WO1998001476A1

WO1998001476A1 - Highly synergistic neutralization of hiv through combinations of monoclonal and polyclonal antibodies

Info

Publication number: WO1998001476A1
Application number: PCT/US1997/011824
Authority: WO
Inventors: Christine V. Sapan
Original assignee: Nabi
Priority date: 1996-07-10
Filing date: 1997-07-08
Publication date: 1998-01-15
Also published as: AU3596197A

Abstract

Compositions for neutralizing HIV include (i) a polyclonal mixture of anti-HIV antibodies derived from a plurality of human donors who carry HIV but are free of clinical symptoms associated therewith and (ii) at least one HIV-neutralizing monoclonal antibody. In preferred embodiments, the MAb is an anti-gp120 MAb, an anti-gp41 MAb, or a combination thereof. The composition is administered to immunocompromised individuals to reduce the quantity of circulating virus, or utilized in vitro to process HIV-infected blood for retransfusion. The utility of the invention stems from the surprising discovery that a combination of a polyclonal anti-HIV mixture with a neutralizing MAb produces a substantial synergistic effect in neutralizing HIV.

Description

HIGHLY SYNERGISTIC NEUTRALIZATION OF HIV

THROUGH COMBINATIONS OF MONOCLONAL

AND POLYCLONAL ANTIBODIES

FIELD OF THE INVENTION

The present invention relates generally to the fields of biochemistry and medicine, and in particular to compositions and methods for enhanced neutralization of the human immunodeficiency virus type l ("HIV-l"), and prevention and treatment of acquired immunodeficiency syndrome ("AIDS").

BACKGROUND OF THE INVENTION

HIV-l has proven highly resistant to therapy and to the normal immunological processes of infected individuals due, in part, to its exceptionally high rate of mutation. The mutating virus escapes the activity of externally administered anti-viral drugs and overwhelms the ability of the immune system to generate an effective response. The variation of HIV-l within a single individual, among individuals and between strains concentrated in different parts of the world has not only prevented the emergence of effective therapy, but has also impeded research. Results obtained with laboratory strains cultivated for research purposes frequently do not apply to wild-type viruses. At present, ten genetic subtypes or clades of HIV- 1 have been identified worldwide.

One intensively explored approach to the treatment of ALDS involves therapies that augment the patient's immune response. In so-called "passive immunity," the patient receives anti-HIV antibodies that act directly against the circulating virus. Some studies suggest that passive immunotherapy may be effective against AIDS (see, e.g., Vittecoq et al., J. Infect. Dis., 165:364-368 (1992); and see generally Zolla-Pazner et al., AIDS 1992, 6: 1235-1247). Human immunoglobulin (Ig) preparations, now formulated for intravenous administration (IVIg), have become widely accepted as therapies against variety of infectious diseases. In a typical approach, a supply of plasma is obtained from donors seropositive for the disease of interest, but who do not exhibit clinical symptoms. The plasma contains high titers of antibodies ("Ab") against the pathogen due to the donors' own immune responses. The demonstrated effectiveness of IVIg preparations against AIDS has thus far been limited to specific therapeutic applications (e.g., to reduce morbidity, although not mortality, in pediatnc AIDS patients by supplementing the Ab immune systems of these immunocompromised subjects).

Each person's immune system produces millions of slightly different Abs, each specific to a certain type of antigen ("Ag"), i.e., a substance, usually foreign to the body, that is capable of binding to the Ab and thereby triggering various immune responses; the presence of the Ag in the body stimulates the production of the Ab or Abs specific to that Ag. A pathogen usually contains more than one Ag, and each Ag may include multiple "epitopes" or immunoreactive sites, so the pathogen can induce the immune system to produce high titers of several Abs collectively specific to all of the Ags (and to the epitopes within the Ags).

The binding of a circulating Ab to a pathogen-borne Ag may not in itself result in destruction of the pathogen. Instead, the Ab-Ag complex may serve as an inducer of additional immune responses that attack the pathogen. For example, pathogens bearing Ag-Ab complexes can be taken up and digested by macrophages, or Ab-coated cells and viruses may be lysed (and thereby destroyed) by killer cells or through the action of complement. In some cases, however, the Ab binds to a site critical to the survival of the pathogen, blocking that site and thereby directly or indirectly causing the pathogen's death. Because pathogens typically contain many Ab-binding sites, these "neutralizing" Abs generally represent a small subset of the population of circulating Abs capable of binding to the pathogen. This is certainly true in the case of HIV-l. Accordingly, only some of the many types of Abs present in an anti-HIV IVIg preparation will actually neutralize the virus, since only a fraction of the HIV binding sites are neutralizing sites.

As a result, researchers have experimented with individual protective Ab "strains," cultivating these in isolation. Such "monoclonal" Abs ("MAbs"), each genetically unique, are homogeneous with respect to both physical characteristics and immunochemical reactivity. MAbs are produced by somatic fusion of the single strain of B lymphocyte responsible for producing the particular Ab of interest with a myeloma cell, creating an immortal biological "factory" for the Ab. One class of neutralizing Ab is specific for gpl20, an envelope glycoprotein of HIV-l (and most prominently, the V3 loop or CD4 binding domain of that glycoprotein). .Another class of HIV-l neutralizing antibody is specific for the transmembrane glycoprotein gp41.

It is frequently found that binding to a single antigenic site is not biologically effective; combinations or "cocktails" of MAbs therefore have been studied as potential neutralizing compositions. Synergistic enhancement of neutralization has been observed, for example, by combining anti-gpl20 and anti-gp41 MAbs (see Tilley et al., AIDS Research and Human Retroviruses, 8:461-467 (1992). Nonetheless, MAbs are highly specific in terms of their affinities, and the AIDS condition is characterized by a rapid and continuous proliferation of HIV mutations. Accordingly, synergistic action by MAbs is unlikely to promote significant and sustained anti-HIV action in infected individuals, since mutating strains unaffected by any of the MAbs will quickly develop and reproduce.

DESCRIPTION OF THE INVENTION

Brief Summary of the Invention

The present invention stems from the surprising discovery that a combination of a purified polyclonal anti-HIV Ab preparation (HIV-IG) with one or more anti- HIV MAbs produces an unexpectedly high synergistic effect on neutralization of various HIV clinical isolates. The extent of this synergistic effect on HTV neutralization is quite unexpected in light of the relatively low neutralization titers obtained against HIV clinical isolates with a given amount of HIV-IG alone or with specific, individual MAbs alone. Previous studies evaluating HIV neutralization synergy by combinations of MAbs have usually shown increased neutralization in the range of 2-5 fold the neutralization effected by the individual MAbs. In accordance with the present invention, the neutralization by a combination of HIV-IG and one or more MAbs is as much as 10-1000 or more times the neutralization obtained with any of the components used alone against several HIV clinical isolates. In addition, previous studies evaluating HIV neutralization synergy by combinations of MAbs have usually utilized laboratory strains of HIV, which are notably easier to neutralize than the HTV clinical isolates used herein.

A surprising and unexpected advantage of the invention is the ability to obtain HTV neutralization across virus clades. Indeed, the synergistic enhancement of neutralization that characterizes the present invention has been observed even with clades that are not significantly neutralized either by the HIV-IG or the MAb (or MAbs) when used alone. These results suggest that the HIV-IG discussed herein, or HIV-IG generated from the plasma of individuals infected with various clades of HTV, may be even more synergistically effective in neutralizing multiple virus clades when combined with certain MAbs expressly selected for their breadth of neutralization across multiple clades, or selected for their ability to neutralize clades of particular importance in a specific geographic area of the world. Although the polyclonal product discussed herein was obtained from donors infected with a single clade of HIV (namely, Clade B), the diversity of Ab to HIV apparently renders the mixture effective against some other HIV clades. Importantly, these results indicate the ability to utilize the combination strategy of the invention to prepare neutralizing compositions against a variety of virus strains using the same primary reagent (i.e., HIV-IG). By combining HIV-IG with a MAb selected for its neutralizing potency against a particular clade or group of clades, enhanced neutralizing synergy may be obtained; this may facilitate the use of relatively low concentrations of the MAb, which is an expensive co-reagent. In effect, the HIV-IG product behaves as a general effector whose neutralization efficacy is sharply enhanced toward a selected, target clade through the action of as few as one judiciously chosen, specific effector (MAb).

Accordingly, in a first aspect, the invention comprises a polyclonal mixture of anti-HIV Abs derived from a plurality of human donors who carry HIV but are free of clinical symptoms associated therewith, and at least one HIV-neutralizing MAb. In preferred embodiments, the MAb is an anti-gpl20 MAb, an anti-gp41 MAb, or a combination thereof.

In a second aspect, the invention comprises a method of preparing a neutralizing composition against HIV. The method comprises, first, producing a polyclonal anti-HIV Ab mixture by identifying a plurality of human donors who carry the virus but are clinically healthy, and withdrawing plasma from the donors. The plasma is processed into a preparation having a high titer of Abs against the p24 core protein of HIV (at least 8000 at a concentration of immunoglobulin G ("IgG") of about 5% by weight, although titers of at least 20,000, and ideally 80,000-160,000, are preferred), and then combined with at least one neutralizing MAb. The MAb or MAbs preferably are present at a concentration of at least 5-10 μg/ml and HIV-IG is preferably present at a concentration of at least 500-1000 μg ml. Ordinarily, the polyclonal mixture will reflect Abs generated against the clade B virus. In this regard, it should be stressed that the titer against the p24 Ag is used solely for selection purposes; the polyclonal product will contain Abs specific for a wide variety HTV Ag. Furthermore, titers of Ab against Ag other than p24 (e.g., gpl20 or gp41) can alternately be used for selection of HIV plasma donors and plasma units.

In a third aspect, the invention comprises a method of treating individuals infected with HIV by reducing the quantity of circulating virus as measured by

ELISA technology. The method comprises intravenously infusing into the individual an effective amount of a polyclonal mixture of anti-HIV antibodies (HIV-IG) derived from human donors who carry HIV but are free of clinical symptoms associated therewith, and at least one HIV-neutralizing MAb. The utility of this aspect of the invention is clearly established by the superior neutralizing capabilities of the mixture as compared with pure HIV-IG, whose therapeutic properties are the subject of intensive and ongoing research. Most significantly, the ability of HIV-IG to suppress maternal transmission of HTV to the fetus and to slow disease progression in pediatric AIDS patients are currently under investigation. See, e.g., Lambert et al., "Safety and Pharmacokinetics of Hyperimmune and anti-HIV Immunoglobulin (HIVIG) Administered to HIV-infected Pregnant Women and their Newborns," J. Infect. Dis. (1996) (hereafter "Lambert et al ," the entirety of which is hereby incorporated by reference); Phase 1 VII Study of HIV-IG in Slowing Progression of Disease in HIV- infected Children, ACTG No. 273 (NIAID). The utility of HIV-IG in preventing the emergence of the AIDS condition following HIV infection has been demonstrated in primate models. See, e.g., Prince et al, AIDS Res. and Hum. Retroviruses 7:917-973 (1991); Emini et al., J. Virol, 64:3674-3678 (1990); Emini et al., Nature, 355:728- 730 (1992); Putkonen et al., Nature, 352:436-438 (1991). HIV-IG (or similar plasma) has also been shown to reduce the HIV viral load in humans, resulting in reduction in the number of opportunistic infections, improvement in T-lymphocyte count, and improvement in the Karnofsky score. See, e.g., Jackson et al., .Lancet, ii:647-651 (1988); Karpas et al., Proc. Nat'l. Acad. Sci. USA, 85:9234-9237 (1988); Karpas et al., Proc. Nat 'l Acad. Sci. USA, 87: 1-5 (1990); Vittecoq et al., J. Infect. Dis., 165:364-368 (1992). The invention offers the prospect of a more general therapeutic approach to prevent, limit or eliminate infection by HIV. Depending on the patient and the condition for which s/he is being treated, it may prove worthwhile to repeat the administration at spaced intervals of time until the deficiency in the patient's immune system is substantially reduced or overcome, or the HIV viral load is significantly reduced. The population of individuals treatable by this approach includes those requiring prophylaxis following accidental exposure (e.g., through needle puncture) or unprotected sexual contact with a partner of unknown HIV status; newborns infected by perinatal transmission; and HIV-infected children and adults requiring therapeutic treatment.

In a variation of this aspect of the invention, the polyclonal/monoclonal mixture is used extracorporeally for therapeutic reduction of the viral load in whole blood or a component thereof, e.g., to process a patient's blood for transfusion if parenteral administration of the mixture is clinically impossible or would present risk. For example, the polyclonal/monoclonal mixture may be bound to a column or other solid support (typically fabricated from glass or latex) over which the patient's whole blood or plasma is passed (e.g., in an apheresis loop); the column sequesters free HIV or infected cells expressing an HTV-induced surface Ag, permitting the return of HIV-free components to the patient. Techniques of binding Ab to solid matrix supports are very well known in the art and readily accomplished without undue experimentation. Alternatively, the mixture may simply be added to whole blood or plasma (e.g., as a prophylactic measure to eradicate virus levels undetected by current screening procedures), since the presence of the mixture poses no health threat.

In a fourth aspect, the invention comprises a method of identifying or locating internal sites of viral accumulation. For example, HIV is known to be sequestered unpredictably in various lymph nodes. By tagging the Abs of the mixture (according to well-known techniques of Ab chemical labeling) with agents, such as radioisotopes or ferromagnetic labels, that may be detected extracorporeally, and then injecting the mixture into an infected individual, the sites of sequestration or concentration can be imaged and identified. Thereafter, these specific sites can be treated or even surgically removed.

Detailed Description of the Preferred Embodiments

The polyclonal anti-HIV mixture employed in the present invention is a hyperimmune globulin isolated from the plasma of HIV-infected persons who are nonetheless asymptomatic. These individuals have developed high concentrations of anti-HIV Abs and are free of detectable replicating virus, at least insofar as the viral presence can be observed; for example, HIV cannot be cultured from their sera, nor can HIV marker Ags be detected therein. Most commonly, the Ag to which immunoassays for HTV are directed is the p24 viral core protein. The absence of this protein from an individual's plasma generally indicates the absence of circulating virus; as discussed below, p24 is also a marker used in assays to measure the effectiveness of an anti-HIV preparation in suppressing the virus and preventing its replication.

In accordance with the invention, the polyclonal anti-HIV mixture is derived from multiple donors whose plasma (i) exhibits an anti-HIV p24 antibody titer of at least 128, (ii) is HIV Ag p24 negative by enzyme immunoassay, and (iii) is HIV- culture negative. The pooled plasma is processed to ensure that the anti-HIV immunoglobulin concentration is sufficient to produce an anti-p24 potency of at least about 8000 at a concentration of about 5% by weight of immunoglobulin; preferably, however, the potency is at least 20,000 and most preferably between 80,000 and 160,000 (as measured in a 5 wt% solution in physiological saline). Any of a variety of assays, well known to those skilled in the art, can be used to detect and measure the presence of the p24 Ag. See, e.g., Paul et al., J. Cell. Biochem., 10 (Suppl A ), 224 (Abstract D130) (1986). Any contaminating viruses in the plasma pool can be removed or inactivated by nanofiltration, solvent-detergent treatment or alcohol fractionation, methods generally known to remove or inactivate HIV by those skilled in the art.

A suitable HIV-IG formulation is manufactured by NABI, Boca Raton, FL. The ensuing discussion is based on use of this material as the polyclonal component.

The MAbs employed in the successful practice of the present invention are directed against gpl20 and gp41 on the HIV. As detailed below, these MAbs are combined with HIV-IG either individually or in combination. It must be emphasized, however, that no exhaustive study of candidate MAbs has been performed; the invention will no doubt prove useful in conjunction with MAbs other than those specifically tested, including different MAbs specific to gpl20 and gp41, as well as neutralizing MAbs binding to viral sites not presently identified.

A preferred anti-gpl20 MAb is the 2G12 MAb described in Katinger, Antibiot Chemother, 46, 5 (1994) (incorporated herein by reference). A preferred anti-gp41 MAb is the 2F5 MAb described in Katinger, Proc. of Sept. Colloque des Cent Gardes, 1992:299-303; Muster et al., J. Virol, 67:6642-6647 (1993); Muster et al., Vaccines 94 at 169 (1994); and Purtscher et al., AIDS Res. and Hum. Retroviruses, 10:1651-1658 (1994), the disclosures of the foregoing references being incorporated herein by reference. The epitope recognized by the 2F5 Ab is the highly conserved sequence of amino acids at positions 662-667 of the HIV-l BH10 isolate. Both the 2G12 and 2F5 MAbs are well characterized in the art, and may be obtained from the Institute of Applied Microbiology, University of Agriculture, Vienna, Austria.

Various concentrations and concentration ratios of monoclonal to polyclonal Ab, as well as overall Ab concentration in the final composition, have been tested. Generally, the onset of synergistic activity is observed at threshold MAb concentrations of at least 5-10 μg/ml and threshold HIV-IG concentrations of at least 500-1000 μg/ml. Above these amounts, a sudden amplification is observed, the degree of amplification depending, as demonstrated below, on HIV clade. Naturally, the minimum concentration of each component varies with the virus studied and the MAb or MAbs employed, and must be optimized for clinical effectiveness in a particular therapeutic context. Given the experience of those skilled in the art with HIV-IG, however, suitable dosages are straightforwardly obtained without undue experimentation. For example, following the approach disclosed in Lambert et al., the mixture may be administered to HIV-infected pregnant women at a dosage of 200 mg/kg body weight by intravenous infusion every 28 days beginning between 20 and 30 weeks of gestation and continuing until delivery. Infants may be given a single infusion of the mixture according to the maternal treatment assignment at the same dose within 12 hours after birth. In the ongoing Phase I II clinical study of pediatric AIDS patients noted above, patients are dosed at four-week intervals for a total of six infusions. One group of 10 patients is dosed at 200 mg/kg body weight, a second group at 400 mg/kg and a third group at 800 mg/kg.

The compositions of the present invention are generally diluted with a pharmaceutically acceptable liquid carrier, such as an aqueous IV fluid, prior to being assayed for bioactivity or administered as a unit dosage. Preservatives commonly added to Ig preparations, such as polysorbate 80, maltose or glycine may be utilized in pharmaceutically acceptable amounts. Ordinarily the compositions are administered parenterally, e.g., by intravenous infusion or injection. Again, the dosage administered to a particular patient will depend on the patient's anatomy and physiology (height, weight, sex, etc.) and the condition for which s/he is being treated.

J. Clade B Viruses

Particularly advantageous results have been obtained with the invention in conjunction with the Clade B virus, the primary strain present in the United States and Europe. This reflects the fact that the HIV-IG component was isolated from individuals infected with Clade B.

The following table summarizes the results of tests conducted with mixtures comprising HIV-IG and 2F5, HIV-IG and 2G12, and HTV-IG with a combination of 2F5 and 2G12; the effectiveness of these compositions was compared with results obtained using isolated HIV-IG, 2F5 or 2G12. All experiments were performed with clinical (primary) isolates of HIV cultured in human peripheral blood mononuclear cells (PMBC); as noted previously, these strains are known to be substantially less susceptible to neutralization than laboratory-adapted (prototype) strains; see, e.g., Mascola et al., J. Infect. Dis., 173(2):340-48 (1996). In particular, each experiment was carried out with the clinical isolates US1, US2, TH/014 and BK132 subtypes of the Clade B strain, and at HIV-IG concentrations of 2500 μg/ml and MAb concentrations of 25 μg/ml. In order to quantify neutrali-zation efficiency, enzyme immunoassays for the p24 viral core protein were performed. The figures reported in the following tables reflect the w-fold decrease in p24 produced by cultured cells after infection with Ab-treated or untreated culture - that is, the reduction of p24 Ag (and hence the virus) measured in terms of a proportionate reduction from the concentration observed in control cultures, so that a reported value n reflects the neutralization capacity of the test Ab; the higher the value, the greater is the neutralization effected by the added Ab.

More specifically, the neutralization assay procedure involved dispensing aliquots of the test mixture into multiple wells of a microtiter plate. Replicate wells containing culture medium with no Ab served as the control for baseline virus growth. An equal volume of virus stock, representing 100 TC-D_S0, was added to each well. After 30 min. at 37 °C, 2xl0⁵ PHA-stimulated PBMC were added and incubated overnight at 37 °C. Cells were then washed extensively to remove exogenous p24 Ag and Ab, and transferred to a microtiter plate with culture medium containing 10% interleukin-2. Inhibition of PBMC infection was assessed by quantitative p24 measurement of cell supematants during the logarithmic growth phase.

Clade B Viruses

Antibody μg/ml US1 US2 TH 014 BK132

HTV-IG 2500

2F5 25 10

2G12 25 18

HIV-IG/2F5 2500/25 53 25 10

HIV-IG/2G12 2500/25 43 14 209

HIV-IG/2F5/ 2500/25/25 142 25 167 3745 2G12

These results demonstrate the significant elevation of neutralization efficiency across viral subtypes that is obtained by combining HIV-IG and one or more MAbs. In all cases, combination with one of the MAbs produced better results than combination with the other, but the identity of the preferred MAb was not consistent across viral subtypes. The most dramatic synergistic results occurred through combination with both MAbs. However, in a large number of cases the elevation in neutralization efficiency is far out of proportion to the effect exerted by HTV-IG and either MAb in isolation. In only a few cases did combinations produce merely additive results.

The following tables provide further quantitative details concerning the results obtained with the US2, BK132 and TH/014 Clade B subtypes. Combination of HTV- IG with the most effective single MAb for each viral subtype or with both MAbs produced neutralization efficiencies ranging from 89% to 99.52%. The US 17 material is a well-characterized human plasma with a high neutralization efficiency that is used as a standard for comparative purposes. In all cases the combination of HIV-IG and one of the MAbs at least equaled the capabilities of US 17, and in most cases substantially exceeded those capabilities.

It should be emphasized that the clinical isolates used in these studies contain a heterogeneous virus population including multiple virus strains and mutations. These clinical isolates are not only far more difficult to neutralize than the laboratory strains used in many in vitro studies of Ab effectiveness, but also bear close similarity to the viral population carried by HIV-infected patients. Accordingly, in vitro performance against these strains is representative of expected performance in vivo, and the studies described herein are predictive of therapeutic results in humans.

US2 (Clade B)

.Antibody p24 % Neutralization n-fold p24 Decrease

(No antibody) 10980

HTV-IG 1859 83%

2F5 1062 90% 10

2G12 6065 45%

HTV-IG/2F5 440 96% 25

HTV-IG/2G12 1504 86%

HIN-IG/2F5/2G12 440 96% 25

US17 448 96% 25

BK132 (Clade B)

Antibody p24 % Neutralization ⁿ-^fold P²⁴ Decrease

(No antibody) 292107

HTV-IG 115882 60%

2F5 65644 78%

2G12 15936 95% 18

HTV-IG/2F5 30600 90% 10

HIV-IG/2G12 1396 99.52% 209

HIV-IG/2F5/2G12 78 99.97% 3745

US 17 73442 75%

TH/014 (Clade B)

Antibody p24 % Neutralization ⁿ-^fold P²⁴ ecease

(No antibody) 186486

HTV-IG 51576 72%

2F5 44838 76%

2G12 44966 76%

HIV-IG/2F5 21042 89%

HTV-IG/2G12 13398 93% 14

HTV-IG/2F5/2G12 1115 99.4% 167

US 17 2328 98.8% 80

2. Clade A Viruses

The following table summarizes the results of tests conducted with mixtures comprising HTV-IG and 2F5, HIV-IG and 2G12, and HIV-IG with a combination of 2F5 and 2G12 against three HIV Clade A clinical isolates. The following tests, as well as those discussed subsequently in connection with other clades, were performed with the same HIV-IG concentrations (2500 μg/ml) and MAb concentrations (25 μg/ml) set forth previously in connection with Clade B. Collectively the tests demonstrate the effectiveness of the approach of the present invention against virus clades other than that from which the HTV-IG was derived. In all cases synergy was observed with at least one combination. Overall, better results against the Clade A HIV clinical isolates DJ258, RW/009 and UG/029 were obtained with the combination of HIV-IG and both MAbs.

Clade A Viruses

Antibody μg/ml DJ258 RW/009 UG/029

HIV-IG 2500 3 5 2

2F5 25 5 17 3

2G12 25 5 6 1

HTV-IG/2F5 2500/25 6 61 3

HLV-IG/2G12 2500/25 9 10 2

HTV-IG/2F5/ 2500/25/25 91 220 21

2G12

The following table provides further quantitative details concerning the results obtained with the DJ258 Clade A subtype, emphasizing the strong synergies obtainable using the MAb combination in conjunction with HIV-IG. For Clade A and the ensuing Clades C, D and E, US 17 usually is not included because it is a Clade B isolate and therefore would not represent a meaningful, high-neutralizing Ab control. DJ258 (Clade A)

Antibody p24 % Neutralization ⁿ-^fold P^{24 Decrease}

(No antibody) 15014

HTV-IG 5963 60%

2F5 2823 81%

2G12 3316 78%

HIV-IG/2F5 2693 82%

HTV-IG/2G12 1699 89%

HIV-IG/2F5/2G12 165 98.9 91

US17 5291 65%

3. Clade C, D andE Viruses

Synergies were less consistently observed in connection with these clades.

However, with the exception of Clade E, substantial synergy was observed with at least a single clade isolate, and in the case of SE365 (Clade D), the neutralization efficiency obtained using the combination of MAbs with HTV-IG was quite

pronounced. Clade C Viruses

Antibody μg/ml SE364 ZB20 BR/025

HIV-IG 2500 12

2F5 25

2G12 25 17

HTV-IG/2F5 2500/25 64

HTV-IG/2G12 2500/25 152

HTV-IG/2F5/ 2500/25/25 ≥ 219 2G12

SE364, ZB20 and BR/025 are Clade C clinical isolates.

Clade D Viruses

.Antibody μg/ml SE365 UG/035

HIV-IG 2500

2F5 25 20

2G12 25

HTV-IG/2F5 2500/25 38

HIV-IG/2G12 2500/25

HTV-IG/2F5/2G12 2500/25/25 1082 17

SE365 and UG/035 are Clade D clinical isolates.

Clade E Viruses

Antibody μg/ml CM235 CM243 NP116

HIV-IG 2500

2F5 25 10

2G12 25

HTV-IG/2F5 2500/25

HTV-IG/2G12 2500/25

HTV-IG/2F5/ 2500/25/25 13 11 2G12

It will therefore be seen that the foregoing represents a highly advantageous approach to in-clade and cross-clade enhancement of viral neutralization. The terms and expressions employed herein are used as terms of description and not of limitation, and there is no intention, in the use of such terms and expressions, of excluding any equivalents of the features shown and described or portions thereof, but it is recognized that various modifications are possible within the scope of the

invention claimed.

Claims

1. A composition for the prevention and treatment of HIV infection comprising, in a pharmaceutically acceptable carrier: a. a polyclonal mixture of anti-HIV antibodies derived from a plurality of human donors who carry HIV but are asymptomatic; and b. at least one neutralizing monoclonal anti-HIV antibody, the composition exhibiting synergistic neutralization against HIV.

2. The composition of claim 1 wherein the carrier is IV fluid.

3. The composition of claim 1 wherein the composition exhibits synergistic neutralization against a plurality of HIV clades.

4. The composition of claim 1 wherein the polyclonal mixture is plasma pooled from different donors.

5. The composition of claim 1 wherein the polyclonal mixture is derived from a plurality of donors whose plasma (i) exhibits an anti-HIV p24 antibody titer of at least about 128, (ii) is HIV Ag p24 negative by enzyme immunoassay, and (iii) is HIV-culture negative.

6. The composition of claim 1 wherein the at least one monoclonal antibody is specific for gp 120.

7. The composition of claim 6 wherein the monoclonal antibody is 2G12.

8. The composition of claim 1 wherein the at least one monoclonal antibody is specific for gp41.

9. The composition of claim 8 wherein the monoclonal antibody is 2F5.

10. The composition of claim 1 comprising monoclonal antibodies specific for gpl20 and gp41.

11. The composition of claim 1 wherein the polyclonal mixture is present in a concentration of at least 500 μg/ml and the at least one monoclonal antibody is present in a concentration of at least 5 μg/ml.

12. A method of preparing a neutraliang composition against HIV, the method comprising: a. producing a polyclonal anti-HIV antibody mixture (i) by identifying a plurality of human donors who carry HIV but are clinically healthy, and (ii) withdrawing plasma from the donors; b. processing the plasma into a preparation having an anti-HIV Ab potency of at least 8000 at a concentration of immunoglobulin G of about 5% by weight; c. adding to the preparation at least one neutralizing MAb against HIV, the polyclonal mixture being present in a concentration of at least 500 μg/ml and the at least one monoclonal antibody being present in a concentration of at least 5 μg/ml.

13. The method of claim 12 wherein the at least one donor is infected with a Clade B virus.

14. The method of claim 12 wherein the donor (i) exhibits an anti-HIV p24 antibody titer of at least about 128, (ii) is HIV Ag p24 negative by enzyme immunoassay, and (iii) is HTV-culture negative.

15. The method of claim 12 wherein the at least one monoclonal antibody is specific for gp 120.

16. The method of claim 15 wherein the monoclonal antibody is 2G12.

17. The method of claim 12 wherein the at least one monoclonal antibody is specific for gp41.

18. The method of claim 17 wherein the monoclonal antibody is 2F5.

19. The method of claim 12 wherein the at least one monoclonal antibody comprises antibodies specific for gp 120 and gp41.

20. A method for the in vitro neutralization of HIV comprising the step of exposing the HIV to a composition comprising, in a pharmaceutically acceptable carrier: a. a polyclonal mixture of anti-HIV antibodies derived from a plurality of human donors who carry HIV but are free of clinical symptoms associated therewith; and b. at least one neutralizing monoclonal anti-HIV antibody, the composition exhibiting synergistic neutralization against HIV.

21. The method of claim 20 wherein the HIV is contained in blood withdrawn from a patient, the blood being reinfused into the patient following the exposure step.

22. The method of claim 21 wherein the HIV is contained in blood plasma, the plasma being separated from the blood by apheresis, the plasma being reinfused into the patient following the exposure step.

23. A method of reducing the HIV load in a patient infected with HIV, the method comprising administering to the patient an effective amount of a composition comprising, in a pharmaceutically acceptable carrier: a. a polyclonal mixture of anti-HIV antibodies derived from a plurality of human donors who carry HIV but are free of clinical symptoms associated therewith; and b. at least one neutralizing monoclonal anti-HIV antibody, the composition exhibiting synergistic neutralization against HIV.

24. The method of claim 23 wherein the composition is administered parenterally.

25. A method for the in vitro sequestration of HIV contained in a fluid, the method comprising the steps of: a. providing a solid support to which is bound: i. a polyclonal mixture of anti-HIV antibodies derived from a plurality of human donors who carry HIV but are free of clinical symptoms associated therewith; and ii. at least one neutralizing monoclonal anti-HIV antibody, the composition exhibiting synergistic neutralization against HIV; and b. passing the fluid over the solid support whereby the antibodies bind the HIV, thereby reducing the amount of HIV in the fluid.

26. The method of claim 25 wherein at least some of the HIV is contained in cells expressing an HIV-induced surface antigen.

27. A method of extracorporeally identifying internal sites of HIV concentration in an HIV-infected patient, the method comprising the steps of: a. providing an antibody mixture comprising: i. a polyclonal mixture of anti-HIV antibodies derived from a plurality of human donors who carry HIV but are free of clinical symptoms associated therewith; and ii. at least one neutralizing monoclonal anti-HIV antibody, the composition exhibiting synergistic neutralization against HIV; and b. tagging antibodies of the mixture with an extracorporeally detectable label; c. administering the mixture to the patient; and d. scanning the patient to locate sites of high antibody concentration.