The present invention is directed to the induction and characterization of a humoral immune response targeting "entry-relevant" gp41 structures. In its broadest aspect, the present invention is directed to methods of raising a neutralizing antibody response to a broad spectrum of HIV strains and isolates. The present invention targets particular molecular conformations or structures that occur at the cell surface of HIV during viral entry into host cells. Such a humoral response can be generated in vivo as a prophylactic measure in individuals to reduce or inhibit the ability of HIV to infect uninfected cells in the individual's body. Such a response can also be employed to raise antibodies against "entry relevant" gp41 structures. These antibodies can be employed for therapeutic uses, and as tools for further illuminating the mechanism of HIV cell entry.

Description of the Invention

SUMMARY OF THE INVENTION

An objective of the present invention is the induction and/or characterization of a humoral immune response targeting "entry-relevant" gp41 structures. In its broadest aspect, the present invention is directed to methods of raising a neutralizing antibody response to a broad spectrum of HIV strains and isolates. The present invention targets particular molecular conformations or structures that occur, or are exposed, following interaction of HIV with the cell surface during viral entry. Such a humoral response can be generated in vivo as a prophylactic or therapeutic measure in individuals to reduce or inhibit the ability of HIV to infect uninfected cells in the individual's body. Such a response can also be employed to raise antibodies against "entry relevant" gp41 structures. These antibodies can be subsequently employed for therapeutic uses, and as tools for further illuminating the mechanism of HIV cell entry.

One aspect of the present invention relates to a method of raising a broadly neutralizing antibody response to HIV by administering to a mammal a peptide or polypeptide comprising an amino acid sequence that is capable of forming a stable coiled-coil solution structure corresponding to or mimicking the heptad repeat region of gp41, (or the N-helical domain of gp41). Peptides of this aspect of the invention are exemplified by P-15 and P-17 described herein.

A second aspect of the present invention relates to a method of raising a broadly neutralizing antibody response to HIV by administering to a mammal a peptide or polypeptide comprising an amino acid sequence that corresponds to, or mimics, the transmembrane-proximal amphipathic .alpha.-helical segment of gp41 (at the C-helical domain of gp41), or a portion thereof. Peptides of this aspect of the invention are exemplified by P-16 and P-18 described herein.

A third aspect of the present invention relates to a method of raising a broadly neutralizing antibody response to HIV by administering to a mammal a composition including one or more peptides or polypeptides which comprise amino acid sequences that are capable of forming solution stable structures that correspond to, or mimic, the gp41 core six helix bundle. This bundle forms in gp41 by the interaction of the distal regions (N-helical domain and C-helical domain) of the transmembrane protein.  This aspect of the invention is also directed to novel mixtures of peptides and polypeptides, including multimeric and conjugate structures, wherein said mixtures and structures form a stable core helix solution structure. A preferred embodiment of this aspect of the invention involves raising antibodies to a physical mixture of N-helical domain peptide and C-helical domain peptide, for example, P-17 and P-18, P-15 and P-16, P-17 and P-16, or P-15 and P-18.

The present invention is also directed to a method of raising a broadly neutralizing antibody response to HIV by administering to a mammal a composition including one or more novel peptides and proteins, herein referred to as conjugates, that mimic fusion-active transmembrane protein structures. These conjugates are formed from two or more amino acid sequences that comprise:

(a) one or more amino acid sequences that are capable of forming a stable coiled-coil solution structure corresponding to or mimicking the heptad repeat region of gp41 (N-helical domain); and

(b) one or more amino acid sequences that correspond to, or mimic, an amino acid sequence of the transmembrane-proximal amphipathic .alpha.-helical segment of gp41 (C-helical domain);

wherein

said one or more sequences (a) and (b) are alternately linked to one another via a bond, such as a peptide bond (amide linkage) or by an amino acid linking sequence consisting of about 2 to about 25 amino acids. These conjugates are preferably recombinantly produced. An example of such a conjugate is described in Example 5.

In a preferred embodiment of this aspect of the invention, one or more of these conjugates folds and assembles in solution into a structure corresponding to, or mimicking, the gp41 core six helix bundle.

The present invention also relates to methods for forming peptides, multimers and conjugates of the invention.

The present invention also relates to pharmaceutical compositions comprising the peptides, multimers and conjugates of the invention and a pharmaceutical acceptable carrier.

The present invention also relates to polyclonal and monoclonal antibodies that are raised to the peptides, multimers and conjugates described in the preceding paragraphs.

The present invention also relates to a method of administering a composition comprising polyclonal or monoclonal antibodies described above to an individual in an amount effective to reduce HIV infection of uninfected cells.

The present invention also relates to a vaccine for providing a protective response in an animal comprising one or more peptides, multimers or conjugates of the present invention together with a pharmaceutically acceptable diluent, carrier, or excipient, wherein the vaccine may be administered in an amount effective to elicit an immune response in an animal to HIV. In a preferred embodiment, the animal is a mammal. In another preferred embodiment, the mammal is a human.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

The transitory-nature of the HIV-entry event, and the structures associated with it, account for the seeming lack of neutralizing epitopes within gp41. These structural components, which form and function only during virus entry, and remain unexposed or are not present in the "native" fusion-inactive envelope complex, constitute a novel set of neutralizing epitopes within gp41. The present invention involves immunization with constructs mimicking these highly conserved, gp41 structures involved in virus entry to elicit the production of broadly neutralizing antibodies targeting these structures. Thus, this invention is the induction of a humoral immune response targeting these "entry relevant" gp41 structures.

One aspect of the present invention relates to a method of raising a broadly neutralizing antibody response to HIV by administering to a mammal a peptide or polypeptide comprising an amino acid sequence that is capable of forming a stable coiled-coil solution structure corresponding to or mimicking the heptad repeat region of gp41 which is located in the N-helical domain as defined herein. Peptides, or multimers thereof, that comprise amino acid sequences which correspond to or mimic solution conformation of the heptad repeat region of gp41 can be employed in this aspect of the invention. The heptad repeat region of gp41 includes 4 heptad repeats. Preferably, the peptides comprise about 28 to 55 amino acids of the heptad repeat region of the extracellular domain of HIV gp41 (N-helical domain, (SEQ. ID NO:1)), or multimers thereof. The peptides can be administered as a small peptide, or conjugated to a larger carrier protein such as keyhole limpet hemocyanin (KLH), ovalbumin, bovine serum albumin (BSA) or tetanus toxoid.

Alternatively, peptides forming a stable coiled-coil solution structure corresponding to or mimicking the heptad repeat region of gp41 can be employed to form polyclonal or monoclonal antibodies that can be subsequently administered as therapeutic or prophylactic agents.

To determine whether a particular peptide or multimer will possess a stable trimeric coiled-coil solution structure corresponding to or mimicking the heptad repeat region of gp41, the peptide can be tested according to the methods described in Wild, C., et al., Proc. Natl. Acad. Sci. USA 89:10537-10541 (1992), fully incorporated by reference herein.

Shown below is the sequence for residues of the HIV-1.sub.LAI gp41 protein that form the N-helical domain of the protein -- see Original Patent.

Two examples of useful peptides include the peptide P-17, which has the formula, from amino terminus to carboxy terminus, of -- see Original Patent.

These peptides are optionally coupled to a larger carrier protein, or optionally include a terminal protecting group at the N- and/or C-termini. Useful peptides further include peptides corresponding to P-17 or P-15 that include one or more, preferably 1 to 10 conservative substitutions, as described below. A number of additional useful N-helical region peptides are described in the section entitled "Peptides."

A second aspect of the present invention relates to a method of raising a broadly neutralizing antibody response to HIV by administering to a mammal a peptide or polypeptide comprising an amino acid sequence that corresponds to, or mimics, the transmembrane-proximal amphipathic .alpha.-helical segment of gp41 (C-helical domain, (SEQ ID NO:4)), or a portion thereof. Useful peptides or polypeptides include an amino acid sequence that is capable of forming a core six helix bundle when mixed with a peptide corresponding to the heptad repeat region of gp41, such as the peptide P-17. Peptides can be tested for the ability to form a core six helix bundle employing the system and conditions described in Chan, D. C., et al, Cell 89:263-273 (1997); Lu, M., et al., Nature Struct. Biol. 2:1075-1082 (1995), fully incorporated by reference herein.

Shown below is the amino acid sequence for residues of the HIV-1.sub.LAI gp41 protein that form the C-helical domain of the protein -- see Original Patent.

Preferred peptides or multimers thereof, that can be employed in this aspect of the invention comprise about 6 or more amino acids, preferably about 24-56 amino acids, of the extracellular C-helical domain of HIV gp41. The peptides can be administered as a small peptide, or conjugated to a larger carrier protein such as keyhole limpet hemocyanin (KLH), ovalbumin, bovine serum albumin (BSA) or tetanus toxoid. This transmembrane-proximal amphipathic .alpha.-helical segment is exemplified by the peptides P-16 and P-18, described below.

Alternatively, peptides or polypeptides comprising amino acid sequences that correspond to, or mimic, the transmembrane-proximal amphipathic .alpha.-helical segment of gp41, or a portion thereof, can be employed to form polyclonal or monoclonal antibodies as therapeutic or prophylactic agents.

Examples of useful peptides for this aspect of the invention include the peptide P-18 which corresponds to a portion of the transmembrane protein gp41 from the HIV-1.sub.LAI isolate, and has the 36 amino acid sequence (reading from amino to carboxy terminus) -- see Original Patent.

These peptides are optionally coupled to a larger carrier protein. Useful peptides further include peptides corresponding to P-18 or P-16 that include one or more, preferably 1 to 10 conservative substitutions, as described below. In addition to the full-length P-18, 36-mer and the full length P-16, the peptides of this aspect of the invention may include truncations of the P-18 and P-16, as long as the truncations is capable of forming a six helix bundle when mixed with P-17. A number of other useful peptides are described in the section entitled "Peptides," below.

A third aspect of the present invention relates to a method of raising a broadly neutralizing antibody response to HIV by administering to a mammal a composition including one or more peptides or polypeptides which comprise amino acid sequences that are capable of forming solution stable structures that correspond to, or mimic, the gp41 core six helix bundle. This bundle forms in gp41 by the interaction of the distal regions of the transmembrane protein, the heptad repeat region and the amphipathic .alpha.-helical region segment roughly corresponding to the N-helical domain and C-helical domain. See FIG. 1 (see Original Patent). The bundle structures that form in native virus are the result of a trimeric interaction between three copies each of the heptad repeat region and the transmembrane-proximal amphipathic .alpha.-helical segment. In the compositions of the present invention, peptide regions interact with one another to form a core six helix bundle. This aspect of the invention is also directed to novel mixtures of peptides and polypeptides, including multimeric and conjugate structures, wherein said structures form a stable core helix solution structure.

This aspect of the invention can employ mixtures of (a) one or more peptides that comprise an amino acid sequence that corresponds to, or mimics, a stable coiled coil heptad repeat region of gp41; and (b) one or more peptides that comprise a region that corresponds to, or mimics, the transmembrane-proximal amphipathic .alpha.-helical segment of gp41. These mixtures are optionally chemically or oxidatively cross-linked to provide additional immunogenic structures that may or may not be solution stable. In addition to physical mixtures, and conventional cross-linking, the peptides (a) and (b) can be conjugated together via suitable linking groups, preferably a peptide residue having at least 2, preferably 2 to 25, amino acid residues. Preferred linking groups are formed from combinations of glycine and serine, or combinations of glycine and cysteine when further oxidative cross-linking is envisioned.

A preferred embodiment of this aspect of the invention involves raising antibodies to physical mixtures of P-17 and P-18, P-15 and P-16, P-17 and P-16 or P-15 and P-18.

The present invention is also directed to a method of raising a broadly neutralizing antibody response to HIV by administering to a mammal a composition including one or more novel peptides and proteins, herein referred to as conjugates, that mimic fusion-active transmembrane protein structures. These conjugates are formed from peptides and proteins that comprise:

(a) one or more amino acid sequences of 28 or more amino acids that are capable of forming a stable coiled-coil solution structure corresponding to or mimicking the heptad repeat region of gp41; and

(b) one or more amino acid sequences that correspond to, or mimic, an amino acid sequence of the transmembrane-proximal amphipathic .alpha.-helical segment of gp41;

wherein

said one or more sequences (a) and (b) are alternately linked to one another via a peptide bond (amide linkage) or by an amino acid linking sequence consisting of about 2 to about 25 amino acids. These peptides and proteins are preferably recombinantly produced.

In a preferred embodiment of this aspect of the invention, one or more of these conjugates folds and assembles into a structure corresponding to, or mimicking, the gp41 core six helix bundle.

Non-limiting examples of the novel constructs or conjugates that can be formed include:

(1) three tandem repeating units consisting of P-17-linker-P-18 (P-17-linker-P-18-linker-P-17-linker-P-18-linker-P-17-linker-P-18),

(2) P-17-linker-P-18-linker-P-17,

(3) P-18-linker-P-17-linker-P-18,

(4) P-17-linker-P-17,

(5) three tandem repeating units consisting of P-15-linker-P-16 (P-15-linker-P-16-linker-P-15-linker-P-16-linker-P-15-linker-P-16),

(6) P-15-linker-P-16-linker-P-15,

(7) P-16-linker-P-15-linker-P-16, and

(8) P-16-linker-P-15;

wherein each linker is an amino acid sequence, which may be the same or different, of from about 2 to about 25, preferably 2 to about 16 amino acid residues. Preferred amino acid residues include glycine and serine, for example (GGGGS).sub.x, (SEQ ID NO:7) wherein x is 1, 2, 3, 4, or 5, or glycine and cysteine, for example (GGC)y, where y is 1, 2, 3 4 or 5. In any of the described constructs, P-15 and P-17 are interchangeable and P-16 and P-18 are interchangeable. An example of such a construct (SEQ ID NO:77) is shown in FIG. 7 (see Original Patent), along with the corresponding nucleic acid sequence (SEQ ID NO:78) used for recombinant expression of the construct.

Alternatively, polyclonal or monoclonal antibodies can be raised against the immunogenic mixtures and conjugates described in this aspect of the invention. Such antibodies can be employed as therapeutic or prophylactic agents.

In preferred aspects of the invention, the methods can be employed to immunize an HIV-1-infected individual such that levels of HIV-1 will be reduced in such individual. In another aspect, the methods can be employed to immunize a non-HIV-1-infected individual so that, following a subsequent exposure to HIV-1 that would normally result in HIV-1 infection, the levels of HIV-1 will be non-detectable using current diagnostic tests.

Immunogen Preparation

Induction and interpretation of a humoral immune response directed against gp41 structural epitopes requires both immunogen preparation and antibody characterization. Synthetic peptides and recombinant proteins can both be used to generate antigenic structures corresponding to gp41 fusion active domains.

In one aspect of the invention, target immunogens model the heptad repeat region delineated by the P-17 peptide (capable of forming a trimeric coiled-coil structure). In another aspect of the invention, target immunogens model the transmembrane-proximal amphipathic .alpha.-helical segment delineated by the P-18 peptide. This region in the absence of the coiled-coil core exhibits random coil solution structure. (Wild, C., et al., Proc. Natl. Acad. Sci. USA 89:10537-10541 (1992); Wild, C., et al., AIDS Res. Hum. Retroviruses 9:1051-1053 (1993); Wild, C., et al., Proc. Natl. Acad. Sci. USA 91:9770-9774 (1994)). In another aspect, combinations of these target imnmunogens are employed for raising antibodies.

In another aspect of the invention the target immunogen is the six helix hydrophobic bundle. This bundle is formed by the specific association of these two distal regions of the ectodomain of gp41 (Chan, D. C., et al, Cell 89:263-273 (1997); Lu, M., et al., Nature Struct. Biol. 2:1075-1082 (1995)). These constructs will mimic entry determinants which form and function during HIV-1 entry.

Synthetic Methods of Immunogen Preparation

Immunogens can be prepared by several different routes. The constructs can be generated from synthetic peptides. This involves preparing each sequence as a peptide monomer followed by post-synthetic modifications to generate the appropriate oligomeric structures. The peptides are synthesized by standard solid-phase methodology. To generate a trimeric coiled-coil structure, the P-17 peptide monomer is solubilized under conditions which favor oligomerization. These conditions include a 20 mM phosphate buffer, pH 4.5 and a peptide concentration of 100 .mu.M (Wild, C., et al., Proc. Natl. Acad. Sci. USA 89:10537-10541 (1992)). The structure which forms under these conditions can be optionally stabilized by chemical crosslinking, for example using gluteraldehyde.

Alternatively, a protocol which makes use of intermolecular disulfide bond formation to stabilize the trimeric coiled-coil structure can be employed in order to avoid any disruptive effect the cross-linking process might have on the structural components of this construct. This approach uses the oxidation of appropriately positioned cysteine residues within the peptide sequence to stabilize the oligomeric structure. This requires the addition of a short linker sequence to the N terminus of the P-17 peptide. The trimeric coiled-coil structure which is formed by this approach will be stabilized by the interaction of the cysteine residues. The trimer is separated from higher order oligomeric forms, as well as residual monomer, by size exclusion chromatography and characterized by analytical ultracentrifugation. These covalently stabilized coiled-coil oligomers serve as the core structure for preparation of a six helix bundle.

To accomplish preparation of a six helix bundle, an excess of P-18 peptide is added to the purified core structure. After incubation the reaction mixture is subjected to a cross-linking procedure to stabilize the higher order products of the specific association of these two peptides. The desired material is isolated by size exclusion chromatography and characterized by analytical ultracentrifugation. The immunogen corresponding only to the P-18 peptide requires no specific post-synthetic modifications. Using this approach, three separate target constructs are generated rapidly and in large amounts.

Recombinant Methods of Immunogen Preparation

Another method for preparing target immunogens involves the use of a bacterial expression vector to generate recombinant gp41 fragments. The use of an expression vector to produce the peptides and polypeptides capable of forming the entry-relevant immunogens of the present invention adds a level of versatility to immunogen preparation.

New and modified forms of the antigenic targets are contemplated as the structural determinants of HIV-1 entry are better understood. The recombinant approach readily accommodates these changes. Also, this method of preparation allows for the ready modification of the various constructs (i.e. the addition of T- or B-cell epitopes to the recombinant gp41 fragments to increase immunogenicity). In addition, a form of the six helix hydrophobic core structure is generated which will not require additional stabilization, since determining the antigenic nature of this structure is important. Finally, these recombinant constructs can be employed as a tool to provide valuable insights into additional structural components which form and function in gp41 during the process of virus entry.

Thus, as part of the invention, novel fusion polypeptides (conjugates) are also provided, as are vectors, host cells and recombinant methods for producing the same. The present invention provides isolated nucleic acid molecules comprising a polynucleotide encoding the conjugates of the invention.

The present invention also relates to recombinant vectors, which include the isolated nucleic acid molecules of the present invention, and to host cells containing the recombinant vectors, as well as to methods of making such vectors and host cells and for using them for production of fusion polypeptides or peptides by recombinant techniques.

The polynucleotides may be joined to a vector containing a selectable marker for propagation in a host. Generally, a plasmid vector is introduced in a precipitate, such as a calcium phosphate precipitate, or in a complex with a charged lipid. If the vector is a virus, it may be packaged in vitro using an appropriate packaging cell line and then transduced into host cells.

The DNA insert should be operatively linked to an appropriate promoter, such as that described herein. Other suitable promoters will be known to the skilled artisan. The expression constructs will further contain sites for transcription initiation, termination and, in the transcribed region, a ribosome binding site for translation. The coding portion of the mature transcripts expressed by the constructs will preferably include a translation initiating at the beginning and a termination codon (UAA, UGA or UAG) appropriately positioned at the end of the polypeptide to be translated.

As indicated, the expression vectors will preferably include at least one selectable marker. Such markers include dihydrofolate reductase or neomycin resistance for eukaryotic cell culture and tetracycline or ampicillin resistance genes for culturing in E. coli and other bacteria. Representative examples of appropriate hosts include, but are not limited to, bacterial cells, such as E. coli, Streptomyces and Salmonella typhimurium cells; fungal cells, such as yeast cells; insect cells such as Drosophila S2 and Spodoptera Sf9 cells; animal cells such as CHO, COS and Bowes melanoma cells; and plant cells. Appropriate culture mediums and conditions for the above-described host cells are known in the art.

Introduction of the construct into the host cell can be effected by calcium phosphate transfection, DEAE-dextran mediated transfection, cationic lipid-mediated transfection, electroporation, transduction, infection or other methods. Such methods are described in many standard laboratory manuals, such as Davis et al., Basic Methods In Molecular Biology (1986).

The polypeptide may be expressed in a modified form, such as a fusion protein, and may include not only secretion signals, but also additional heterologous functional regions. For instance, a region of additional amino acids, particularly charged amino acids, may be added to the N-terminus of the polypeptide to improve stability and persistence in the host cell, during purification, or during subsequent handling and storage. Also, peptide moieties may be added to the polypeptide to facilitate purification. Such regions may be removed prior to final preparation of the polypeptide. The addition of peptide moieties to polypeptides to engender secretion or excretion, to improve stability and to facilitate purification, among others, are familiar and routine techniques in the art.

The fusion protein can be recovered and purified from recombinant cell cultures by well-known methods including ammonium sulfate or ethanol precipitation, acid extraction, anion or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, affinity chromatography, hydroxylapatite chromatography and lectin chromatography. Most preferably, high performance liquid chromatography ("HPLC") is employed for purification. Depending upon the host employed in a recombinant production procedure, the polypeptides of the present invention may be glycosylated or may be non-glycosylated. In addition, polypeptides of the invention may also include an initial modified methionine residue, in some cases as a result of host-mediated processes.

A bacterial expression vector (kindly provided by Dr. Terrance Oas, Duke University) was developed specifically for the expression of small proteins. This plasmid, pTCLE-G2C, is based on pAED4, a T7 expression vector. A modified TrpLE (Yansura, D. G., Methods Enzymol. 185:161-166 (1990)) fusion peptide (provided by Dr. Peter Kim) was inserted after the T7 promoter (Studier, F. W., et al., Methods Enzymol. 185:60-89 (1990)). There is an in frame Nde I site at the end of the TrpLE peptide that encodes a methionine cyanogen bromide (CNBr) cleavage site. This vector was used in an earlier study to express a recombinant form of the P-17 peptide (Calderone, T. L., et al., J. Mol. Biol. 262:407412 (1996)) and has been modified to expresses the P-18 peptide.

To generate a six helix hydrophobic core structure, several combinations of the heptad repeat (for example, P-17 or P-15) region and the amphipathic .alpha.-helical (for example, P-16 or P-18) segment of gp41 are separated by a flexible linker of amino acid residues. For example, (GGGGS).sub.3 (SEQ ID NO:7) can be encoded into the vector. This is accomplished by standard PCR methods. The (GGGGS).sub.3 (SEQ ID NO:7) linker motif is encoded by a synthetic oligonucleotide which is ligated between the P-17 and P-18 encoding regions of the expression vector.

All constructions are characterized by multiple restriction enzyme digests and sequencing. The success of this approach to attain multicomponent interactions has been recently demonstrated (Huang, B., et al., J. Immunol. 158:216-225 (1997)).

Examples of the novel constructs or conjugates that can be formed by the method are described above.

Based on the parallel orientation of the subunits of the coiled coil core and the antiparallel orientation of the amphipathic .alpha.-helical segment in the six helix bundle, these constructs fold to generate the desired structures. Following expression, the recombinant gp41 fragments are isolated as inclusion bodies, cleaved from the leader sequence by cyanogen bromide, and separated from the leader by-product by size exclusion chromatography step (SUPERDEX 75). This protocol has been successfully used in the purification of large quantities of a modified form of the P-17 peptide (Calderone, T. L., et al., J. Mol. Biol. 262:407-412 (1996)). Recombinant constructs (2) and (3) are mixed in equal molar quantities under non-denaturing conditions to generate a six-helix hydrophobic core structure. Constructs (1) and (4) will fold either intra- or intermolecularly to generate the same or similar structures (see FIG. 3 (see Original Patent) for the folding process). The desired product is purified by size exclusion chromatography on a SUPERDEX 75 FPLC column and characterized by molecular weight under using a Beckman Model XL-A analytical ultracentrifuge.
 

Claim 1 of 16 Claims

1. A conjugate polypeptide formed from two or more amino acid sequences that comprise: (a) a first gp41 polypeptide having an amino acid sequence corresponding to a polypeptide comprising the amino acid sequence of SEQ ID NO:2 or SEQ ID NO:3 and is capable of forming a stable coiled-coil structure; and (b) a second gp41 polypeptide having an amino acid sequence corresponding to a polypeptide comprising the amino acid sequence of SEQ ID NO:5 or SEQ ID NO:6 and is capable of forming an amphipathic .alpha. helical segment; wherein at least three polypeptides of (a) and (b) are alternately linked to one another via a peptide bond to form the conjugate polypeptide or at least two polypeptides of (a) and (b) are linked by an amino acid linking sequence consisting of about 2 to about 25 amino acids to form the conjugate polypeptides.
 

____________________________________________
If you want to learn more about this patent, please go directly to the U.S. Patent and Trademark Office Web site to access the full patent.