The present invention features antibodies and antibody fragments that specifically bind a CD4-inducible HIV gp120 epitope that is enhanced by binding a co-receptor for HIV, such as CCR5 or CXCR4, and pharmaceutical compositions comprising the antibodies or antibody fragments. The invention also features nucleic acids encoding the antibodies or antibody fragments, pharmaceutical compositions comprising the nucleic acids encoding the antibodies or antibody fragments, vectors comprising the nucleic acids, and cells comprising the vectors. The invention further features methods of identifying antibodies or antibody fragments with broadly neutralizing activity against HIV. The invention also features methods of inhibiting HIV entry into cells and methods of inhibiting replication of HIV in mammals, using the antibodies and nucleic acids of the invention.

SUMMARY OF THE INVENTION

We have discovered that purified complexes containing HIV Env together with the cell-surface HIV receptor CD4 and an HIV co-receptor, e.g., CCR5 or CXCR4, can be used to identify and isolate antibodies, and active fragments thereof, which display broadly neutralizing activity against multiple genetic subtypes of HIV. Such antibodies can be used as inhibitors of HIV infection and for development of HIV vaccines.

In a first aspect, the invention relates to an isolated antibody or antibody fragment that specifically binds a CD4-inducible epitope on Human Immunodeficiency Virus (HIV) Env that is enhanced by the binding of Env to a co-receptor for HIV, wherein the CD4-inducible epitope is distinct from the HIV co-receptor binding site on gp120.

In one embodiment of the first aspect of the invention, the antibody or antibody fragment is selected by virtue of its ability to specifically bind to a CD4-inducible epitope on HIV Env that is enhanced by binding a co-receptor for HIV.

In a second aspect, the invention relates to an isolated antibody or antibody fragment that is selected by virtue of its ability to specifically bind to a CD4-inducible epitope on Human Immunodeficiency Virus (HIV) Env that is enhanced by the binding of Env to a co-receptor for HIV, and wherein the CD4-inducible epitope is distinct from the HIV co-receptor binding site on gp120.

In various embodiments of the first and second aspects of the invention, the epitope can be on gp120, on gp41, or on gp120-gp41 (Env).

In a third aspect, the invention relates to an isolated antibody or antibody fragment that is selected by virtue of its ability to specifically bind to a complex comprising HIV gp120, CD4, and a co-receptor for HIV. In one embodiment of the third aspect of the invention, the complex also comprises gp41.

In various embodiments of the first, second, and third aspects of the invention, the HIV co-receptor can be CCR5 or CXCR4; the isolated antibody or antibody fragment can have broadly neutralizing activity against HIV, e.g., HIV-1; the isolated antibody or antibody fragment can be monoclonal; the isolated antibody or antibody fragment can be human or humanized; and/or the isolated antibody or antibody fragment can be isolated from a phage display library.

In other embodiments of the first, second, and third aspects of the invention, the antibody or antibody fragment can comprise the heavy chain of the Fab fragment X5 (SEQ ID NO: 3), the light chain of the Fab fragment X5 (SEQ ID NO: 2), or both chains of X5.

In still other embodiments of the first, second, and third aspects of the invention, the isolated antibody or antibody fragment can comprise the CDR3 region (SEQ ID NO: 5) of the heavy chain of the Fab fragment X5 and/or the CDR3 region (SEQ ID NO: 8) of the light chain of the Fab fragment X5. The antibody or antibody fragment can also comprise any of the other CDR and/or FR regions found in the heavy or light chain of antibody Fab fragment X5, in any combination.

In yet other embodiments of the first, second, and third aspects of the invention, a fusion polypeptide comprising a heavy chain or light chain of the antibody or antibody fragment can comprise a soluble CD4 (sCD4) domain. Such a polypeptide can further comprise an amino acid sequence corresponding to that of the peptide T20, which is a synthetic peptide derived from the HIV gp41 amino acid sequence.

In a fourth aspect, the invention relates to an isolated polypeptide comprising the heavy chain of antibody Fab fragment X5 (SEQ ID NO: 3).

In a fifth aspect, the invention relates to an isolated polypeptide comprising the light chain of antibody Fab fragment X5 (SEQ ID NO: 2).

In a sixth aspect, the invention relates to an isolated polypeptide comprising the CDR3 region (SEQ ID NO: 5) of the heavy chain of antibody Fab fragment X5.

In a seventh aspect, the invention relates to an isolated polypeptide comprising the CDR3 region (SEQ ID NO: 8) of the light chain of antibody Fab fragment X5.

In an eighth aspect, the invention relates to an isolated polypeptide comprising the CDR3 region (SEQ ID NO: 5) of the heavy chain of antibody Fab fragment X5 and the CDR3 region (SEQ ID NO: 8) of the light chain of antibody Fab fragment X5. For example, the polypeptide can be a single chain antibody or a single chain antibody fragment, such as a single chain variable fragment (ScFv).

In a ninth aspect, the invention relates to an antibody or antibody fragment that is an amino acid sequence variant of the Fab fragment X5, wherein the sequence variant of X5 comprises at least one amino acid substitution in the heavy chain or light chain of X5, wherein the sequence variant of X5 binds a complex comprising gp120, CD4, and an HIV-co-receptor with an affinity that is about equal to or greater than the affinity by which X5 binds the comprising gp120, CD4, and an HIV-co-receptor.

In one embodiment of the ninth aspect of the invention, the sequence variant of X5 has broadly neutralizing activity against HIV-1. In other embodiments, the amino acid substitution is in the CDR3 region of the heavy chain and/or light chain of X5. The amino acid substitution can also be in any other region of the heavy or light chains, e.g., in any of the CDR, FR, or CH1 regions; for example, the amino acid substitution can be in CH1, and the sequence variant of X5 can comprise SEQ ID NO: 11.

In another embodiment of the ninth aspect of the invention, the sequence variant of X5 is selected by virtue of its ability to specifically bind to a complex comprising HIV gp120, CD4, and a co-receptor for HIV. In another embodiment, the sequence variant of X5 is selected by virtue of its ability to specifically bind to a CD4-inducible epitope on HIV Env that is enhanced by binding a co-receptor for HIV.

In a tenth aspect, the invention relates to an isolated polypeptide comprising an amino acid sequence variant of a CDR3 region of antibody Fab fragment X5, wherein an antibody or antibody fragment comprising the amino acid sequence variant of the CDR3 region of X5 binds HIV gp120 with an affinity that is about equal to or greater than to the affinity by which X5 binds gp120. The CDR3 region may be from the heavy chain or the light chain of antibody X5.

In one embodiment of the ninth and tenth aspects of the invention, the amino acid sequence variant is selected by virtue of its equivalent or increased affinity for gp120 relative to the affinity of X5 for gp120.

In an eleventh aspect, the invention relates to an isolated nucleic acid that encodes SEQ ID NO: 3.

In a twelfth aspect, the invention relates to an isolated nucleic acid that encodes SEQ ID NO: 2.

In a thirteenth aspect, the invention relates to an isolated nucleic acid that encodes SEQ ID NO: 5.

In a fourteenth aspect, the invention relates to an isolated nucleic acid that encodes SEQ ID NO: 8.

In a fifteenth aspect, the invention relates to an isolated nucleic acid that encodes an antibody or antibody fragment comprising the CDR3 region (SEQ ID NO: 5) of the heavy chain of antibody Fab fragment X5 and the CDR3 region (SEQ ID. NO: 8) of the light chain of antibody Fab fragment X5.

In a sixteenth aspect, the invention relates to an isolated nucleic acid that encodes an antibody or antibody fragment comprising the heavy chain of antibody Fab fragment X5 (SEQ ID NO: 3) and the light chain of antibody Fab fragment X5 (SEQ ID NO: 2). For example, the isolated nucleic acid can comprise the nucleotide sequence set forth in SEQ ID NO: 4.

In a seventeenth aspect, the invention relates to an isolated vector comprising the isolated nucleic acid of aspects eleven through sixteen above. The vector can be, for example, an expression vector for expression of the peptide or polypeptide encoded by the isolated nucleic acid.

In an eighteenth aspect, the invention relates to an isolated cell comprising the isolated vector of the seventeenth aspect of the invention. The cell can be a prokaryotic cell or a eukaryotic cell.

In a nineteenth aspect, the invention relates to a pharmaceutical composition comprising the isolated antibody or antibody fragment of the first three aspects and the ninth aspect of the invention, and a pharmaceutically acceptable carrier. The pharmaceutical composition can further comprise soluble CD4.

In a twentieth aspect, the invention relates to a pharmaceutical composition comprising a nucleic acid that encodes the isolated antibody or antibody fragment of the first three aspects and the ninth aspect of the invention, and a pharmaceutically acceptable carrier. In one embodiment of the twentieth aspect of the invention, the nucleic acid is within an expression vector.

In a twenty-first aspect, the invention relates to a method of selecting an antibody or antibody fragment with broadly neutralizing activity against HIV, comprising detecting an antibody or antibody fragment that specifically binds a CD4-inducible epitope on HIV Env that is enhanced by the binding of Env to a co-receptor for HIV, wherein the CD4-inducible epitope is distinct from the HIV co-receptor binding site on gp120. For example, the antibody or antibody fragment can be selected by virtue of its binding to a complex comprising HIV gp120, CD4, and a co-receptor for HIV.

In a twenty-second aspect, the invention relates to an antibody produced by the method of the twenty-first aspect of the invention.

In a twenty-third aspect, the invention relates to a method of inhibiting entry of HIV into a cell, comprising administering to the cell an effective amount of an isolated antibody or antibody fragment that specifically binds a CD4-inducible epitope on HIV Env that is enhanced by the binding of Env to a co-receptor for HIV, wherein the CD4-inducible epitope is distinct from the HIV co-receptor binding site on gp120, thereby inhibiting entry of HIV into the cell.

In various embodiments of the twenty-third aspect of the invention, the cell can be any cell susceptible to HIV infection, e.g., but not limited to, a T cell, a B cell, a monocyte, a macrophage, or a microglial cell. In another embodiment of the twenty-third aspect of the invention, the cell is within a mammal that is susceptible to infection by HIV and the isolated antibody or antibody fragment is administered to the mammal.

In a twenty-fourth aspect, the invention relates to a method of inhibiting replication of HIV in a mammal that is susceptible to HIV infection, comprising administering to the mammal an effective amount of an isolated antibody or antibody fragment that specifically binds a CD4-inducible epitope on HIV Env that is enhanced by the binding of Env to a co-receptor for HIV, wherein the CD4-inducible epitope is distinct from the HIV co-receptor binding site on gp120, thereby inhibiting replication of HIV in the mammal.

In various embodiments of the twenty-third and twenty-fourth aspects of the invention, the isolated antibody or antibody fragment is administered to the mammal by administering a nucleic acid encoding the isolated antibody or antibody fragment to the mammal.

In other embodiments of the twenty-third and twenty-fourth aspects of the invention the mammal is a primate, for example, a human or a non-human primate.

In all of the above embodiments of the invention, the HIV can be HIV-1 or HIV-2.

In all of the above embodiments of the invention, the co-receptor can be, e.g., CCR5 or CXCR4.

DETAILED DESCRIPTION OF THE INVENTION

Binding of the HIV envelope glycoprotein (also known as Env or gp120-gp41) to CD4 and the co-receptor CCR5 or CXCR4 initiates a series of conformational changes that are the heart of the fusion machinery leading to viral entry. The elucidation of the nature of the Env conformational changes is not only a clue to the mechanism of HIV-1 entry but also provides new tools for the development of inhibitors and vaccines.

Described herein is a novel approach for the identification of broadly cross-reactive antibodies that neutralize multiple genetic subtypes of HIV. This approach involves the use of purified Env-CD4-co-receptor complexes for screening libraries of antibodies or antibody fragments that specifically bind to receptor-inducible HIV epitopes. Such antibodies can be used for treating, inhibiting, and/or preventing HIV infection by providing passive immunity to treated individuals. Currently there are known only three well characterized monoclonal antibodies with broadly neutralizing activity, and none of these antibodies is directed against a receptor-inducible epitope.

Using this approach, a novel human antibody Fab fragment, denoted "X5", was identified by screening a phage display library, as described in Example I below. The epitope recognized by the X5 antibody is inducible by CD4 and exposure of the epitope is enhanced by the major HIV-1 co-receptor CCR5. The antibody neutralizes R5 and R5X4 viruses, including primary isolates, and to lesser extent, X4 viruses. Sequence variants and antibody fusion proteins based on X5 are also described herein, and will be apparent to those of ordinary skill in the art.

Antibodies

The term "antibodies" is used herein in a broad sense and includes both polyclonal and monoclonal antibodies. In addition to intact immunoglobulin molecules, also included in the term "antibodies" are fragments of immunoglobulin molecules and multimers of immunoglobulin molecules (e.g., diabodies, triabodies, and bi-specific and tri-specific antibodies, as are known in the art; see, e.g., Hudson and Kortt, J. Immunol. Methods 231:177 189, 1999), fusion proteins containing an antibody or antibody fragment (e.g., a fusion protein containing a fragment of CD4, e.g., sCD4 (Salzwedel et al. J. Virol. 74:326 333, 2000), which are produced using standard molecular biology techniques, single chain antibodies, and human or humanized versions of immunoglobulin molecules or fragments thereof, as long as they are chosen for their ability to broadly neutralize HIV (e.g., multiple genetic subtypes of HIV-1 or HIV-2) by binding a CD4-inducible HIV epitope that is enhanced by binding an HIV co-receptor, as described herein. The antibodies are tested for their desired activity using the in vitro assays described herein, or by analogous methods, after which their in vivo therapeutic and/or prophylactic activities are tested according to known clinical testing methods.

The term "monoclonal antibody" as used herein refers to an antibody or antibody fragment obtained from a substantially homogeneous population of antibodies or antibody fragments, i.e., the individual antibodies within the population are identical except for possible naturally occurring mutations that may be present in a small subset of the antibody molecules. The monoclonal antibodies herein specifically include "chimeric" antibodies in which a portion of the heavy and/or light chain is identical with or homologous to corresponding sequences in antibodies derived from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chain(s) is identical with or homologous to corresponding sequences in antibodies derived from another species or belonging to another antibody class or subclass, as well as fragments of such antibodies, as long as they exhibit the desired antagonistic activity (See U.S. Pat. No. 4,816,567 and Morrison et al., Proc. Natl. Acad. Sci. USA, 81:6851 6855 (1984)).

Monoclonal antibodies of the invention can be prepared using hybridoma methods, such as those described by Kohler and Milstein, Nature, 256:495 (1975). In a hybridoma method, a mouse or other appropriate host animal is typically immunized with an immunizing agent to elicit lymphocytes that produce or are capable of producing antibodies that will specifically bind to the immunizing agent. Alternatively, the lymphocytes may be immunized in vitro, e.g., using the HIV Env-CD4-co-receptor complexes described herein.

The monoclonal antibodies may also be made by recombinant DNA methods, such as those described in U.S. Pat. No. 4,816,567 (Cabilly et al.). DNA encoding the monoclonal antibodies of the invention can be readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes that are capable of binding specifically to genes encoding the heavy and light chains of murine antibodies). Libraries of antibodies or active antibody fragments can also be generated and screened using phage display techniques, e.g., as described in U.S. Pat. No. 5,804,440 (Burton et al.) and U.S. Pat. No. 6,096,441 (Barbas et al.). Recombinant antibodies, antibody fragments, and fusions and polymers thereof can be expressed in vitro or in prokaryotic cells (e.g., bacteria) or eukaryotic cells (e.g., yeast, insect, or mammalian cells) and further purified, as necessary, using well known methods (see, e.g., Sambrook et al. Molecular Cloning: a Laboratory Manual, Cold Spring Harbor Laboratory Press (1989); and Ausubel et al., Current Protocols in Molecular Biology, John Wiley & Sons, New York, N.Y., 2001, which is updated quarterly).

In vitro methods are also suitable for preparing monovalent antibodies. Digestion of antibodies to produce fragments thereof, particularly, Fab fragments, can be accomplished using routine techniques known in the art. For instance, digestion can be performed using papain. Examples of papain digestion are described in WO 94/29348 published Dec. 22, 1994 and U.S. Pat. No. 4,342,566. Papain digestion of antibodies typically produces two identical antigen binding fragments, called Fab fragments, each with a single antigen binding site, and a residual Fc fragment. Pepsin treatment yields a fragment that has two antigen combining sites and is still capable of cross-linking antigen.

Any antibody or antibody fragment of the invention, whether attached to other sequences or not, can also include insertions, deletions, substitutions, or other selected modifications of particular regions or specific amino acids residues, provided the activity of the antibody or antibody fragment is not significantly altered or impaired compared to the non-modified antibody or antibody fragment. These modifications can provide for some additional property, such as to remove/add amino acids capable of disulfide bonding, to increase its bio-longevity, to alter its secretory characteristics, etc. In any case, the antibody or antibody fragment must possess a bioactive property, such as specific binding to its cognate antigen. Functional or active regions of the antibody or antibody fragment may be identified and/or improved by mutagenesis of a specific region of the protein, followed by expression and testing of the expressed polypeptide. For example, amino acid sequence variants of antibodies or antibody fragments can be generated and those that display equivalent or improved affinity for antigen can be identified using standard techniques and/or those described herein. Methods for generating amino acid sequence variants are readily apparent to a skilled practitioner in the art and can include site-specific mutagenesis or random mutagenesis (e.g., by PCR) of the nucleic acid encoding the antibody or antibody fragment (Zoller, M. J. Curr. Opin. Biotechnol. 3:348 354, 1992). Both naturally occurring and non-naturally occurring amino acids (e.g., artificially-derivatized amino acids) may be used to generate amino acid sequence variants of the antibodies and antibody fragments of the invention.

As used herein, the term "antibody" or "antibodies" can also refer to a human antibody and/or a humanized antibody. Many non-human antibodies (e.g., those derived from mice, rats, or rabbits) are naturally antigenic in humans, and thus can give rise to undesirable immune responses when administered to humans. Therefore, the use of human or humanized antibodies in the methods of the invention serves to lessen the chance that an antibody administered to a human will evoke an undesirable immune response.

Human Antibodies

The human antibodies of the invention can be prepared using any technique. Examples of techniques for human monoclonal antibody production include those described by Cole et al. (Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, p. 77, 1985) and by Boerner et al. (J. Immunol., 147(1):86 95, 1991). Human antibodies of the invention (and fragments thereof) can also be produced using phage display libraries (Hoogenboom et al., J. Mol. Biol., 227:381, 1991; Marks et al., J. Mol. Biol., 222:581, 1991; and C. F. Barbas, D. R. Burton, J. K. Scott, G. J. Silverman, Phage Display: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 2001).

The human antibodies of the invention can also be obtained from transgenic animals. For example, transgenic, mutant mice that are capable of producing a full repertoire of human antibodies, in response to immunization, have been described (see, e.g., Jakobovits et al., Proc. Natl. Acad. Sci. USA, 90:2551 255 (1993); Jakobovits et al., Nature, 362:255 258 (1993); Bruggermann et al., Year in Immunol., 7:33 (1993)). Specifically, the homozygous deletion of the antibody heavy chain joining region (J(H)) gene in these chimeric and germ-line mutant mice results in complete inhibition of endogenous antibody production, and the successful transfer of the human germ-line antibody gene array into such germ-line mutant mice results in the production of human antibodies upon antigen challenge. Antibodies having the desired activity are selected using Env-CD4-co-receptor complexes as described herein.

Humanized Antibodies

Antibody humanization techniques generally involve the use of recombinant DNA technology to manipulate the DNA sequence encoding one or more polypeptide chains of an antibody molecule. Accordingly, a humanized form of a non-human antibody (or a fragment thereof) is a chimeric antibody or antibody chain (or a fragment thereof, such as an Fv, Fab, Fab', or other antigen-binding portion of an antibody) which contains a portion of an antigen binding site from a non-human (donor) antibody integrated into the framework of a human (recipient) antibody.

To generate a humanized antibody, residues from one or more complementarity determining regions (CDRs) of a recipient (human) antibody molecule are replaced by residues from one or more CDRs of a donor (non-human) antibody molecule that is known to have desired antigen binding characteristics (e.g., a certain level of specificity and affinity for the target antigen). In some instances, Fv framework (FR) residues of the human antibody are replaced by corresponding non-human residues. Humanized antibodies may also contain residues which are found neither in the recipient antibody nor in the imported CDR or framework sequences. Generally, a humanized antibody has one or more amino acid residues introduced into it from a source which is non-human. In practice, humanized antibodies are typically human antibodies in which some CDR residues and possibly some FR residues are substituted by residues from analogous sites in rodent antibodies. Humanized antibodies generally contain at least a portion of an antibody constant region (Fc), typically that of a human antibody (Jones et al., Nature, 321:522 525 (1986), Reichmann et al., Nature, 332:323 327 (1988), and Presta, Curr. Opin. Struct. Biol., 2:593 596 (1992)).

Methods for humanizing non-human antibodies are well known in the art. For example, humanized antibodies can be generated according to the methods of Winter and co-workers (Jones et al., Nature, 321:522 525 (1986), Riechmann et al., Nature, 332:323 327 (1988), Verhoeyen et al., Science, 239:1534 1536 (1988)), by substituting rodent CDRs or CDR sequences for the corresponding sequences of a human antibody. Methods that can be used to produce humanized antibodies are also described in U.S. Pat. No. 4,816,567 (Cabilly et al.), U.S. Pat. No. 5,565,332 (Hoogenboom et al.), U.S. Pat. No. 5,721,367 (Kay et al.), U.S. Pat. No. 5,837,243 (Deo et al.), U.S. Pat. No. 5,939,598 (Kucherlapati et al.), U.S. Pat. No. 6,130,364 (Jakobovits et al.), and U.S. Pat. No. 6,180,377 (Morgan et al.).

Administration of Antibodies

Antibodies of the invention are preferably administered to a subject in a pharmaceutically acceptable carrier. Suitable carriers and their formulations are described in Remington: The Science and Practice of Pharmacy (19th ed.) ed. A. R. Gennaro, Mack Publishing Company, Easton, Pa. 1995. Typically, an appropriate amount of a pharmaceutically acceptable salt is used in the formulation to render the formulation isotonic. Examples of the pharmaceutically acceptable carrier include, but are not limited to, saline, Ringer's solution and dextrose solution. The pH of the solution is preferably from about 5 to about 8, and more preferably from about 7 to about 7.5. Further carriers include sustained release preparations such as semipermeable matrices of solid hydrophobic polymers containing the antibody, which matrices are in the form of shaped articles, e.g., films, liposomes or microparticles. It will be apparent to those persons skilled in the art that certain carriers may be more preferable depending upon, for instance, the route of administration and concentration of antibody being administered.

The antibodies can be administered to the subject, patient, or cell by injection (e.g., but not limited to, intravenous, intraperitoneal, intradermal, subcutaneous, intramuscular), or by other methods such as infusion that ensure its delivery to the bloodstream in an effective form. Local or intravenous injection is preferred.

Effective dosages and schedules for administering the antibodies may be determined empirically, and making such determinations is within the skill in the art. Those skilled in the art will understand that the dosage of antibodies that must be administered will vary depending on, for example, the subject that will receive the antibody, the route of administration, the particular type of antibody used and other drugs being administered. Guidance in selecting appropriate doses for antibodies is found in the literature on therapeutic uses of antibodies, e.g., Handbook of Monoclonal Antibodies, Ferrone et al., eds., Noges Publications, Park Ridge, N.J., (1985) ch. 22 and pp. 303 357; Smith et al., Antibodies in Human Diagnosis and Therapy, Haber et al., eds., Raven Press, New York (1977) pp. 365 389. A typical daily dosage of the antibody used alone might range from about 1 .mu.g/kg to up to 100 mg/kg of body weight or more per day, depending on the factors mentioned above.

Following administration of an antibody for treating, inhibiting, or preventing an HIV infection, the efficacy of the therapeutic antibody can be assessed in various ways well known to the skilled practitioner. For instance, one of ordinary skill in the art will understand that an antibody of the invention is efficacious in treating or inhibiting an HIV infection in a subject by observing that the antibody reduces viral load or delays or prevents a further increase in viral load. Viral loads can be measured by methods that are known in the art, for example, using polymerase chain reaction assays to detect the presence of HIV nucleic acid or antibody assays to detect the presence of HIV protein in a sample (e.g., but not limited to, blood or another body fluid) from a subject or patient, or by measuring the level of circulating anti-HIV antibodies in the patient. Efficacy of the antibody treatment may also be determined by measuring the number of CD4+ T cells in the HIV-infected subject. An antibody treatment that delays or inhibits an initial or further decrease in CD4+ T cells in an HIV-positive subject or patient, or that results in an increase in the number of CD4+ T cells in the HIV-positive subject, is an efficacious antibody treatment.

The broadly-neutralizing antibodies of the invention can also be administered prophylactically to patients or subjects who are at risk for being exposed to HIV or who have been newly exposed to HIV. Such patients include, but are not limited to, healthcare workers; fetuses, neonates, or infants (e.g., nursing infants) whose mothers are infected or at risk for being infected; intravenous drug users; recipients of blood transfusions, blood products, or transplantation tissue; and other individuals who have been exposed to a body fluid that contains or may contain HIV.

In subjects who have been newly exposed to HIV but who have not yet displayed the presence of the virus (as measured by PCR or other assays for detecting the virus) in blood or other body fluid, efficacious treatment with an antibody of the invention partially or completely inhibits or delays the appearance of the virus or minimizes the level of the virus in the blood or other body fluid of the exposed individual.

Nucleic Acid Approaches for Antibody Delivery

The broadly neutralizing anti-HIV antibodies and antibody fragments of the invention can also be administered to patients or subjects as a nucleic acid preparation (e.g., DNA or RNA) that encodes the antibody or antibody fragment, such that the patient's or subject's own cells take up the nucleic acid and produce and secrete the encoded antibody or antibody fragment, thereby treating, inhibiting, or preventing HIV infection.

Nucleic Acid Delivery

In the methods described above which include the administration and uptake of exogenous DNA into the cells of a subject (i.e., gene transduction or transfection), the nucleic acids of the present invention can be in the form of free DNA or RNA, or the nucleic acids can be in a vector for delivering the nucleic acids to a cell and expressing the encoded polypeptide within a cell, whereby the antibody-encoding DNA fragment is under the transcriptional regulation of a promoter, as well as an other necessary and/or desirable components to regulate and/or enhance transcription and/or stability of the mRNA and to regulate and/or enhance translation of the encoded polypeptide, as would be well understood by one of ordinary skill in the art. The vector can be a commercially available preparation, such as a plasmid or an adenovirus vector (Quantum Biotechnologies, Inc. (Laval, Quebec, Canada).

Delivery of the nucleic acid or vector to cells can be via a variety of mechanisms. As one example, delivery can be via a liposome, using commercially available liposome preparations such as LIPOFECTIN, LIPOFECTAMINE (GIBCO-BRL, Inc., Gaithersburg, Md.), SUPERFECT (Qiagen, Inc. Hilden, Germany), and TRANSFECTAM (Promega Biotec, Inc., Madison, Wis.), as well as other liposomes developed according to procedures standard in the art. Delivery can also be by injection (e.g., but not limited to, intravenous or intramuscular) of naked DNA, e.g., in a plasmid or viral vector. In addition, the nucleic acid or vector of this invention can be delivered in vivo by electroporation, the technology for which is available from Genetronics, Inc. (San Diego, Calif.) as well as by means of a SONOPORATION machine (ImaRx Pharmaceutical Corp., Tucson, Ariz.).

As one example, vector delivery can be via a viral system, such as a retroviral vector system which can package a recombinant retroviral genome (see e.g., Pastan et al., Proc. Natl. Acad. Sci. U.S.A. 85:4486, 1988; Miller et al., Mol. Cell. Biol. 6:2895, 1986). The recombinant retrovirus can then be used to infect and thereby deliver to the infected cells nucleic acid encoding a broadly neutralizing antibody (or active fragment thereof) of the invention. The exact method of introducing the altered nucleic acid into mammalian cells is, of course, not limited to the use of retroviral vectors. Other techniques are widely available for this procedure including the use of adenoviral vectors (Mitani et al., Hum. Gene Ther. 5:941 948, 1994), adeno-associated viral (AAV) vectors (Goodman et al., Blood 84:1492 1500, 1994), lentiviral vectors (Naidini et al., Science 272:263 267, 1996), pseudotyped retroviral vectors (Agrawal et al., Exper. Hematol. 24:738 747, 1996). Physical transduction techniques can also be used, such as liposome delivery and receptor-mediated and other endocytosis mechanisms (see, for example, Schwartzenberger et al., Blood 87:472 478, 1996). This invention can be used in conjunction with any of these or other commonly used gene transfer methods.

For example, if the antibody-encoding nucleic acid of the invention is delivered to the cells of a subject in an adenovirus vector, the dosage for administration of adenovirus to humans can range from about 10.sup.7 to 10.sup.9 plaque forming units (pfu) per injection but can be as high as 10.sup.12 pfu per injection (Crystal, Hum. Gene Ther. 8:985 1001, 1997; Alvarez and Curiel, Hum. Gene Ther. 8:597 613, 1997). A subject can receive a single injection, or, if additional injections are necessary, they can be repeated at six month intervals (or other appropriate time intervals, as determined by the skilled practitioner) for an indefinite period and/or until the efficacy of the treatment has been established.

Parenteral administration of the nucleic acid or vector of the present invention, if used, is generally characterized by injection. Injectables can be prepared in conventional forms, either as liquid solutions or suspensions, solid forms suitable for solution of suspension in liquid prior to injection, or as emulsions. A more recently revised approach for parenteral administration involves use of a slow release or sustained release system such that a constant dosage is maintained. See, e.g., U.S. Pat. No. 3,610,795, which is incorporated by reference herein. For additional discussion of suitable formulations and various routes of administration of therapeutic compounds, see, e.g., Remington: The Science and Practice of Pharmacy (19th ed.) ed. A. R. Gennaro, Mack Publishing Company, Easton, Pa. 1995.
 

Claim 1 of 27 Claims

1. An isolated antibody or antibody fragment comprising SEQ ID NO: 5 or SEQ ID NO: 8.
 

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