA is then introduced into the target cell by direct fusion of the -2apposing viral and plasma membranes (Maddon et al., Cell, 54:865-874 (1988).

The DNA sequence of the CD4 has been disclosed by Maddon et al. (Cell 42.:93-104 (1985)). From this it was deduced that the mature CD4 protein consists of an extracellular region containing four immunoglobulin-like domains (V1-V4), a transmembrane domain, and a charged intracellular region of approximately 40 amino acids (Maddon et al., supra).

Recombinant soluble derivatives of CD4, such as sT4, comprise proteins in which the transmembrane and cytoplasmic domains which have been deleted, yet retain the ability to bind gpl20. sT4 has also been shown to inhibit both viral infectivity and HIV mediated syncytia formation (Deen et al., Nature 331: 82-84 (1988)). This inhibition results from the association of the sT4 protein with the HIV gpl20 envelope protein, thereby competing with the native CD4 protein on the cell surface. The mechanism by which CD4 and soluble CD4 inhibits viral infectivity and syncytia formation is not completely understood. In vitro, it has been shown that the addition of soluble CD4 to HIV-l or HIV-l infected cells appears to induce the release of gpl20 without a concomitant increase in other viral proteins. This implicates CD4 as an active rather than passive inhibitor of HIV-l.

However, in vivo, the sT4 protein and similar soluble CD4 proteins are rapidly eliminated from serum. This rapid elimination severely limits the clinical application of soluble CD4 as an inhibitor of HIV function.

To prolong the serum clearance time, several soluble CD4 chimeras have been constructed. Traunecker et al. (Nature 331:84-86 (1988)) disclosed CD4 chimeras (V1V2 and V1V2V3V4) in which the carboxy terminal portion of the protein consists of a murine immunoglobulin light chain constant region. Traunecker et al. further disclosed that these constructs were expressed in myeloma cells.

Traunecker et al. (Nature 339:68-70) (1989) subsequently disclose CD4 chimeras (V1V2) fused to murine IgM -3or IgG2 heavy chain constant regions. These constructs were also disclosed to form pentamers.

Seed (EP-A-325,262, published July 26, 1989) and Capon et al. (W089/02922, published April 6, 1989) disclose CD4 chimeras fused to human IgG heavy and light chain constant regions. The proteins disclosed by Seed and Capon et al. were all expressed in mammalian systems, i.e. COS, BHK (baby hamster kidney) and CHO cells. As a result, the disclosed CD4 chimeras are expensive to produce and the overall production capacity is limited, relative to alternate expression systems.

It is thus an object of the present invention to produce CD4 chimeras in a bacterial host system to reduce the cost of production as well as to produce CD4 chimeras which have an increased serum half-life and/or potency against HIV infections relative to soluble CD4.

It is common for proteins produced by recombinant DNA techniques to be fused with a portion of a protein produced by the host cell or other protein. These extra portions typically do not add to the function of the desired protein but their presence can hinder folding or processing of the desired protein. It is thus desirable to be able to produce proteins which are as near as possible to the native protein so these effects can be avoided or reduced. Accordingly, it is also an object of the invention to produce CD4 derivatives, chimeras and other proteins in bacterial host systems that do not contain amino acids derived from host system proteins or that contain only a minimal number of amino acids added to the authentic protein sequence. It is a further object of the invention to provide vectors for producing CD4 chimeras and proteins and other proteins that result in chimeras that do not contain amino acids derived from host system proteins or that contain only a minimal number of amino acids added to the authentic protein sequence. Summary of the Invention The present invention provides nucleic acid sequences and DNA vectors useful for the production of CD4 chimeric proteins, as well as other heterologous proteins, in — 4 — Streptomyces. The nucleic acid sequences of the invention comprise the coding sequence for the signal peptide of the Streptomvces lonqisporus tyrosine inhibitor (LTI) gene operatively linked with a propeptide sequence consisting essentially of an amino acid sequence coding for from one to about 6 amino acids, the sequence of said amino acids selected to result in the formation in Streptomvces of a protein product having a homogeneous amino terminus after processing to remove said signal peptide formed on the protein product during synthesis of the protein product. In an alternative embodiment of the invention, the propeptide is omitted and the LTI signal peptide is operatively linked with a nucleic acid sequence coding for a heterologous protein which has been modified to code for the sequence lys-ala- at the 3'end.

In other aspects the present invention provides Streptomvces cells transformed with a nucleic acid sequence or vector of the invention, and methods of using the nucleic acid sequences of the invention to produce heterologous proteins in Streptomvces.

This invention is more particularly pointed out in the appended claims and is described in its preferred embodiments in the following description.

Detailed Description of the Invention The present invention provides nucleic acid sequences and DNA vectors useful for the production of CD4 chimeric proteins, as well as other heterologous proteins, in Streptomyces. The nucleic acid sequences of the invention comprise the coding sequence for the signal peptide of the Streptomyces lonqisporus tyrosine inhibitor (LTI) gene operatively linked with a propeptide sequence consisting essentially of an amino acid sequence coding for from one to about 6 amino acids, the sequence of said amino acids selected to result in the formation in Streptomvces of a protein product having a homogeneous amino terminus after processing to remove said signal peptide formed on the protein product during synthesis of the protein product. Preferably the amino -5acid sequence of the propeptide is selected to cause processing of the protein product at a position between the end of the nucleic acid sequence coding for the signal peptide and the beginning of the propeptide sequence. In preferred embodiments of the invention, the amino acid sequence of the propeptide is thr-, thr-pro-ala-ala- (SEQ ID NO: 1), or thrpro-ala-ala-ala- (SEQ ID NO: 2). The propeptide sequence thris more preferred, as it is only one amino acid and provides good yield of protein product. In these embodiments of the invention, the nucleic acid sequence of the propeptide preferably comprises the sequence ACC (coding for thr); ACC CCG GCC GCT (SEQ ID NO:3) (coding for thr-pro-ala-ala, SEQ ID N0:l) or ACC CCG GCC GCT GCT (SEQ ID NO:4) (coding for thrpro-ala-ala-ala, SEQ ID N0:2). The nucleic acid sequences of the invention are preferably operably linked with other DNA sequences to form an expression vector which may then be inserted in Streptomyces for production of the heterologous protein. Heterologous proteins thus formed will have a homogeneous N-terminus comprising the sequence of the propeptide.

In an alternative embodiment of the invention, the propeptide is omitted and the LTI signal peptide is operatively linked with a nucleic acid sequence coding for a heterologous protein which has been modified to code for the sequence lys-ala- at the 3’end. The nucleic acid sequence is preferably operably linked with other DNA sequences to form an expression vector which may then be inserted into Streptomyces for production of the heterologous protein. In this embodiment of the invention, signal sequence is cleaved from the heterologous protein at the end of the signal sequence such that the heterologous protein is formed having an N-terminus of lys-ala-X, where X is the remainder of the heterologous protein. This embodiment of the invention is particularly advantageous for production of CD4 derivatives and chimeric proteins.

Applicants have surprisingly and unexpectedly found that by altering only one amino acid at position 2 near the -6N-terminus of CD4 (in the Vl region), a heterologous protein could be formed that is efficiently secreted and correctly processed to remove the entire LTI signal sequence, but which still retained substantial gpl20 binding capacity. By altering the nucleic acid sequence coding for the CD4 derivative or chimera such that it codes for lys-ala- at the amino terminus, the product coded for contained an N-terminus amino acid sequence similar to the sheep L3T4 (CO analogue) receptor, which has an N-terminus of lys-ala-.

When it is desired to use this embodiment of the invention to prepare heterologous proteins other than CD4 derivatives or chimeras, the nucleic acid sequence coding for the heterologous protein may be modified to code for lysala-at the amino terminus by deleting the bases coding for the two amino acids at the N-terminus and substituting a sequence coding for lys-ala for the deleted bases, or a sequence coding for lys-ala may simply be added to the 3'end of the coding sequence for the LTI signal peptide. Thus heterologous proteins having a modified N-terminus having the amino acid sequence lys-ala- will be formed in Streptomyces.

Because of the possible deleterious effects of additional amino acids on the function of heterologous proteins, Applicants desired to produce heterologous proteins that have the sequence of the native protein (or portions of it) with few or no additional amino acids derived from host system proteins, as is not the case when heterologous proteins are formed as fusion proteins. Applicants have surprisingly found that the pro-peptide of the LTI gene can be deleted or modified and heterologous proteins formed that have nearly authentic N-termini and are also efficiently secreted from the Streptomyces host cell.

It became apparent the amino acid sequence surrounding the signal peptide cleavage site can have dramatic effects on signal peptide processing. There are at least two parameters which can influence processing and consequently, secretion and production levels: the physico-chemical nature -Ιοί amino acids and the net charge within the region surrounding the cleavage site.

A statistical analysis of eukaryotic and prokaryotic species signal peptides has shown clear preferences for specific classes of amino acid at specific residues within the region surrounding the signal peptide cleavage site (Vonlleijne, FEBS Letters 244:439-446 (1989)). For example, amino acids residues at positions -3 and -1 are generally small, uncharged residues. Alanine is the most frequent amino acid at these positions. Positions -3 and -1 also show a clear bias against certain classes of amino acids; for example, aromatic, charged, hydrophobic residues and proline were not found at position -1 within 78 eukaryotic signal peptides. Within the amino terminus of the mature protein, proline and glycine are seldom found at position +1.

Both experimental data and statistical analysis indicate a clear preference for a net neutral or negative charge within the region surrounding the cleavage site (Li, et al., Proc. Natl. Acad. Sci. USA 85:7685-7689 (1988), Yanane and Mizushima, 263:19690-19696 (1988), Summers, et al. , J. Biol. Chem. 264:20082-20088 (1989), Von Heijne, J. Mol. Biol. 192:287-290 (1986) of bacterial signal peptides. Li, et al. supra have shown that when the amino terminus of E. coli alkaline phosphate was mutagenized such that the net charge increased from 0 to +2, the total production of alkaline phosphatase dropped 50 fold. In addition, the time required for the precursor processing into the mature product changed from undetectable for the wild type protein to 30 minutes for the mutant with the net charge of +2. The severity of this defect was somewhat decreased by reducing the net charge to +1. Summers, et al. supra described a variety of hybrid proteins between the B-lactamase signal peptide and chicken muscle triosephosphate isomerase. The native triosephosphate isomerase is not secreted into the periplasm of E. coli. This protein could be exported into the periplasm, however, when the arginine residue at position 3 of the triose phosphate isomerase was changed to a proline or serine residue. These -8observations lend credence to Von Heijne's statistical analysis which showed a clear preference for a net negative charge within the cleavage site region.

It has been found that the first two amino acid 5 residues of CD4 (in the VI region) lys-lys- are absolutely required for gp 120 binding activity. In attempts to provide CD4 derivatives and chimeras having authentic N-termini (e.g. KK-V1V2), it was found that CD4 derivatives and chimeras could not be produced.

When the CD4 V1V2 regions are fused to the LTI signal sequence, the resulting product is processed in at least two forms. Applicants surprisingly and unexpectedly found that moving the positively charged lysine residues away from the signal peptide cleavage site by inserting up to five amino acids resulted in a CD4 heterologous protein that has a homogenous N-terminus, was efficiently transported into the culture medium, and still retained gpl20 binding capacity. Even more surprisingly, Applicants found that by changing lysine at position 2 of the CD4 molecule to alanine, a homogeneous CD4 protein having a nearly authentic N-terminus could be produced despite the presence of lysine at the Nterminus end. In addition, this heterologous protein was also efficiently secreted into the medium, and retained gpl20 binding capacity. Heterologous proteins produced using the nucleic acid sequences and vectors of the invention have homogeneous N-termini, i.e., the N-terminus of the product has the same amino acid sequence which gives them several advantages over heterologous proteins that are produced as a mixture of forms. Production costs are lower since fewer separation steps are needed in order to obtain the desired product in a purified form. A single product rather than a mixture of heterologous proteins is preferred for regulatory approval. Additionally, natural folding and function of the protein product is encouraged by the absence of amino acids derived from host system proteins.

In other embodiments the invention is directed to DNA vectors for expressing heterologous proteins in -9Streptomvces which comprise a coding sequence for the heterologous protein operatively linked to a promoter and a nucleic acid sequence coding for the signal sequence of the Streptomvces lonqisporus tyrosine inhibitor gene operatively linked with a propeptide sequence consisting essentially of an oligonucleotide coding for from one to about 6 amino acids, the sequence of said amino acids selected to result in the formation in Streptomvces of a heterologous protein having a homogeneous amino terminus after processing to remove the signal peptide formed on a heterologous protein during synthesis of the heterologous protein. Preferably the coding sequence for the heterologous protein comprises a sequence coding for a HIV gpl20 binding region.

In other embodiments, the invention is directed to DNA vectors for expressing heterologous proteins in Streptomvces which comprises a coding sequence for the heterologous protein operatively linked to a promoter and the Streptomvces lonqisporus trypsin inhibitor gene signal sequence, wherein the sequence coding for the heterologous protein is modified at its 5' end by adding bases coding for the amino acid sequence lys-ala, or by deleting bases coding for the two amino acids at the 5’ end and substituting for the deleted sequence a sequence coding for the amino acid sequence lys-ala. Preferably the coding sequence for the heterologous protein comprises a sequence coding for a HIV gpl20 binding region.

Additional embodiments of the invention are directed to methods for using the DNA vectors of the invention, which comprise the steps of introducing a vector of the invention into a Streptomvces host cell, and growing the host cell in a suitable culture medium.

Further embodiments of the invention are directed to Streptomvces cells transfected with the nucleic acid sequences or DNA sequences of the invention.

The CD4 T lymphocyte receptor protein, herein referred to as CD4, is a surface glycoprotein that interacts with the HIV envelope protein, gpl20. This high affinity -10interaction occurs between gpl20 and the extracellular domain of CD4. The extracellular domain of CD4 comprises 4 regions of limited homology with the immunoglobulin variable (V) regions. The approximate boundary domains for the CD4 variable-like regions (V1-V4) are, respectively, amino acids 100-109, amino acids 175-184, amino acids 289-298, and amino acids 360-369. Thus, as used herein, VI refers to amino acids 1-183 (approximately) and so on.

Removal of the transmembrane and cytoplasmic domains 10 of CD4 results in a soluble receptor protein. Such soluble CD4 proteins, consisting of all or portions of the external region of the human CD4 receptor, have been shown to inhibit HIV-l infection and virus-induced cell fusion (see e.g., Deen et al., Nature 331: 82-84 (1988). As used herein, the term HIV gpl20 binding domain refers to any soluble human CD4 molecule (i.e., lacking the transmembrane and cytoplasmic domains) that binds HIV gpl20 with essentially the same or greater affinity than the full-length CD4 receptor protein. The binding affinity of CD4 for gpl20 can be assayed as described by Arthos et al., (Cell 57:469-481 (1989).

The CD4 molecule of the present invention comprises the minimal HIV gpl20 binding region found in domain VI (amino acids 41-55). Preferably, it includes the V1V2 domains of CD4 or substantially the same sequence (i.e., differs by no more than 15 amino acids). Hence a preferred embodiment of the present invention is V1V2 joined to a human immunoglobulin constant region.

The DNA coding sequence for CD4 is disclosed by Maddon et al. (Cell 42:93-104 (1985) and incorporated by reference herein with the noted exception that the nature amino terminal of human CD4 is Lys-Lys and not Gln-Gly-LysLys as reported by Maddon et al. (see, Littman et al., Cell 55:541 (1988). DNA encoding for CD4 is available from various sources, for example, plasmid pT4B (Maddon et al., supra).

Alternatively, cDNA molecules encoding the CD4 sequence can be derived from mRNA of T lymphocytes which express CD4 using — 11— known techniques (e.g., polymerase chain reaction) or can be synthesized by standard DNA synthesis techniques.

In addition to those proteins exemplified below, one of skill in the art can readily construct DNA molecules which encode further CD4 derivatives. For example, it is within the art to construct amino acid additions, substitutions, deletions, rearrangements (e.g., V1V3, V1V4) and chemical modifications thereof. Such derivatives, however, must retain the ability to bind HIV gpl20.

An inherent advantage of the CD4-Ig chimeras of the present invention is the potential to increase the potency for inhibition/inactivation of an HIV infection relative to soluble CD4. This can be accomplished through the acquisition of Fc effector functions as described more fully below.

The effector functions, which lie primarily within the Fc portion of the immunoglobulin, include: prolonged serum clearance time (Nakamura et al., J Immun 100:376-383 (1968); protein A binding (Deisenhofer et al. Biochem 20:2361-2370 (1981); Fc receptor binding (and Antigen-dependent cellular cytotoxicity (ADCC): mediated through the binding of antibody to Fc receptors on cytotoxic T-cells)? complement fixation (e.g., Clq binding); dimerization (Davies et al., Annu Rev Immunol 1:87 (1983); and placental transfer (Morgan et al., Adv Immunol 40:61-134 (1987).

However, when expressing portions of the immunoglobulin heavy chain, some of the effector functions will be lessened or eliminated. An advantage of the present invention, therefore, is that effector functions which are desired may be conferred back to the molecules of the present invention by the addition of amino acids which are associated with those functions. For example, crystallographic studies of Fc fragments of human IgG indicates that dimerization occurs within the hinge region and CH3 domain (see, e.g., Deisenhofer et al. , J Biochem 20:2361-2370 (1981). Therefore, by adding amino acids from the hinge region or CH3 domain, one may produce IgG fragments that have the ability to dimerize. -12Furthermore the Fc effector functions disclosed above may be derived from any appropriate immunoglobulin constant region or isotope. For example, IgGl, IgG2, IgG3, IgG4, IgM, IgA or IgE, but preferably IgGl. Human IgGl is preferred since this immunoglobulin subclass has been shown to be the most efficient at mediating cell killing by both complement and ADCC (Bruggeman et al., J Exp Med 166:13511361 (1987). The IgGl constant region is comprised of several domains: the CHI, Hinge (h) , CH2 and CH3 as disclosed by Ellison et al. (Nar 10: 4071-79 (1981)), and incorporated by reference herein.

The immunoglobulin constant region of the present invention may include all or a portion of a human immunoglobulin heavy chain in which the variable region has been deleted and replaced with CD4 or an HIV gpl20 binding fragment thereof. In addition, the gpl20 binding fragment thereof. In addition, the gpl20 binding domain can be joined directly (i.e., synthesized or expressed as a single polypeptide) or indirectly (i.e., coupled after synthesis) to the immunoglobulin constant region.

One embodiment is a human immunoglobulin IgG constant region lacking most (i.e., at least 51%) or all of the CH3 domain. Preferably the immunoglobulin constant region comprises most or all of the CH2 domain, that is the CH2 domain plus or minus the hinge (h) region. Preferred embodiment include -CHlhCH2, -hCH2 and -CH2. However, the present invention need not be limited to a single constant region domain.

Another embodiment of the present invention is an aglycosylated human immunoglobulin constant region, comprising at least one domain. Preferred embodiments include, but are not limited to, -CHlhCH2CH3, -hCH2CH3, -CH2CH3, -hCH2, -CH2 and -CH3. The most preferred embodiments include, but are not limited to, -hCH2 and -CH2. Preferably the protein of this particular embodiment is produced in a bacterial host system. It is more preferable that the host cell is from the genus Streptomvces. -13 — Methods for the preparation of DNA which encodes the heavy or light chain constant region of immunoglobulins are taught, for example, by Robinson et al., PCT Application Publication No. W087/02671, published May 7, 1987. Moreover, one skilled in the art can prepare a DNA molecule encoding the human IgGl sequence from any cell line expressing IgGl, for example, ARH-77 (from the ATCC) using known techniques (e.g., polymerase chain reaction). Alternatively, the DNA molecule can be synthesized by standard DNA synthesis techniques.

In addition to those proteins specifically exemplified herein, one of ordinary skill in the art can readily construct DNA molecules which encode further Ig derivatives. For example, amino acid additions, substitutions, deletions, rearrangements and chemical modifications thereof. Other examples include, the substitution of deletion of cysteine residues, mutagenesis to increase Fc receptor binding and/or Clq binding, or the introduction of Fc regions from other immunoglobulin molecules. Such derivatives possess some or all of the Fc effector functions as disclosed below.

Depending on which regions of IgG are expressed, the resulting chimeric protein may bind to antibodies to human IgG, Protein A, complement (specifically Clq), and Fc receptors on appropriate cells of the immune system, e.g. macrophages. Furthermore, the binding of complement (and other effector functions) appears dependent on other factors than simply the presence of relevant sequences on the heavy chain constant region, since different subclasses of human IgG which contain a complement binding sequence differ in their complement binding capacity. Thus, other structural features may be involved.

The recombinant DNA molecule and nucleic acid sequences of this invention may comprise additional DNA sequences, for example, a vector comprising a regulatory element, one or more selectable markers, and sequences that code for replication and maintenance functions. The regulatory region typically contains a promoter found upstream — 14 — from the coding sequence of this invention, which functions in the binding of RNA polymerase and in the initiation of transcription. In other words, the regulatory element or region is operatively linked to the recombinant DNA molecule of this invention. It will be appreciated by one of skill in the art that the selection of regulatory regions will depend upon the host cell employed.

The selectable marker system can be any of a number of known marker systems such that the marker gene confers a selectable new phenotype on the transformed cell. Examples include Streptomvces drug resistance genes such as thiostrepton resistance ribosomal methylase (Thompson et al., Gene 20:51 (1982)) and neomycin phosphotransferase (Thompson et al., supra).

DNA sequences coding for replication and maintenance functions include, for example, those derived from the Streptomvces. pIJlOl derivatives (see, e.g., Keiser et al., Mol Gen Genet 185:223 (1982)) or the Streptomvces SLP1 derived vectors (see, e.g., Bibb et al., Mol Gen Genet 184 : 230 (1981)). The vector of this invention may also contain a marker which permits gene amplification. Such markers which serve to amplify gene copy number in Streptomvces include the gene for spectinomycin resistance (llornemann et al., J Bacteriol 169:2360 (1987)) and arginine auxotrophy (Altenbuchner et al., Mol Gen Genet 195:134 (1984).

The present invention also relates to a host cell transformed with the recombinant DNA molecule of this invention. Such host cell is capable of growth in a suitable culture medium and expressing the protein encoded by the recombinant DNA molecule of this invention. Such host cell is prepared by the method of this invention, i.e., by transforming a desired host cell with the plasmid of this invention. Such transformation is accomplished by utilization of conventional transformation techniques. Preferred host cells belong to the genus Streptomvces. For example such hosts include, but are not limited to, S. lividans. S. coelicolor. S. albus. and S. lonqisporus. Preferred — 15 — embodiments include S. lividans and S. lonqisporus. Other host cells which may be suitable include, but are not limited to, mammalian cells, insect cells, yeast and other bacterial cells (e.g., E. coli. Salmonella. Bacillus). Thus, this invention and the products thereof need not be limited to any specific host cells.

For expression of heterologous proteins in Streptomyces. there are a variety of promoters available. Examples include the galactose-inducible promoter of the Streptomyces galactose operon (Fornwald et al., Proc. Natl. Acad. Sci. USA 84.: 2130 (1987)), the constitutive promoter of the S. lividans /?-galactosidase gene (Eckhardt et al., J. Bacteriol 169:4249 (1987) or Brawner et al., U.S. Patent 4,717,666), the S. lonqisporus trypsin inhibitor gene (see EP-A-264,175, published April 20, 1988), or a temporarily regulated promoter such as that reported in M. echinosporsa (Baum et al., J. Bacteriol 170:71 (1988)). Regions for transcription termination in Streptomyces are derived from the 3' end of several Streptomyces genes, for example the termination signal at the end of the Streptomyces galactose operon or that found at the end of the S. fradiae neomycin phosphotransferase gene (Thompson et al., Proc Natl Acad Sci USA 80.:5190 (1983)). Sequences for protein export in Streptomyces include those isolated from the S. lividans LEP25 10 gene and the S. lonqisporus trypsin inhibitor (LTI) gene (see EP-A-264, 175, published April 20, 1988). Preferably the export or signal sequence is derived from but not limited to the LTI gene. More preferably, the signal sequence is modified (e.g., additions, substitutions, deletions and/or rearrangements) as described in the Examples section.

For the initial expression studies, the CD4-Ig chimeras as more fully described in the Examples, were fused to the LTI prepro sequence. The Streptomyces replication functions for this expression vector were provided by plasmid pIJ351, which is a derivative of PIJ101 (Keiser et al., Mol Gen Genet 185:223 (1982)). The host strain used was wildIE 912416 -16type S. llvidans 1326 (Bibb et al., Mol Gen Genet 184 :230 (1981)) .

The present invention also relates to a method of producing the protein encoded by the recombinant DNA molecule of this invention which comprises culturing the transformed host of the invention in an appropriate culture media and the isolation of such protein. By appropriate culture media is meant that media which will enable such host to express the coding sequence of the invention in recoverable quantity. It will be appreciated by one of skill in the art that the appropriate culture media to use will depend upon the host cell employed. The isolation of the protein so produced is preferably from the host's culture medium, that is, the protein of the invention is preferably exported into the culture medium.

The protein(s) of the present invention may be isolated and purified in accordance with standard techniques. For example, extraction, selective precipitation, column chromatography, affinity chromatography or electrophoresis. For example, the IgG chimeric proteins may be purified by passing a solution containing said chimeric protein through a column which contains immobilized protein A or protein G which selectively binds the Fc portion of the fusion protein. See for example, Reis et al., J Immunol 132:3098-3102 (1984). The chimeric protein may then be eluted by treatment with a chaotropic salt or by a change in pH (e.g. , 0.3M acetic acid) . Alternatively, the protein of the invention may be purified on anti-CD4 antibody columns, or anti-immunoglobulin antibody columns.

The protein and protein product of the present invention may be used for the treatment of HIV viral infections. As a prophylactic, the CD4-Ig chimeras are administered to individuals at high risk to AIDS or individuals who show exposure to HIV by the presence of antibodies to HIV. Administration of an effective amount of the chimeric protein at an early stage of the disease or prior to its onset would act to inhibit infection of CD4* 17lymphocytes. As a therapeutic, administration of CD4-Ig chimeras to individuals infected with HIV would inhibit extracellular spread of the virus.

The dosage ranges for the administration of the 5 chimeric protein of the invention are those to produce the desired effect whereby symptoms of HIV or HIV infection are ameliorated. This amount administered is selected so as to maintain an amount which suppresses or inhibits secondary infection by syncytia formation or by circulating virus throughout the period during which HIV infection is evidenced such as by presence of anti-HIV antibodies, presence of culturable virus and presence of -24 antigen in patent sera. The presence of anti-HIV antibodies can be determined through use of standard ELISA or western assays for example, anti15 gpl20, anti-gp41, anti-tat, anti-p55, anti-pl7 antibodies, etc. The dosage will generally vary with age, extent of the infection, and counterindications, if any, for example, immune tolerance. The dosage can vary from 0.01 mg/kg/day to 50mg/kg/day, but preferably 0.01 to 1.0 mg/kg/day. The chimeric molecule can be administered intravenously, intraperitoneally, intramuscularly, or subcutaneously. If administered parenterally, it can be by single injunction (e.g., bolus) or by gradual perfusion over time.

The proteins of the invention may be used in combination with other agents, for example, in association with agents directed against other HIV proteins, such as reverse transcriptase, protease or tat. An effective therapeutic agent against HIV should prevent virus medicated as well as cell to cell transmission of infection. The proteins may also be used in combination with other antiviral agents, for example, azidothymide (AZT).

The proteins of this invention can also be used as reagents to identify natural, synthetic or recombinant molecules which act as therapeutic agents or inhibitors of CD4+ cell interactions. For example, the proteins can be used in screening assays, such as assays for protein interaction -18measured by ELISA based methodologies to assay for competitors of the CD4 receptor surface domain.

Based on in vitro data showing that soluble CD4 proteins bind to cells expressing HIV env proteins, the proteins of the present invention can also serve as selective targeting molecules for HIV infected cells in vivo. As a target specific carrier protein, CD4-Ig proteins can serve, for example, as the carrier protein for delivery of cytotoxic agents to the infected cells, including the delivery of liposome formulations.

The examples which follow are illustrative, and not to be construed as limiting of the present invention.

EXAMPLES Enzymes used in the genetic manipulations were 15 obtained from commercial sources and were used substantially in accordance with the vendor’s instructions. Except where otherwise indicated, procedures were carried out substantially as described by Maniatis et al., Molecular Cloning. Cold Spring Harbor Laboratory, 1989.

Example 1 Preparation of CD4-IqG Chimeras Using recombinant DNA manipulations, the coding sequence for the V1V2 domains of CD4 are followed by the Hinge-CH2 or Hinge-CH2-CH3 regions of human in various plasmids, which function in various host cells.

The V1V2 region contains amino acids 1-183 (see Maddon et al., Cell 42 : 93-104 (1985) and Littman et al., Cell, 55:541 (1988)). The Hinge-CH2 and Hinge-CH2-CH3 regions comprise IgGl amino acids. 97- 228 and 97-330, respectively (see Ellison et al., NAR 10::4071-79 (1982).

A) VlV2-hCH2DHFR and VlV2-hCH2CH3DHFR (CHO cells): The expression vector ST4184.DHFR, disclosed in EPA-331,356 (published September 6, 1989), was modified to create plasmid V1V2183DHFR: Plasmid ST4184.DHFR was cut with EcoRI and Nhel. and a 682 base paid fragment, containing nucleotide 1-682 of the CD4 DNA was isolated. This fragment was ligated to a synthetic linker encoding CD4 amino acids -19177-183 followed by a ΤΛΑ termination codon. In addition, the synthetic linker created a Hindlll site by changing nucleotides 693 (G to A) and 696 (C to T) of the CD4 sequence. These nucleotide changes do not alter the amino acid sequence.

The resulting fragment, which is flanked by EcoRI and Xbal ends, was ligated into the EcoRI and Xbal site of another ST4184.DHFR plasmid. The sequence of the synthetic linker is substantially as follows: ' CTAGCTTTCCAGAAAGCTTCCTAAT 3' 3’ GAAAGGTCTTTCGAAGGATTAGATC 5' The resulting plasmid, V1V2183DHFR, contains the mouse 0-globin promoter, DHFR gene, SV4 0 poly A region and the CD4 coding sequence for amino acids 1-183.

V1V2183DLHFR was subsequently linearized by Hindlll. which 15 cuts after the V1V2 coding sequence.

Next, a Banll-Aval fragment which encodes the HingeCH2 region (nucleotides 299-677, Ellison et al., supra) was isolated from human IgGl cDNA. (The Hindlll site was introduced by PCR mutagenesis.) Each fragment was ligated into the Hindlll site of a different V1V2183DHFR plasmid with synthetic linkers: i) A Hindlll-Banll linker; to ligate the 3’ end of the V1V2 gene with the 5’ end of Hinge CH2 or Hinge-CH2CH3.

' AGCTTCCAAGGTGGAGCC 3' 3' AGGTTCCACC 5' ii) An Aval-Hindlll linker; used to introduce a stop codon after the CH2 coding sequence and to join the 3' end of the Hinge-CH2 fragement with the 5'end of the V1V2 sequence.

' CCGAGAGTAGTGACTGCAGA 3 ’ 3' CTCATCACTGACGTCTTCGA 5' These constructs maintain the correct reading frame without the addition of non-CD4 or non-IgGl amino acid residues at the Hindlll junction and are herein referred to as VlV2-hCH2DHFR and VlV2-hCH2CH3COS, respectfully.

B) VlV2-hCH2COS and VlV2-hCH2CH3COS (COS Cells): The vectors VlV2-hCH2DIIFR and VlV2-hCH2CH3DLHFR were digested with Nhel. Fragments of 1,817 and 2,147bp, — 20 — respectfully, were isolated which comprises the carboxy end of V2 the complete Hinge-CH2 or Hinge-CH2CH3 coding region, the bovine polyA region, the mouse 0-globin promoter, and part of the mouse DHFR coding region.

The vector V1V2183COS2 (described below) contains the Rouse sarcoma virus LTR, the SV40 early promoter, V1V2, the SV40 poly A early region, and the mouse 0-globin promoter operatively linked to the mouse DHFR coding region. When V1V2183COS2 was digested with Nhel, a 1,723 bp fragment containing the carboxy end of V2 to the mouse DHFR coding region was removed. The Nhel fragments from VlV2-hCH2DHFR and VlV2-hCH2CH3DHFR were then ligated into the Nhel sites of vector V1V2183COS2, which restores, in the correct reading frame, the carboxy end of V2 and the DHFR gene, respectively.

The resultant vectors are herein referred to as VlV2-hCH2COS and VlV2-hCH2CH3COS, which was used to transect COS cells.

Construction of V1V2183COS: To create plasmid V1V2183COS2, plasmid V1V2183COS2, plasmid V1V2183DHFR was digested with EcoRI and Xbal. The EcoRI site was filled in, and a 707 bp fragment consisting of the V1V2 coding region was isolated. This fragment was ligated to plasmid Rst4COS2 (described below), which had been cut with Smal and Xbal to delete the sT4 coding region. The resulting plasmid was V1V2183VOS2.

Construction of Rst4COS2: To create plasmid Rst4COS2, plasmid st4DHFR (Maddon et al., PCT/W088/01304, published February 25, 1988) was digested with Smal and EcoRI. The EcoRI site was filled in and a 338 bp fragment containing the SV40 early promoter was isolated. This fragment was ligated into Rst4DHFR (see below) , which had been cut with BamHI and the sticky ends filled in. The resulting plasmids were screened for orientation, and one in which the SV40 early promoter is in the opposite orientation to RSV LTR was selected as plasmid Rst4COS2.

Construction of RST4DHFR: Plasmid TND (Connors et al., DNA. 7: 651-661 (1988) was digested with Bglll and Hindlll. A 600 bp fragment containing the Rous Sarcoma Virus -21(RSV) LTR was isolated. Using a commercially available linker (New England Biolabs, Beverly, MA) consisting of a Hindlll sticky end, a Smal site, an a EcoRI sticky end, the RHV LTR fragment was ligated to plasmid ST4DHFR (Maddon et al., supa) which had ben digested with Bglll and EcoRI to delete the SV40 early promoter.

C) OmpAvlV2-hCH2 and OmpAVlV2-hCH2CH3 (E. coli) The vectors VlV2-hCH2DHFR nd VlV2-hCH2CH3DHFR were digested with Af1III and Xbal. Isolated were fragments of 746 and l,041bp; respectfully which contains part of VI (approximately 73 amino acids) and the complete Hinge-CH2 or Hinge-CH2CH3 coding regions. These Af111-XbaI fragments replaced the Af111-XbaI fragments of plasmid 0mpAVlV2, disclosed in EP-A-331,356 (published September 6, 1989), to create a vector containing the lambda pL promoter and the OmpA signal sequence fused to either VlV2-hCH2 or VlVl-hCH2CJI3. The vectors, which function in E. coli are here in referred to as OmpAvlV2-hCH2 and OmpAVlV2-hCH2CH3.

D) VlVl-hCH2-Tkk, VlV2-hCH2-KA, V1V2-hCHLStrept and VlV2-hCH2CH3strept (Streptomyces) The vectors VlV2-hCHL2DHFR and VlV2-hCH2CH3DHFR were digested with Af1III and Xbal. Isolated were fragments of 746 and l,041bp, respectfully which contains part of VI and the complete Hinge-CH2 or Hinge-CH2CH3 coding regions. These Af1III and Xbal. fragments replaced the AFlII -Xbal fragments of plasmid 12B1. Plasmid 12B1 contains V1V2 operatively linked to the Streptomyces lividans longisporus trypsin inhibitor (LTI) promoter and signal sequence (see, EP-A264, 175, published April 20, 1988), as well as Streptomyces replication functions as found on plasmid pIJ351 (Keisert et al., Mon Gen Genet 185:223 (1982)). The resultant vectors herein referred to as VlVl-hCH2strept and VlV2-hCH2CH3strept, comprise the LTI signal sequence fused or linked to V1V2Hinge-CH2 or VlV2-Hinge-CH2CH3. These vectors are designed to function in Streptomyces and export VlV2-hCH2 or V1V2hCH2CH# into the culture medium. -22However, the naturally occurring LTI protein is expressed as a prepro protein, that is, the signal sequence is cleaved upon secretion and the pro-sequence is cleaved extracellularly. As a result, the LTI protein isolated can have heterologous amino terminal ends. The expression of VlV2-hCH2 or VlV2-hCH2CII3 from VlV2-hCH2strept and V1V2hCH2CH3strept, respectively, produce this anomaly; i.e. proteins with heterologous amino terminal ends. The ratio of pro-protein to mature protein i.e. lacking all signal sequence amino acid residues can be shifted by variations in culture conditions, e.g. pH, dissolved 02 etc.

As an alternative, the wild-type LTI prepro sequence i.e. signal sequence plus pro-peptide) has been modified to produce proteins with homogeneous amino terminal ends. The wild-type prepro sequence comprises the following amino acids: Met-Arg-Asn-Thr-Ala-Arg-Trp-Ala-Ala-Thr-Leu-Ala-Leu-Thr-AlaThr-Ala-Val-Cys-Gly-Pro-Leu-Thr-Gly-Ala-Ala-Leu-Ala-I-ThrPro-Ala-Ala-Ala-Pro-Ala-Ser; wherein I denotes the cleavage site between the signal sequence and pro-protein. In one construct, the pro-protein sequence was deleted and replaced with Thr (T). (The amino terminus of the mature CD4 protein is KK) . Hence the amino terminus of this chimeric CD4Immunoglobulin protein is Thr-Lys-Lys (TKK) . In another construct, the pro-peptide sequence and the first two amino acids of the mature chimeric protein were deleted and replaced with Lys-Ala (KA). The vectors that encode VlV2-hCH2 are hereby referred to as VlV2-hCH2-TKK and VlV2-hCH2-KA, respectively. These vectors encode VlV2-hCH2 proteins having a homogeneous amino terminus of Thr-Lys-Lys or Lys-Ala, respectively.

The complete nucleotide sequence for vector V1V2hCH2-KA and the corresponding amino acid sequence for the chimeric protein VlV2-hCH2-KA is disclosed as follows: -231 GCGOOCAATA CGCAAAXX CTCTOCOOQC GCXTIO3XG A1TCATTAAT XAGCTGGCA CGACAGGITT CCOGACIGGA AAGCGOXAX TCAGCXAAC GCAA1TAA1G TGAiTITAGCT 121 CACICATEAG GCAXOCATX CTITTCACTT TA'IGCTJCCG GClCGTAlGr TG1GIXAAT 181 IGTGAGCQGA TAACAAITIC ACACAXAWL CAXTA1OC CA1GA1TACG AA1TCGAGCT 241 cxocagctc ctcxaagaga toctooxca googgocqos aaxagccx cagcitcict 301 CGITGCICTC TCGEAA1CAT GTAATCAXA 1TACTOOX GGAXATGAA CGCAAXX3 361 TGCQGGGGAG TOOOGOGACA GCTCAAXGG AATGITTCAG ΧΧΕΑΤΓΑΑΕ TAAGXCAG3 421 AAATOGOXA CTIGQCTGCT TCXQGCGAIC AAGAAOOGCT CAGTICCAGG GGICATCXG 401 TOGAACTCIG TGACTICGX CCACIGA1TC AACACGCAAG GTEACTGAAA CACATCXGGT 541 CGAGGTGTIT lTCOGCXCG GEACA1XGT GOGACTOGOG CTCX300GGIC CGOCACCAAA 601 CCQGAACQX TOOXACAX CICGAATOCT GGGGAAGGAT OOCAO ATC 003 MC TCC 1» Me t Arg Asn Thr 660 GGG OX 103 GGA OX AOG CIC OX CIC TCG OX AX OX GTC IX XA OX 5> A 1 a Ar g Trp Al a Al a Thr Leu Al a Leu Thr Al a Thr Al a Va 1 Cy» Gly Pr o 711 CIC ACC G3A OX QGG CIC GX AAG OX GIG GIG CIG OX AAA AAA OX OT 22*Leu Thr Gl y A 1 a Ala Leu Ala Ly» Al a Val Val Leu Gly Ly» Ly» Gl y Asp 762 KA GIG CPA CIG AX TOT KA GGT TOC CAG AAG AAG AX ΑΊΑ CAA TIC GC 39*Thr Val Gl u Leu Thr Cys Thr A 1 a Ser Gl n Ly» Lys Ser 1 1 e Gl n Phe Hi s 013 TOG AAA AAG TOG AAC CAG ΑΊΆ AAG ΛΤΓ CIG QA AAT CAG OX IX TIC TIA 56»Trp Lys Asn Ser Asn Gl n 1 1 e Lys 1 1 e Leu Gly Asn Gl n Gly S e r Phe Leu 864 ACT AAA QGT OA TOG AAG CIG AAT OT OX XT GC TCA AO 70 AX CTT 73» Thr Ly s Gl y Pro Ser Ly s Leu Asn Asp Arg A 1 a Asp Ser Ar g A r g Ser Leu 915 TOG OC CAA QA AAC nc ax CX ATC ATC AAG AAT CTT AAG AHA OA OC 90» Tr p Asp Gl n Gly Asn Phe Pr o Leu 1 1 e 1 1 e Ly» Asn Leu Ly» 1 1 e Gl u Asp 966 TCA GAT ACT TAC ATC TOT OA GIG OG oc CAG AAG OG OG GIG CAA nc 107»Ser Asp Thr Tyr 1 1 e Cy » Gl u Val Gl u Asp Gl n Ly» Gl u Gl u Va 1 Gl π Leu 1017 CIA GIG TIC OA TIG AGT ax AAC 1CT GAC AX GC CIG CIT CAG OX CAC 124 » Leu Val Phe Gl y Leu Thr A 1 a Asn Ser Asp Thr Hi s Leu Leu Gl n Gl y G 1 n 1068 AX CTC AX CIG AX TIG OG MC OX OCT GOT AGE AX ex 1CA GIG CAA. 141>Ser Leu Thr Leu Thr Leu Gl u Ser Pr o Pr o Gly Ser Ser Pr o S e r Val Gl n 1119 TOT AX AGT OA AGG GGT AAA MC ΑΊΑ CAG OX OX AAG AX CIC TCC GIG 158»Cy s Ar g Ser Pr o Ar g Gly Ly s Asn 1 1 e Gl n Gly Gly Ly» Thr Leu Ser Val 1170 TCT CAG CIG GAG CIC GC GAT ACT OX AX 103 ACA IX TCT GTC TIG OG 175» Ser Gl n Leu Gl u Leu Gl n Asp Ser Gly Thr Trp. Thr Cy » Thr Val Leu G 1 n 1221 MC CAG AAG AAG GIG GAG TIC AAA A3A OC ATC GIG GIG CIA OCT TTC CAG 192» Asn Gl n Ly » Ly s Val Gl u Phe Ly » 1 1 a Asp 1 1 e Val Val Leu A 1 a Phe Gl n 1272 AAA XT TOG AAG GIG GAG ax AAA TCT 1ΟΓ GC AAA ACT CAC KA IX QO·. 209» Ly s A 1 a Ser Ly s Val Gl u Pr o Ly» Ser Cy» Asp Ly» Thr Hi s Thr Cy» Pr o 1323 CHG TOG OCA GA XT CAA CIC CIG OX OO OX TCA GTC TIC CTC nc OX 226»Pr o Cf s Pr o Al a Pro Gl u Leu Leu Gly Gl y Pro Ser Val Phe Lou Phe Pr o 1374 OCA AAA OX AAG GC TCC CIC AIG ATC IX OX AX XT GC G1C AO IX 243» Pr o Ly» Pr o Ly s Asp Thr Leu Met 1 1 e Ser Ar g Thr Pr o Gl u Va 1 Thr Cy s 241425 GIG GIG GIG GTC GIG 260>Val Val Val Asp Val 1476 GIG GTC GGC GIG CTG 277>Val Asp Gly Val Gl o 1527 TTC TTC TGC TO3 TTC 294>Tyr Asn Ser Thr Tyr 1570 TCG CIG ΑΛΤ OT TTG 311>Trp Leu Asn Gly Lys TOC CAC GPA GTC OCT GTG Ser HI s Gl u Asp Pro Gl u GIG στ TTT OT TTG TCA Val Hl s Asn A 1 a Ly» Thr OT GIG GIC TGC GIC C1C Ar g Val Val Ser Val Leu GTG TTC WG TOC ATG GIC Gl u Tyr Lys Cy s Lys Val GIC TTG TTC TTC TCG TTC Val Ly s Phe Asn T r p Tyr TTG OT OT CTG CTG CTG Ly s Pr o Ar g Gl u Gl u Gl n TGC GIC CIG CTC CTG CTC Thr Val Leu Hi s Gl n Asp TCC TTC TTA OT CTC OTt Ser Asn Ly s Al a Leu P r o 1629 Q3C CCC A1C GTG ATA TOC AIC TCC AAA GCC AAA 328>Ata Pro lie Glu Lye Thr lie Ser Lys Ala Lys 1600 TGTCOGAAGC TTOCTAATCT TGAGICGACC TCCTGCOGGG 1740 GGOQGAAOOG TOCIUTGACC TOCQGOOOGA GTITCGTCTO 1000 TOCATTIOGT CTCGIGACGT ATCTOGGOGA GOGPCTCOGG 1060 GOCTOGOGCT GGOGOOQGGC CICGTTCGOC CGCIGGOQGC 1920 OGGCICOQGC CGACGOOGGA TTGCTIGAIC TOOGTGOOGC 1980 COGTOGGOGT COCTOGGGGT GGrGGIGCTC ATCGCQGACG 2040 T7COGAGGOG AGIOGGGCQC GTGGIACOGO CIGCAOGATG 2100 GGOGTGTAAT COCTGGATTA CICCCGOGQC TtOGAOOOOG 2160 CAOOGTCOOC CGOOGETCGT CTCOGGGATC AOGTAOGGCG 2220 ATGTOGGOOC ACTIGOGAGC OGGGOGATGT GCOGGGOGGC 2280 CGICGTOGTC CTOGICGIOG TOCTCCTOIO GCOGIOGQOG 2340 GCTGGGOGTG GGOQGOGAGO ATCTCQGOGT ACGOCIOGCC 2400 OCAOCTOTCT GTOGOOOGOG TO3GOCTGOG OCTGGTOOIC 2460 OOQOOGGATC CTOGTIGCOG TOCTIGOCLT CGOCGGCOOG 2520 CG0QGCQ3GT GTOCGOGIGC CATCOGIOCT OGGIOAOGGC 2500 CGOOGTOGOC GTOGGCCGOO TGGTGCTGAT GGAGGICGTC 2640 CGTGCCCGTG CATCIGCCGC TGGIAGOGOG ΊΌΟΑΠΟΟΛΤ 2700 GOC&7IOGEA CIOGIGQCTG OGCGTGTGGT TOCTOTOCTG 2760 OGIOCTCGGT CATGCOGCCG GTCTGG1OQC OGATCCGTOC 2820 OGTTOCOGOC GGTOGOOGIC TTOTGGICGG COCCCCCG1G 2880 OGTOCTQGGT CTIGGOGATG TTCTOGGCGA GGICGTIGGC 2940 GCTIGAAGTC GTCGOOGIGC COGTOGICGG GOGIGATGGC 3000 OGGCGGICCA CACGGTOCGC CTGTCCCCCT GCCACTOGIG 3060 OGATGGTQGC GACGATCTGC TTO30GGTCC GCTCCOOCTC 3120 CGATOGOGIG GATGIGOGGG TGCCTGOCGT TGATCTGCOC 3180 GGATCATOOC GTOGTTOCOG ATCGGOICIC GGATGCOCTC 3240 OGTOCTTOGC CCGGCGICCG GCCCTCGIGC OGOOCGIGAT 3300 GOOGCCGGCG GCIGTOCGGC GIOTIOCOGG TGCOCTCGTG 3360 GGOQGTCOGT GTGOOCATGG CGGGGOGIGA TGGIGTOCTG 3420 TCATOCTCTG GTOCTCGGCG GCGGTCATOT CCLCGGCCOG 3480 CGCTGACCGG GCTGTGOCTG ATCCSCCCGC ACOGCATCTG 3540 OGGOOGOOGT CTCGGCGACG ATGACGOCGG TGGCAGGGIC 3600 OCITCCTCGC GGCGIOCCQG CIGATCCGOC TCAGCGIOCG 033 CTG CCC CGA. GO TTG Gly Gin Pro Arg Glu ·· GGIOOGGCTG GGCOGGTGCG GIGAOGGAAT GGATCGCTCC ACGOCTCGGC GGTCTGGA'IO AGGTGOGGOG GCTGI'AG'ICC TCCTGGCGCA GTGCTTCGCG TOOGEPCGOG GCTCGTOTCG TGOCGGCGGG GCCGTCCCCG GCCGCCGTCG CCGOGTACGT GGGGGGTGCG TGTETGTCCG OOGCTCCTCG OGGIOGTOGG TGCTGTIGCT TCCIOGCGGC CACCTOCCCC GCCGTCTGCA COGCTCCCGT TCGCCCTCGG GGTOGCCTCG TGGIOGTGGG GTCCCCGGOC OGCTGCIOCC GGCCTCGGIG TCGCCGCCGT GGOOOGGOGT CCCCOT7ITC CGTGCCGTCC CCGGOCCCGT GPGGTGTTCG AACGGGGCGA TTOGTGGGOG GGCGCCTICC GICGOGCTCG GTCTOCTGCC GOGGTTCTGC TTGCOCTGGG GTGQGOGGCG CCGGTCGGCT GGICCGGOOG OCGTCCTGGA CffiQGm TCGGICGCGC GOGGTOGGCG GCCCGGIOCC CTGTOGCTCG TAGGCGCCCG GOOGIOCATC TGGICCGCGA GETGGCGGIC CCCCCGCGCT CITGPGCOaj ATOgIOG_Gj GOCCAGGACC ACGGACGITC CATCAGGGCG OGGCOGCTGC G0GG0GGC1G TACCCGGCGG 25CmOCOCfiG T0QGQCA3CC TOGGTOOQOG CICTOGGGCT CTCDCCATOC AOCOCCIDCG icrcTCTTCT ttctitcxca MjGtgtidcc GCGOGATCGC CCGCTTOGCT QCCATOOGCC TCOOCGGIGT GTQQGCAATr GOGGIDOOGC GCAATCCCTC GGCAIDCCTC CGTACTCTGG A7Q3O3OGCG A7ICTCQGN3 CQGACQCGIC GGTCIDG1OG GAATCX’IGGC AZCAATOQQG CCTTOGOGCT GGIUIGACTT GGH333JCGA ITCTCGTCCG GTCMDCCT GTG1DQ00GA GCIDCGCDGA AGIDGCCCCA GTCQCAfiOCG OCGTIGGCGr CCTC3GAXAG CTIDAOGGIC ATC37IDQSIO TCDTC3GTDGG TO3QQ3CQ3G GCCTICGIDC GGTCN3QCGC CGMCCGQOG G3OCATO3QG CGGTDGOGGT OOGTOGID7D CICIDAOCCA ADCIDTACGT GGACGCIOPG GCTCDGGCGC GOOQCTGGCG CGICCTCOQG TCN3GC7WG CCO3OOCOC CITC1TC7DG ACOOQGTTGA GOOGITCCAC COCGCITCfiC QTAACGT1CC GADOdTIDC ZGGffiGQGGC TIDATCTPCC TIOOGGAGOC CITCIOGGCC GGAGOGAOOG COGICAOCTD GTIDAIGOGG GOGATCAGOG GGOGCAOCTC CITCICAAIC TGCTGCOOCA AOGGCTICAG CGCCGOCICT TOCGICATCA AGICA1CCTG AOCGACTCTG TITGCGGT7C AACriTOCCT AOG1CAICAA COCADGQOOC GGOOGAOGOT OCTG1CTIDG GOGCACAOGA CGGGGGOGGC ATCGGTGGGC GOGCATCGOC GITCGIDCTOG CDGACAGGQC ADGCGGOOGT C1CAGOGCCT GICAGOCICC ICAGOCGCGT GAGGOGACCC TGAGOOCCGT GATCAGGIDG CGGOOGICGA OTUJKM CT3CCCGA1C GT3GT3GGCA AGAICOOGGC 1DOQQCGQGC CGOGGGCGGG CCTCOGEAGA CGGOQGGOCA CAGGOOCAGG AGCTCCOGCA O333CAGCAC TC333GCTAAC G0CT3GTCXT 00GGICA1CA AGGOGAATAC TICATAICOG AGAGOGGGGA GCITTOOCAG AGAGOGAOGA GGCAGCCAIC CGCCATOGTC GCGTAGG3T3 AGCAGCCGGT AGGAOGACCA TGACIDAGIT GGT3CAGCGG AICAIDGATG TCACCAZGOZ CGAOGAOGIC GAGOOCETCA TGCACAGCAT acgigctidc ttcitoccag aggciuicgc GAGAAAOOGC AGGIDGGAGG GjIGOGGGAA 1ITIGOCTOG GGCGGCAIDT CGCGICACAC AOOQGIUICA GCAGTAGATA CGCGGCOGIT PGKX£X£G G3G000GG0G GGCCGATCTC GCAOGAGCAA CGTTOCTGIC TCGCGOGGCT GGAGGTCOGA AGICOGGOCC GTTGCTCTCT OCAGAGG1TC OCIDOGCCAG 1DCCGACGCC 1WO&XCC QOOQGGTAIC CGGOGGGGCT GOGATGQOCA OGAGGOCCTG GAAGOCGAGC GGCICAGOGC AGTQGGCGGA CCtGC&CCG COCICGGICA GXGTCCGIC GAAGTOGCTC gggocagcag gaagtccogg cgccoogogg GDGGCG30G GCGADCAGGC OG1DGGOGGC GQOGGTGOGG TD030QGCGG CCCAGTCCTG GGOGACAGGA ICGAGGTCGG GGAGICOGIC GACICAICAC TOGCCCATTC AGAAGOCCCG TOCAGAOGAC GTGGITCTCCG GGGGITGCCC OOOOCGCAAC CQGGTCTGAC CTCCGAAGIT OCTACGOGTC CGOQOGCGAG GAAGCCAITT TOOGOCTIDG COCACGCOOG GGCOGIDTID OQGTAGGGGA CCIDCATOCG GAOOGOCICC TOGTCCAGCT CO3JGAOGAG CITGAICCGC GOOICGGCOC GGATC7ICGOC CGGIGTCTIT 1CAGICTG0G CAACT2GTIC AGGICOGITT GGOGGOCOGC GAGCGGGCCG CGCGGCCCGG COOOGCIDCG GOOOQGCCGC OGAOOGGCCC GGFITAOGTG G3GCCIGCIC OGCOGACIUZ AGOGGGGAGG GGTGGGGGAC ICGOGGCOCT OGGPGGCCCG TACOCOCGOC GTCGOGGTGC CGIGGGTTGC TGGGGPGCAC CTGCTCCCGC ogtccgoctc cgicgiotc ggicaiccgg GGAACCGGOG GACCIDGACC GCGACGGCTA G3GCGAGGGC GGGCGCCATG CCGACCGCCA CGAICAGOGT COCGCOGAGC AGGAGCAI7TC GGIOCOGGTC CTGGTGGTGC ATCPGICCTC GQGATOGAGC G0G03GGIDC CGGAGGGGGA CTTCCCCTTC CGTTCGTGAT IGCCGGICAG ICACACOCCA GGAAIDGOGT CACTCAAZAC GA0ACCATC GCAAATCCGT CCGAT0CCGC GTCGCGATCC AACA1AAAGA CAACGTTCAT CGCDGOCGGG GIDGAGITCA IDGAGGIYJI’A OGGCAGOGAC AGGAGTOCTT TTCCATCIGA AGCQGFOOQC CPCATOGACT CCTOGATOGT CAAGACATIC GtjCATCGCCC GOGICOCTOG OGGTOGGGAC GT0GTOGITC TOGAOGQGGT A0GGAGGTO3 CTOGOGCTOG GAGOGTOGGG CAIUXGGAG CGGOGICTOC AAAGQQGCAG TCICTCOGGT CGCX3AGGAGG OCATOGOCIT QCIGAAGGOG GAIGGCGACA TTIOOG1GAA CrrXTCTTC GGGAGOGAAA M3O3P333DC CTOGGTl'IOC ATOOOGATGA TGAGOCAGAC AATOGCGCIG CAGGAGAGGA TOGACAGGAA OCIOGGAOGC TOGGTKjCTC GAGCIOGATT AAGGOGTTIG CQQGTOCCGG TOGGCGQOQG TCIGOGACQG GOOQGAOGQC AGGGOGACOG TGGCACIGGC CGTOGTITIA CAAOCTOG1G ATOQOCTIGC AGGAGATCOC OCTiTCDOCA ATODGCCTTC RSSTCT1W4PM POCAAGAGIT GOOGTATTIT CTOCTEACGC ATCTGDQOQG TACAATCIGC TCTGAIGCOG CAIAGTEAAG CQOGOCCTGA CGQGCTIGTG TQCTOOOGGC 0G3GPGCTQC A'lGTGIGAGA GGinTCACC OCTCGIGAIA CGOCEATYIT TAIAGGTTAA AGGTOGCACT TTIOGQOGAA AIGIGCGOGG T1GAAATATG TATOOOCTCA TGAGAGAATA AAGGAAGAGT AIGAGEATTC AACATTIOGG TIGOCTICCT GTiTTIGCTC ACCCAGAAAC GIT3GGT3CA CGAGIGQGIT AGATCGAAGT TmCGOOOC GAAGAACGTT TIGCAATGAT GGTATTAIGC CGTAITGAOG OOGGQCAAGA GAATGACITG GTIGAGEACT CACCAGTCAC AAGAGAAITA TGCAGTGCIG CCATAACCAT GACAACGATC GGAGGAOCGA AGGAGCl’AAC AACICGOCIT GATOGTTCGG AAGOGGAGCT CAGCACGATG CCIGTAGCAA IGGCAACAAC TAGICEAGCT TOCCGGCAAC AATEAAIAGA AGTTGTGCGC TOGGCOCLTG CGGCTQQCIG GOGIGGGTCT CGOGOEATCA TIGCAGCACT AGTEATCEAC AOGAGGQGGA GTCAGGCAAG GATAGGTGOC TCACIGAITA AGCATIGGTA TTAGATIGAT TIAAAACTTG ΑΓ1ΤΓΙΑΑΊΤ TAATCIGATG ACCAAAATCC CTTAAGGiGA AGAAAAGATC AAAGGATdT CTTGAGAKX GITGCTO3AT CIGTGOGGQC GGCAGAAGAT CAACCAGTTG TTGAAGGGGG AGOOGAAGGC OOOQGCCAGG ITQQGOGATA TCGCGAGCCG GAAGATOGIG GGGAACATOG GCCOGATAGT GAICATOCTG GTOGAGAGTG ACATCAGCAG COGAGGITAC GICITCT00C TTCOOGICGT cattcqogac agoqgtaigc agcigatgac GGAACTOGQG GAGAATCOOG ATCGGCIGGC TTOOGACCIG TTOGAGGAGG OGTCTIOOGC OGAGTCTGTG AACdTIOOG TI'ICCCTGGG TCTOGOGQOC AACOGATAAG CGOCICIGIT CGTCAGTGAT GATCACCCOG ACAGOGGATC GOQQGQGAQG CAGGACCOGC CGACGCIGCC QCOGCOOXO GAGCTGCAGG CATGCAAGCT ACTQGGAAAA OOCTGGCGIT AGOCAAGTEA GCIGGCGEAA TAGOGAAGAG (GOOGCACCG GCDCAOOCIG AATGGCGAAT GGCGCCIGAT TMTTCACAC OGCAIATGGT GCACTCTCAG OCAGCOOCGA CAOCOGCCAA CACOOGCPGA ATCQGCTEPC AGACAAGCIG TGAGOGPOIG GTCATCAGOG AAAGGCGCGA GAOGAAAGGG TGICATGATA AEAATOGITT CTEAGAGGTC AACCCCEATT ΤΕΠΤΕΑΤΙΊΤ TCTAAATACA AGOCIGATAA ATOCTIOAAT AATATTGAAA TGTOGCOCTT ΑΠΟΧΤΠΤ TIGOGGCATT GCTGGTGAAA GTAAAAGAIG CTGAAGAICA GGATCTCAAC AGOGGEAAGA TCdTGAGAG GPGCACTTIT AAAGTTCIGC TAIGIGGOGC GCAACTOQGT CGCOGCATAC ACTATTCTCA AGAAAAGCAT CTEAGGGATG GCATGACAGT GAGIGATAAC ACIGOGGOCA ACTEAGTICT OGCTmTIG CAGAAGATGG GGGATCA'IGT GAATGAAGOC AEAOCAAAOG ACGAGOGIGA GriGCGCAAA CTATCAACTG GCGAACTACT CTGGATGGAG GOGGAIAAAG TIGCAGGACC GTITATIGCT GATAAATCTG GAGCCGGIGA G03GCCAGAT QGEAAGCOCT CCCGTATOGT TAIGGATGAA CGAAATAGAC AGATCGCPGA Af.TGIGAGAC CAAGTTrACT CATAEATACT TAAAAGGATG TAGGIGAAGA TOXTITITGA GrrrrcGrrc cactgagogt cagaccccgt TlTTmCIG OGOGEAATVr GCTGCl’IGCA -278700 APCAAAAAAA CCAGOGCTAC CAGCGGIGGT TIGITIGOOG GAIGAAGAX TAOCAACICT 8760 TTTIGCGAAG GTAACTOGCT TCAGCAGAGC GCAGATAOCA AATAGIGTOC TIUIAGIGTA 8820 GOOGIAGTEA GGOCAGCACT IGAAGAACTC IGl’AGCAOOG CC17GATAGC IGGGICIGCT 8880 AATOCTGTTA CCAGIGGC1G Cn-jCCAGIGG CGATAAGIGG TGICTIACOG GGTIGGACTC 8940 AAGAOGATAG 1TAGGGGATA AGGOQCAGOG GTOGGGCTGA AGG3GQGGIT CGTGCAG^GA 9000 GCCCAGCITC GAGOGAACGA OCEACAOOGA ACIGAGATAC CTAGAGCGIG AGCFATCAGA 9060 AAGOGCCAOG CTIGOCGAAG GGAGAAAGQC GGACAGGTAT OCQGTAAGOG GCAGGGIGGG 9120 AACAGGAGPG CGCAGGPGGG AGCTIGCAGG GGGAAAOGOC TGGEATCT1T ATAGICCTGT 9180 COOCTTIGQC CAOCTOIGAC T1GAGCGIGG ATTITIGIGA TGCTOGTCAG GGGGGCGGAG 9240 OCTAIGGAAA AAOGGCAGCA ACGCGGCCTT TTIACGGITC CTGGCCTTtT GCTCGCCTIT 9300 IGCTCACATG TTCTTIOCTG OGTIMGOOC TGA1TC1GIG GATAACCGEA TTAODGCCIT 9360 IGAGTGAGCT GATAOOQCTC GOCGCAGOOG AAOGAOCGAG CGCAGCGAGT CAGTGAGCGA 9420 GGAAGOGGAA G\ Wherein the signal sequence begins at nucleotide 648 and ends at nucleotide 731; VI begins at nucleotide 732 and ends at nucleotide 1286, the Hinge region begins at nucleotide 1287 and ends at nucletotide 1331, CH2 begins at 1332 and continues to the end of the coding sequence.

Example 2 Expression of CD4-IqG Chimeras A) VlVl-hCH2DHFR and VlV2hCH2CI.H3DHFR in CHO cells: VlV2-hCH2 was transfected into CHO cells via electroporation. 15Mg ofVlV2-hCH2 DNA suspended in 15 μΐ phosphate buffered sucrose (PBSucrose) (272mM sucroe, 7 mM sodium phospate pH7.4, 1 mM MgCl2, sterile filtered) was mixed with 1.0 x 10 7 CHO cells (0.8ml) and incubated on ice for 15 min in a Gene Pulser cuvette (Bio-Rad, Richmond, Calif.). The cuvette was then placed in the electrode chamber of the Gene Pulser (Bio-Rad) and one pulse of 700 volts/3/iFd (time constant=0.7) was delivered to the cells. The cells were placed on ice for 10 minutes and then diluted to 10 ml with Growth medium (containing lxITS [insulin, transferrin, selenium; Collaborative Research, Bedford, MA], lx Lipides [Gibo, Grand Island, NY]). Cells were then plated onto 96 well microtiter plates at 3 χ 103 cells/well on a total of 5 plates. After 48 hours the medium was changed to Selection — 28 — medium (i.e., nucleoside-free Growth medium) for DHFR selection.

Cells were maintained on Selective medium for approximately 4 weeks. Western blot analysis was performed using 15% SDS-PAGE gel electrophoresis and anti-sT4 polyclonal antibodies. The VlV2-hCH2 gene product was identified by a single positive band at a molecular weight of approximately 40,000 daltons. Cells from positive wells were moved to 24 well plates and maintained on Selective medium, until ready for scale up. The two clones (3F8 and 2F6) were chosen for scale up. Clone 3F8 was amplified in Selection medium containing 50 nM methotrexate. Cells were replated onto 96 well microtiter plates at 3 x 103 cells/well. Amplification was started at methotrexate levels of 50 nM and were to be increased on each successful round of amplification according to standard protocols.

Similarly, the VlV2-hCH2CH3 vector was transfected into CHO cells by electroporation. Positive clones were selected by growing the cells on nucleoside-free medium and screening the supernatant of healthy wells by Western blot analysis using anti-sT4 antibodies. The resultant positive clones were then amplified using increasing amounts of methotrexate.

B) VlV2-hCH2COS and VlV2-hCH2CH3COS in COS cells: COS cells were obtained from the ATCC (Rockville, MD) and grown according to ATCC recommended protocols. The cells were then transfected with 10 pg of plasmid DMA in 2.5% Nu serum (Collaborative Research, Cambridge, MA) in DMEM (Dulbecco’s minimal essential medium) )GIBCO Grand Island, NY with 400 g/ml DEAE-dextran (Pharmacia) and lOOuM chloroquine (Seed et al., Proc Natl Acad Sci 84:3365-3369 (1987)). Following a 4 hour incubation at 37°C, the medium was removed and the cells were treated with 2ml of 10% dimethylsufoxide in PBS (phosphate buffered saline) for 3 min at 25°, the medium was removed and replaced with DMEM.

Both VlV2-hCH2 and VlV2-hCH2CH3 were expressed and exported into the culture media. In a standard transfection -29in a 6 mm dish (about 106 cells) , 20Mg of VlV2-hCH2 had accumulated int he medium at 70 hrs post-transfection. Approximately 0.4^ig of material was present in the nucleifree cell lystate. For comparison, the corresponding values for sT4, a soluble CD4 protein which had been expressed at high levels in several mammalian cell types were 20pg in the supernatant and lMg in the cell lysate. The VlV2-hCH2 protein in the medium migrated as two closely spaced fragments. Under nonreducing conditions, the VlV2-hCH2 migrated principally a monomer although approximately 10% of the sample appeared to migrate as a dimer. The VlV2-hCH2 protein in the medium bound to gP120 coupled to a sepharose resin.

VlV2-hCH2CH3 was analyzed in the same manner as described above. This protein also expressed well as a secreted product, accumulating to 20-25/xg in the medium and l-2μg in the cell lysate. The protein in the medium migrated as a single fragment of approximately 50 kD by SDS/PAGE under reducing conditions, while the cell lysate sample migrated as two equivalent bands of approximately 40 and 50 kD. Under nonreducing conditions the VlV2-hCH2CH3 migrated principally as a single fragment with a molecular weight consistent with dimer formation. This protein bound to gpl20 coupled to sepharose.

C) OmpAVlV2-hCH2 and ΟπιρΑνΐν2-)ι(:Η2(:Η3 in E. coli The E. coli lambda lysogens, AR58 (Debouck et al., EP-A0,216, 747, published Aril ‘1, 1987) and Arl20 (Mott et al., Proc Natl Acad Sci 82: 88-92 (1985)) were transformed with OPMAVlV2-hCH2 and OmpAvlV2-hCH2CH3, respectively, using standard procedures. Expression of VlV2-hCH2 and V1V230 hCH2CH3 in strain AR58 was accomplished by raising the temperature of the culture media from 32*C to 42 °C (see for example, Rosenberg et al., Meth Enzymol 101:123 (1983)).

The expression of VlV2-hCH2 and VlV2-hCH2CH3 in strain AR120 was accomplished by the addition of nalidixic acid (Nal) to the culture media (see for example, Mott et al., Proc Natl Acad Sci 82:88-92 (1985) as follows. A culture of AR120 was grown at an optical density (at 650nm) of 0.4 -30absorbance units at 37 °C whereupon nalidixic acid was added to a final concentration of 50/ig/ml. The culture was maintained at 37 °C in a shaker incubator for approximately 5 hours at which point the cells were centifuged and subsequently chilled to stop the induction.

For both the heat and Nal inductions, the cell pellets and the clarified culture media (i.e. centrifuged media) were assayed for VlV2-hCH2 and VlV2-hCH2CH3 expression. Both chimeric proteins expressed well, however only a small percentage of VlV2-hCH2CH3 was detected in the culture media (i.e. exported).

D) VlV2-hCH2-TKK, VlVl-hCH2-KA, VlV2-hCH2 strept and VlV2-hCH2CH3strept in Streptomyces Plasmids VlV2-hCH2-TKK, VlV2-hC2strept and VlV2-hC3strept were transformed into S. lividans strain 1326 (Bibb et al., Mol Gen Genet 184:230 (1981)) using standard procedures (see Hopwood et al., Genetic Manipulation of Streptomyces - A Laboratory Manual. F. Crowe & Sons, Ltd., Norwich, England (1985)). Transformants were selected by overlaying the transformation plates with agar (0.4%) containing 100gg/ml thiostrepton. Colonies which express the protein(s) of interest are then grown in trypticase soy broth media supplemented with 5 /xg/ml thiostrepton. All of the chimeric proteins were secreted into the culture media, however the VlV2-hCII2 constructs were expressed at a higher level than the VlV2-hCII2CH3 construct. VlV2-hCH2-KA was expressed at approximately 14 mg/L.

Example 3 Characterization of CD4-IqG Chimeras A) Subunit Structure: The native molecular form of secreted VlV2-hCH2ΚΛ and VlV2-hCH2CH3 were analyzed by electrophoresis on a 15% SDS-polyacrylamide gel under non-reducing conditions. The protein bands were identified by Western Blot analysis and the results are shown in Table I.

B) Antibody Recognition: All of the chimeric proteins made by the present invention are recognized by both anti-CD4 antibodies (Deen -31et al., Nature, 331:82-84 (1988)) and anti-human IgG Fc receptor antibodies (Cappel, Malvern, PA.) See Table I.

C) gpl20 Binding: The chimeric proteins made in S. llvidans, COOS and 5 CHO cells all exhibited gpl2O binding (refer to Table I) either by the immunoprecipation assay as described by Arthoos et al. (Cell, 57.:469 (1989)) or by binding to gpl2O immobilized to sepharose as described below: Samples are diluted to a total volume of 300μ1 with 10 precipitation (ppt) buffer (phosphate buffered saline containing 0.5% dry milk and 0.1% NP40) . A 50% slurry of gpl20 sepharose beads (ΙΟΟμΙ, described below) of wa added to the diluted sample and incubated at 4C for 60 min. The sample was spun (30 sec) in a microfuge to precipitate the chimeric protein/gpl20-Sepharose complex. The complex was washed 5 times with 400μ1 of ppt buffer and once with ice cold phosphate buffered saline. The complex was spun down again and resuspended in 60μ1 loading buffer (Laemmli, Nature, 227: 680 (1970)), boiled for 5 minutes and loaded onto a 15% polyacrylamide gel followed by Western blot analysis. The chimeric proteins expressed in E. coli were not tested for gpl20 binding. gpl20 was obtained from in-house sources. It is also available commercially, e.g., American BioTechnology, Cambridge, ΜΛ. The sepharose beads were prepared as follows: 1.0 liter of Sepharose C1-6B (Pharmacia Fine Chemicals, Piscataway, NJ) was reacted with 52.5 ml epibromohydrin in a basic solution of 0.5N NaOH/30% tetrahydrofuran (THF) at 40’C for four hours. The activated sephrose was collected by filtration on a sintered glass funnel and was washed extensively with 30% THF to remove unreacted epibromohydrin. The product was further washed with water until the washing was neutral. This gel was then resuspended with ethylenediamine (50ml) overnight at room temperature. The gel was filtered and unreacted ethylenediamine was removed by washing the gel with 0.1M acetic acid followed by water. The gel was resuspended in 1.0 liter of water, then was reacted -32with succinic anhydride (25 gm) at pH 6.0. The gel was further washed with 1.0 liter sodium carbonate solution (0.2M) followed by water until the washing was found to be neutral. This gel was finally washed with isopropyl alcohol and stored at 4’C for further use as a moist powder.

D) Protein A & Protein G Binding: The VlV2-hCH2 chimeric proteins expressed in S. lividans and COS cells do not bind to protein A or protein G. In contrast, the VlV2-hCH2CH3 chimeric protein expressed in S. lividans and COS cells do exhibit binding affinity to both proteins A and G. albeit at different affinities. The VlV2-h CH2CH3 produced in Streptomyces has a lower affinity for proteins A and G compared to the VlV2-hClI2CH3 produced in COS ce411s. The results are summarized in Table I. rt ro φ Ul c Φ p o p (X ϋ •rd P 0) E •r| oi c c rl -r| Φ X) o -p c Ρ O Pu p BQ Cu Φ P o o c CJ Η XI C α·Η O U O c rl •P ro N •rl P QJ P ϋ ro P ro ro rH JQ ro H •H ϋ p b. •H 1 c •rH tPP o c ϋ ro Φ Oi O •H P c < P Φ Φ P P P Ρ E P •H 3 φ Φ -H Φ c P B Β Ό E P 3 o o O o Φ 43 3 C C XI c E 3 P O o c o •r| in P B E ro E XI in ω c ro Ό Ul •rl c > ro •r| XJ rH w •H o > • a •H in r—1 in Cl Cl o K w • u u u W n n 43 43 w 33 1 1 1 u I a CJ CJ CJ Cl o Cl > > > S3 > 33 rH rH H U rH U > > > 43 > 43 in o -34Example 4 - Construction of fusion plasmids TKK-VIV2, TPAAAA-VIV2 AND and KA-VIV2.

Fusion plasmids TKK-VIV2, TPAA-VIV2, TPAAA-VIV2 AND KA-VIV2 were constructed as follows from plasmid 12D1.

Construction of plasmid 12B1. Plasmid 12B1 contains VIV2 operatively linked to the Streptomyces lonqisporus trypsin inhibitor (LTI) promoter and signal sequence (see ΕΡ-Λ-264, 175, published April 20, 1988) as well as Streptomyces replication functions as found on plasmid pIJ351 (Keiser et al. Mol Gen Genet 185:223 (1982).

The parental plasmid 12B1 was constructed as follows. The Bbvl cleavage site within the coding sequence for the CD4 signal peptide (between nucleotides 148 and 149 of the CD4 DNA sequence? Maddon, et al. Cell 42.: 93-104 (1985)) was moved by site directed mutagenesis (Kunkel, Proc.

Natl. Acad. Sci. USA 82 : 488-492)) such that the Bbvl cleavage site was placed between nucleotides 150 and 151. This mutation (named 1478) was inserted into a sCD4 minigene which contained the coding sequence for amino acid residues 1 - 129.

An EcoRI + Hindlll fragment containing the 1478 mutation was transferred from M13mpl8 into pUC18 to generate pUCVlpV2(1478). Following Bbvl digestion of pUCVlpV2(1478), it was treated with the Klenow fragment of DNA polymerase I to fill-in the 5' single-stranded sequence, then digested with Hindlll. The Hindlll-blunt end fragment resulting from these manipulations was cloned into pLTI450 which had been digested with AccI. treated with the DNA polymerase I Klenow fragment and digested with Hindlll. The resulting plasmid, 12B1/1477, contains a sCD4 minigene (amino acid residues 1-129) fused to the coding sequence of the LTI signal sequence such that the expressed V1V2 protein will contain at its amino terminus the 6 amino acid LTI pro peptide plus residues 1 and 2 of the mature LTI protein. A Streptomyces replicon and selectable marker were cloned into 12B1/1477 by inserting pIJ351 (Kieser, et al., Mol. gen. Genet. 185:223-238 (1982) using the unique Pstl site within both plasmids. To create a complete V1V2 minigene (amino acid residues 1-183) in the 12B1/1477 plasmid 35an AfIII + Xbal fragment from DHFR V1V2 183#7 in Example 1 was inserted into 12B1/1477 which had been digested with AfIII and Xbal. The resulting plasmid was 12B1.

Construction of plasmid PLTI450: A 0.92 kb Sacl-Kpnl fragment containing the LTI gene was inserted into pUC18 which had been digested with Sacl and Kpnl. This plasmid, pLTI520, was partially digested with Eagl and totally digested with Sail then ligated to a synthetic linker (2x stranded) which had Eagl and Sail ends: '-GGCCGCCGCCCCCGCG (SEQ ID NO:5) CGGCGGCGGGGGCGCAGCT-5' (SEQ ID NO:6) Plasmids obtained from this ligation where screened for insertion of the synthetic linker into the Eagl site located approximately 0.5 kb from the Sacl site. This Eagl site is located at base pair 86 with respect ot the 5' end of the LTI gene. The resulting plasmid contains the LTI promoter and the coding sequence for the signal peptide and signal peptide cleavage site.

To create the fusion plasmids TKK-VIV2, TPAA-VIV2, TPAAA-VIV2 and KA-VIV2 oligonucleotide mutagenesis was used to delete the LTI pro peptide coding sequence and create the amino acid coding sequence of choice following the signal peptide cleavage site. The M13 single stranded DNA used for these mutagenesis was mpl8VlV2/12Bl which was created by inserting a 1.1 kb EcoRI-Xbal fragment from 12B1 into M13mpl8 digested with EcoRI and Xbal. Summarized in Table 3 are the oligonucleotides used for site directed mutagenesis.

Table 3 - Oligonucleotides used to create V1V2 derivatives Derivative Oligo- Oligonucleotide Nucleotide Sequence 'GCCCAGCACCACTTTCTTGGTGGCGAGCGCGGCTCC-3' ’-GCCCAGCACCACTTTCTTAGCGGCCGGGGTGGC-3' '-GCCCAGCACCACTTTCTTAGCAGCGGCCGGGG-3' '-GCCCAGCACCACGGCCTTGGCGAGCGCGGCTCC-3' I, nucleotide sequence 2214 is SEQ ID NO:7; nucleotide sequence 2253 is SEQ ID NO:8; nucleotide seuqence 2254 is SEQ ID NO:9; nucleotide sequence 2216 is SEQ ID TKK-V1V2 2214 TPAA-V1V2 2253 TPAAA-V1V2 2254 KA-V1V2 2216 In Table NO:10. — 36 — A 0.7kb EcoRI-AfIII fragment isolated from the RF form of the mutagenized mpl8VlV2 was exchanged with a 0.75kb EcoRIAf III fragment from 12B1 to generate plasmids pVlV2-2214 (or pTKK-VlV2), pVIV2-2253 (or pTPAA-VIV2) , pVlV2-2254 (or pTPAAA5 VIV2) and pVlV2-2216 (or pKA-VlV2). pVlV2-2214, also named PTKK-V1V2, was created using oligonucleotide 2214 as shown in Table 3 and contains the LTI signal sequence linked to the modified propeptide sequence threonine, and the modified propeptide sequence threonine is linked to V1V2. pVlV2-2253, also named pTPAA-VIVl was constructed using oligonucleotide 2253 as shown in Table 3 and contains the LTI signal sequence linked to the modified propeptide sequence thr-pro-ala-ala. pVlV2—2254, also named pTPAAA-VlV2 was constructed using oligonucleotide 2254 as shown in Table 3 and contains the LTI signal peptide sequence linked with the modified propeptide sequence thr-pro-ala-ala-ala, which is in turn linked with V1V2. pVlV2-2216, also named pKA-VlV2 was constructed using oligonucleotide 2216 as shown in Table 3 and contains the LTI signal peptide sequence which is in turn linked with V1V2.

Example 5 - Construction of flgal *KK-V1V2 fusion plasmid 0gal *KK-VIV2 was constructed by mutagenesis from plasmid p/?gal sT4/7. p/?gal sT4/7 was constructed as follows. Construction of pj3qalsT4/7: Plasmid p*galsT4/7 was constructed from plasmids pUCst4, pIJ702 and p3SSXMCP. p3SSXMCP is a pUC9 (Viera and Messing, Gene 19:259(1982) derivative which contains the /3-galactosidase promoter and signal sequence. Twenty-six base pairs downstream of the coding sequence for the signal peptide cleavage site is an XmnI site (Eckhardt, et al.. J. Bacteriol. 169:4249(1987)).

Inserted into this site was a synthetic linker which contains a BamHI recognition sequence (New England Biolabs, Beverly, Massachusetts) to generate plasmid p3SSX10. This vector was treated with BamHI and reverse transcriptase followed by Xhol digestion. Ligated to this vector was a fragment which had an Ncol end treated with reverse transcriptase to generate a blunt end and a Sail end. Ligation of the filled-in BamHI -37site with the filled-in Ncol site recreated both the BamHI and Ncol sites. The resulting plasmid was p3SSXMCP. pUCsT4 was treated with Xbal and reverse transcriptase followed by Ncol digestion. This 1.1 kb Ncol-Xbal (RT) fragment was then inserted into p3SSXMCP which had been treated with Sacl and T4 DNA polymerase I followed by Ncol digestion. The Streptomyces replication functions were provided by plJ702 which was inserted into p3SSXsT4 via the unique Bglll site on both plasmids. The resulting plasmid was p/3galsT4/7.

A 1.6kb EcoRI-Af1111 fragment from p/?gal sT4/7 was exchanged with the 0.75kb EcoRI-AfIII fragment from mpl8VlV22214 to generate mpl8BgalVlV2. Site directed mutagenesis was used to delete the coding sequence for amino acid residues 9 to -1 of the CD4 signal peptide, change the arginine residues at positions -3 and -4 of the 0gal signal peptide to aspartamate and glutamate, respectively, and delete the coding sequence for residues 1 to 8 of the mature /3-galactosidase protein. These mutations were made using oligonuclotide 2256 whose sequence is ’-GCCCAGCACCACTTTCTTCGCCGCGTCCTCTACGGCGCCTG-3' (SEQ ID NO: 11).

A 1.6kb (EcoRI-AfIII fragment from the RF form of mpl8/?galVlV2-2256 was exchanged with the 0.75kb EcoRI-Af III fragment from plasmid 12B1. The resulting plasmid, pVlV2/?gal25 2256 (/3gal *KK-V1V2) has the following characteristics: (1) V1V2 expression is directed by the Bgal promoter, (2) the coding sequence of the 0gal signal peptide has been altered such that amino acid residue -4 is a glutamate and -3 is an aspartamate, and (3) V1V2 is fused to the /?gal signal sequence such that the predicted N-terminal amino acid sequence of V1V2 is Lys-Lys.

Example 6 - V1V2 Expression in S. lividans Plasmids pVlV2-2214, pVlV2-2253, pVlV2-2254, pVlV2-2216, and pVlV2Bgal-2256 from Example 4 were transformed into S. lividans 1326 (Bibb et al., Mol. Gen. Genet. 184: 230-240 (1981) using standard procedures (Hopwood, et al., Genetic -38Manipulation of Streptomyces - A Laboratory Manual, F. Crowe & Sons Ltd., Norwich, England (1985). Transformants were selected by overlaying the R2YE transformation plates with 3ml of 0.4% agar + 100μg/ml thiostrepton. Transformants which express the protein of interest were grown in trypticase soy broth or MEI + 5% HycaseSF (40g/liter glucose, 50g/liter HycaseSF, 50g/liter Hysoy, lg/liter yeast extract, lg/liter CaCO4, and O.OOlg/liter CoC12 supplemented with 5μ9/πι1 thiostrepton. Cell-free supernatants used for the gpl20 binding assay and the gpl20 affinity chromatography were harvested from cultures grown in MEI + 5% Hycase.

The V1V2 derivatives when expressed in S. lividans were initially characterized by immunoblotting.

Immunoblot Procedure - Cell free supernatants were 15 separated on a 15% polyacrylamide (30:0.8 acrylamide:bis)sodium dodecyl sulfate gel (Laemmli (1970) Nature 227: 680685), then transferred to nitrocellulose (Towbin, et al. (1979) Proc. Natl Acad. Sci. USA 76: 4350-4354). The nitrocellulose filter was processed to detect V1V2 (Brawner et al. (1985) Gene 40: 191-201) using rabbit anti-serum prepared against denatured sCD4 protein. The bound antibody was detected with 125I-protein A. The immunoreacting proteins were visualized by autoradiography.

The TKK-VIV2, TPAA-VIV2, TPAAA-VIV2, KA-VIV2 and 0gal*KK derivatives were produced as a single, immunoreacting band. A doublet was produced from the KK-V1V2 derivative. One of the KK-V1V2 proteins co-migrated with V1V2 produced from CHO cells. The other band, which migrated with a slower mobility than the V1V2 reference protein, may be the result of incomplete processing of the LTI signal peptide or processing at an alternative cleavage site within the LTI signal peptide. Because of the heterogenous nature of the product expressed by the KK-V1V2 derivative no additional studies were undertaken.

The biological activity of these V1V2 derivatives was then determined by gpl20 binding. As judged by quantitative gpl20 immunoprecipitation (Arthos, et al., Cell 57.: 469-481 (1989), -39the Streptomyces produced V1V2 proteins were fully active. Results of the immunopreciptation assay are shown in Table 2. All of the VIV2 derivatives tested produce gpl20 binding of greater than 90%.

V1V2 production of the TKK-V1V2, TPAA-V1V2, TPAAA-V1V2, and fl-gal*KK derivatives was compared to that obtained from plasmid 12B1 which produces V1V2 at levels equal to or greater than 100 mg/1. V1V2 expression levels of the TPAA-V1V2 and TPAAA-V1V2 derivatives were comparable to that obtained from 12B1; KA-V1V2 expression was 70% of that obtained from 12B1; TKK-V1V2 expression was 30% of that from 12B1; Bgal*KK expression was approximately 1 mg/1.

Example 7 - N-terminal amino acid analysis of heterologous proteins produced by plasmids PVIV2-2214, PVIV2-2253, pVIV215 2254, PVIV2-2216 and pVIV2 flgal-2256.

A. Purification of VIV2 from Streptomyces media for Nterminal sequencing by using GP-120 sepharose affinity column 1. Preparation of gpl20 Sepharose Purified gpl20 was immobilized on Sepharose CL-6B (Pharmacia) through amino group using active ester chemistry. Approximately 0.25 mg of gpl20 was coupled to 1ml of resin and binding capacity for sT4 was determined as 20 μ9/π»1. 2. Sample preparation from media.

Fermentation media containing VIV2 constructs was diluted -fold in column equilibration buffer (see below), centrifuged for 15 minutes at 20,000 x g and filtered through 0.2μ Acrodisc low protein binding filter (Gelmen). 3. Gpl20 affinity chromatography Gpl20 Sepharose column (1.6 cm x 6 cm) was equilibrated with 50mM Hepes pH 7.5, 150mM NaCl. The prepared media sample was applied to the column, washed with equilibration buffer until the absorbance reached to baseline, washed again with 50mM Hepes pH7.5, 500mM NaCl to remove any nonspecifically bound impurities. V1V2 was eluted with 0.1 M acetic acid. 4. Removal of peptide impurities from affinity purified V1V2 by using reverse phase HPLC -40V1V2 eluted from affinity column was made to 0.05% TFA solution and applied to C3 RP-IFPLC column (DuPont Pro 10/300, 4.6cm x 250 cm) equilibrated in 0.05% TFA. The column was eluted at lml/min for 60 minutes with linear gradient of 05 60% acetonitrile in 0.05% TFA. The V1V2 product was eluted as a single peak at 50 minutes. Pooled fractions were concentrated in Centricon 3 (Amicon) and used for N-terminal sequencing and amino acid analysis.

B. N-Terminal Sequence The N-terminal amino acid sequence was identified to determine if signal peptides processing had occurred at the predicted cleavage site. The V1V2 proteins were purified from culture supernatant using gpl20 sepharose affinity chromatography and reverse phase HPLC. The partially purified V1V2 preparations were further purified by polyacrylamideSDS gel electrophoresis followed by electroelution into a polyviny1idene difluoride (PVDF) membrane in preparation for N-terminal amino acid sequencing (Matsudaria, J. Biol. Chem. 262:10035-10038 (1987)). The N-terminal amino acid sequence was determined using a gas-phase protein sequenator. This analysis showed that the V1V2 proteins TKK-V1V2, TPAAKK-V1V2, TPAAAKK-V1V2 and KA-V1V2 fused to the carboxy terminus of the LTI signal peptide were correctly processed at the LTI signal peptide cleavage site. The N-terminal amino acid sequence is summarized in Table 2. The N-terminal end of the V1V2 peptide is LYS-LYS. The Bgal*KK-VlV2 derivative, however, was not processed at the natural signal sequence cleavage site. In this derivative, the amino acids preceeding LYS-LYS are derived from the coding sequence at the 3' end of the μ-gal * signal peptide. Signal sequence cleavage for the R(4)E/R(-3)D signal peptide mutation occured within the 0gal signal sequence between positions -8 and -7. -41Table 2 - Characteristics of the VIV2 derivatives Derivative TKK-V1V2 TPAAKK-V1V2 TPAAAKK-V1V2 TPAAAKK-V1V2 KA-V1V2 Bgal*KK-VlV2 N-terminal Amino Acid Sequence Thr-LYS-LYS--gpl2O Binding >90% Thr-Pro-Ala-Ala-LYS-LYS--- >90% Thr-Pro-Ala-Ala-Ala-LYS-LYS--- >90% LYS-LYS ND (1) LYS-Ala--- >90% Ala-Ala-Val-GLu-Asp-Ala-Ala-LYS-LYS >90% (1) Not Determined.

The above description and examples fully disclose the invention including preferred embodiments thereof. However, it is appreciated that the invention is not limited to the particular embodiments described above. Modifications of the methods described above that are obvious to those of ordinary skill in the art are intended to be within the scope of the following claims.

Claims (24)

Claims

1. A nucleic acid sequence comprising a nucleic acid sequence coding for the signal sequence of the Streptomyces lonqisporus tyrosine inhibitor 5 gene operatively linked with a propeptide sequence consisting essentially of an oligonucleotide coding for from one to about 6 amino acids, the sequence of said amino acids selected to result in the formation in Streptomyces of a heterologous protein having a homogeneous amino terminus after processing 10 to remove the signal peptide formed on said heterologous protein during synthesis of said heterologous protein.

2. The nucleic acid sequence of claim 1 wherein said propeptide sequence causes processing of said heterologous protein at a position between the end of the 15 nucleic acid sequence coding for said signal sequence and the beginning of said propeptide sequence.

3. The nucleic acid sequence of claim 2 wherein said propeptide codes for the amino acid threonine.

4. The nucleic acid sequence of claim 2 wherein 20 said propeptide codes for the amino acid sequence thr-proala-ala (SEQ ID NO:1).

5. The nucleic acid sequence of claim 2 wherein said propeptide codes for the amino acid sequence thr-proala-ala-ala (SEQ ID N0:2) 25

6. The nucleic acid sequence of claim 1 wherein said nucleic acid sequence coding for the signal sequence of the Streptomyces lonqisporus tyrosine inhibitor gene comprises the sequence TGC GGA AGG ATG CAC ACA ATG CGG AAC ACC GCG CGC TGG GCA GCC ACC CTC GCC CTC ACG GCC ACC GCC GTC TGC GGA CCC CTC ACC GGA GCC GCG CTC GCC, - 43 or derivative thereof capable of acting as a signal peptide in Streptomvces.

7. The nucleic acid sequence of claim 6 wherein said propeptide sequence comprises the sequence ACC. 5

8. The nucleic acid sequence of claim 6 wherein said propeptide sequence comprises the sequence ACC CCG GCC GCT (SEQ ID NO:3).

9. The nucleic acid sequence of claim 6 wherein said propeptide sequence comprises the sequence ACC CCG GCC 10. GCT GCT (SEQ ID N0:4).

10. A nucleic acid sequence comprising a nucleic acid sequence coding for the signal sequence of the Streptomvces lonqisporus tyrosine inhibitor gene operatively linked with a nucleic acid sequence coding for a polypeptide 15 modified at the 3'end to code for lys-ala-.

11. A DNA vector for expressing heterologous proteins in Streptomvces which comprises a coding sequence for the heterologous protein operatively linked to a promoter and a nucleic acid sequence of claim 1. 20

12. The DNA vector of claim 11 wherein said coding sequence for the heterologous protein is a sequence coding for a HIV gpl20 binding region.

13. The DNA vector of claim 11 wherein said coding sequence for the heterologous protein is a sequence coding 25 for a HIV gpl20 binding region joined to a portion of a human immunoglobulin constant region encoding a polypeptide lacking most or all of the CH3 domain.

14. A DNA vector for expressing heterologous proteins in Streptomvces which comprises a coding sequence - 44 for the heterologous protein operatively linked to a promoter and the Streptomyces lonqisporus trypsin inhibitor gene signal sequence, wherein said sequence coding for the heterologous protein is modified at its 3 ' end by adding 5 bases coding for the amino acid sequence lys-ala, or by deleting bases coding for the two amino acids at the 3' end and substituting for the deleted sequence a sequence coding for the the amino acid sequence lys-ala.

15. The DNA vector of claim 14 wherein said coding 10 sequence for the heterologous protein is a sequence coding for a HIV gpl20 binding region and wherein the bases coding for the two amino acids at the 3· end have been deleted and a sequence coding for the amino acids lys-ala have been substituted for the deleted dequence. 15

16. The DNA vector of claim 14 wherein said coding sequence for the heterologous protein is a sequence coding for a HIV gpl20 binding region joined to a portion of a human immunoglobulin constant region encoding a polypeptide lacking most or all of the CH3 domain and wherein the bases coding 20 for the two amino acids at the 3' end have been deleted and a sequence coding for the amino acids lys-ala have been substituted for the deleted sequence.

17. A method for using the DNA vector of claim 12 comprising the steps of introducing the vector of claim 12 25 into a Streptomyces host cell, and growing said host cell in a suitable culture medium.

18. A method for using the DNA vector of claim 14 comprising the steps of introducing the vector of claim 14 into a Streptomyces host cell, and growing said host cell in 30 a suitable culture medium.

19. A Streptomyces cell transfected with the nucleic acid sequence of claim 1. - 45

20. A Streptomyces cell transfected with the nucleic acid sequence of claim 6.

21. A Streptomyces cell transfected with the DNA vector of claim 7. 5

22. A Streptomyces cell transfected with the DNA vector of claim 10.

23. A nucleic acid sequence or expression vector for the expression of heterologous proteins, substantially as described herein.

24. A process for obtaining a nucleic acid sequence or expression vector, as claimed in any one of the preceding claims, substantially as described herein by way of example.