GB2281300A - Anti-HIV antibody oligomer - Google Patents

Abstract

Oligomers, particularly dimers, of a broadly neutralizing monoclonal antibody specific for HIV increase affinity and avidity, rendering the oligomers useful in the prophylaxis, treatment and therapy of AIDS or ARC. The antibodies are conjugated by a cross-linking agent.

Description

TITLE OF THE INVENTION BROADLY NEUTRALIZING ANrI,l-HIV ANTIBODY OLIGOMER W1TH HIGH AVIDITY BACKGROUND OF THE INVENTION A netrovirus designated human immumoldeficiency virus (HIV) is the etiological agent of the complex disease Ihat includes progressive destruction of the immune system (acquired immune deficiency syndrome; AIDS) and degeneration of the central and peripheral nervous system. This virus was previously known as LAV, HmV-lll, or ARV. AIDS is a transmissable deficiency of cellular immunity characterized by opportunistic infections and certain malignancies.A similar disease, AIDS-related complex (ARC), shares many of the epiderniological features and immune abnormalities with AIDS, and often precedes the clinical manifestations of AIDS.

AIDS is a disease of a virus with a unique collection of attributes. HIV itself targets the immune system; it possesses a reverse transcriptase capable of turning out highly mutated progeny; it is sequestered from the immune system and it has a hypervariable sequence in the (env) region. See, e.g., Hilleman, M.R., Vaccine 6, 175 (1988); Barnes, D.M. Science 240, 719 (1988).

One consequence of these attributes is the diversity of HIV serotypes. The principal neutralizing determinant of HIV is an epitope residing in a hypervariable region of the env region. As a result, neutralizing antibodies directed against this epitope are generally extremely t'rrpe-specific; that is, they neutralize only the parental virus and not other variants. Appropriate immunological therapies for AIDS require special consideration of this serological diversity. In particular, one likely AIDS vaccine will be polyvalent and comprise HIV determinants corresponding to each serotype.

Neutralization is now regarded as one of the key features in the successful design of an HIV immunological therapy. When a virusspecific antibody neutralizes its virus, it blocks continued replication of the virus, but the precise mechanism is not fully characterized and is thought to vary with virus and target cell. See, e.g., Dimmock, NJ., Trends in Biochem. Sci 12,70(1987).

Recently'a broadly neutralizing monoclonal antibody against HIV has been discovered. This "447 antibody"binds to about 80% of all known HIV serotypes and neutralizes HIV, It was isolated from a human patient.

Applicants have now made oligomers, including dimers and trimers, of this broadly neutralizing anti-HIV antibody, by covalently linking two to five monomers. The object is to increase the affinity or avidity of the antibody. An increase in affinity or avidity is readily detected in vitro. Further, substantial increases in HIV neutralization result.

BRIEF DESCRIPTION OF THE INVENTION Oligomers of a broadly neutralizing monoclonal antibody specific for HIV are described. Increases in affinity and avidity result, rendering the polymers useful in the prophylaxis, treatment and therapy of AIDS or ARC.

ABBREVIATIONS AND DEFINITIONS ARC AIDS-related complex Oligomer a polymerized antibody having 10 or fewer antibody combining sites. For example, the dimer of IgG has 4 antibody combining sites.

M monomeric unit or monomer of an antibody, e.g., IgG3, or fragment thereof containing functional antibody combining site n number of antibody combining sites in oligomer.

v valency of antibody DETAILDE DESCRIPTION OF THE INVENTION HIV-specific oligomers of a broadly neutralizing monoclonal antibody are described. These oligomers are useful in the treatment, prevention and prophylaxis of AIDS and related ARC. The oligomers of the present invention are defined as (M)x wherein M is a monomer of HlV-specific monoclonal antibody. The "x" is the degree of polymerization, and may involve a dimer, trimer, and so on. The degree of polymerization varies from 2-5, but the number of antibody combining sites (n) must be equal to or less than 10.

Thus, for example, since a monoclonal IgG has two antibody combining sites, a dimer, trimer, tetramer, or pentamer may be formed.

The monomer of this invention is any antibody molecule containing at least one antibody combining site. Typically, the monomer is an IgG immunoglobulin, but particular subclasses and other classes of immunoglobulins are readily adaptable and suitable to this invention. Thus, the following oligomers are included: X n 1 IgG1 2 2 4 2 2 3 6 3 2 4 8 4 u 2 2 4 5 23 6 6 2 4 8 7 8 2 2 4 8 2 3 6 9 2 4 8 10 IgA 2 2 4 11 2 3 6 12 2 4 8 13 4 2 8 14 IgM 5 2 10 As the Table indicates, the present invention includes, e.g., dimers, trimers, or tetramers of IgG3; or dimers of IgM molecules having a valency of 5.

The monomer of this invention also includes recombinant antibody molecules, such as the 447 antibody employed in this application for illustrative purposes. The 447 antibody herein, mAb 447, is a recombinant IgG1 version of the parental IgG3 antibody and its associated combining site. Other antibody molecules suitable for this invention include conventional fragments with functional antibody combining sites, e.g., Fab, Fv, as well as single H-chain antibodies.

The 447 antibody binds to the V3 loop of gp120. The unusual characteristic of the 447 antibody is its capacity to neutralize a large variety of HIV variants, i.e., it is broadly neutralizing. Because of the great diversity of HtV, this invention is limited to oligomers of broadly neutralizing monoclonal antibodies. Other monoclonal antibodies with different antigenic specificities for HIV are suitably adapted to the present invention, for example, specificities for CD4, the N-terminus of gp41, and other determinants of gp120 or gp160.

OLIGOMERIZATION Covalenthly linking purified monoclonal antibody or mmamus is conveniently carried out by faction wifh a hetero- bifunctional cross-linking reagent. Such reagents include, but are not limited to, the following: 2-iminothiolane hydrochloride (2-IT or Traut's reagent), 3-maleimidobenzoic acid N-hydroxysuccinimide ester, 3-maleimidobenzoic acid-3-sulfo-N-hydroxysuccinimide ester(sulfo MBS), gamma-maleimidobutyric acid N-hydroxysuccinimide ester(GMBS), gamma-maleimidobutyric acid 3-sulfo-N hydroxysu ccinimi de ester(sulfo-GMBS), epsilon-maleimidocaproic acid N-hydoxysuccinimide ester, 4-(N-maleimidomethyl)cyclohexane- 1 -carboxylic acid 3 sulfo-N-hydroxysuccinimide ester, beta-maleimidopropionic acid-N-hydroxysuccinimide ester, 4-succinimidylxycarbonyl-alpha-methyl-alpha-(2 pyridyldithio)-toluene (SMPT), sulfosuccinimidyl-6-[alpha-methyl-alpha(2 pyridyldithio)toluamido]hexanoate (sulfo-LC SMPT), N-succinimidyl-3-(2-pyridyldithio)proprionate(SPDP), succinimidyl 6[3-(2 pyridyldithio)proprionamido]hexanoate (LC-SPDP), sulfosuccinimidyl 6[3-(2 pyridyldithio)propionamido]hexanoate (sulfo-LC SPDP), N-succinimidy[4-iodoacetyl)amino benzoate (SIAB), sulfosuccinimidyl(4-iodoacetyl)amino benzoate (Sulfo SIAB), succinimidyl 4-(paramaleimidophenyl) butyrate(SMPB), or sulfosuccinimidyl 4-(paramaleimidophenyl)butyrate (sulfo SMPB).

This type of reagent cross-limks enzymes or receptors, Madison, L.D. et al., J.Biol. Chem. 259, 14818 (1984). GMBS and sulfo-GMBS are reagents of choice for the purposes of the present invention, since they rninimize immunogenicity at the linkage site in the formed dimer, trimer or other oligomer.

In one preferred embodiment, separate aliquots of monoclonal antibody are reacted with 2-11 or GMBS. The 2-IT-treated antibody has free sulfhydryls, the GMBS-treated antibody has maleimide groups introduced. Subsequent mixing of these separately treated aliquots gives a high proportion of dimers formed by thioether linkages.

Alternatively, a wide variety of homo-bifunctional crosslinking reagents can be employed, e.g., N, N'-bis(3-maleimido- propionyl)-2-hydroxy-1,3-propanediamine (which is specific for sulfhydryls), disuccinimidyl suberate (specific for amino groups), or disuccinimidyl glutarate (amino group).

Chromatography for desalting purposes, ion exchange for separation by charge, and molecular sieve chromatogrphy for molecular weight fractionation are conveniently performed at any step in the process of oligomerization, and are readily carried out by the skilled artisan. Reaction conditions for oligomerization are routine in nature, and include temperature, concentration of reactants, buffer characteristics, and the like.

BINDING AFFINITY MEASUREMENT Numerous methods are available for measuring the affinity of a monoclonal antibody. See, e.g., Stanley, C. et al., J. Immunol.

Methods 64, 119(1983); Karlsson, R. et al., J. brimunol. Methods 145, 229 (1991). Traditional and classic techniques typically measure equilibrium values. More recently, kinetic measurements Cf antibody antigen interactions allows a more detailed view of ire effective binding of synthesized oligomers. The most suitable technique for kinetic analysis is the measurement of changes in in the refrartive index close to a metal surface by changes in surface plasm on resonance (SPR). See, e.g., Karlsson, R. et al., supra.

The SPR system allows a quantitative analysis of molecular interaction in real time. Therefore, association and dissociation rate constants are readily calculated. The system uses surface plasmon resonance (SPR) [Kreischmann, E. et al., Z. Naturforsch., Teil A23, 2135 (1968)], a quantum mechanical phenomenon, which detects changes in optical properties at the surface of a thin gold film on a glass support (sensor surface) [Lofas, S. et al., J. Chem. Soc., Chem.

Commun. 21, 1526 (1990)]. The sensor surface carries a carboxylated dextran matrix on which one of the two molecules (a peptide in this assay) is covalently attached. The other (an antibody in this assay) is introduced in a flow passing over the surface. Resonance occurs at a specific angle of incident light. The resonance angle depends on the refractive index in the vicinity of the sensor surface and is monitored continuously, thus allowing the association or dissociation of molecules from the sensor surface to be followed in real time. No labeling of the ligands is required.

ANTTBODY USE AND FORMULATION When admisitered to humans infected with HIV-1, or at risk for HIV infections, the antibody oligomers of the present invention can provide therapeutic or prophylactic benefits. Such individuals particularly at risk are known in the art and include health care workers who have been exposed via a needle stick to HIV-1.

The oligomers of the present inventions are useful in the neutralization of HIV, the prevention of infection by human immunodeficiency vinus (HIV) and the treatment of consequent pathological conditions snch as AIDS. Treating AIDS or preventing or treating infection by HIV is defined as mcludig, but not limited to, treating a wide range of states of HIV infection: AIDS,ARC (AIDS Related complex), borh symptomatic and asymptomatic, and actual or potential exposure to HIV. For example, the oligomers of this invention are useful in treating infection by HIV after suspected past exposure to HIV by e.g., blood transfusion, exchange or body fluids, bites, accidental needle stick, or exposure to patient blood during surgery.

The oligomers of this invention are also useful in the preparation and execution of diagnostic as says for HIV. For example, the oligomers of this invention are useful for determining if a patient has been exposed to, or infected with, HIV-1. Thus the oligomers of the present invention are commercial products to be sold for those purposes.

The antibodies are also useful for analyzing the expression of HIV proteins for which they are specific.

In the present invention the HIV-specific human mAb dimer, trimer, or other oligomer, can be used to treat individuals infected by HIV or suffering from AIDS. The antibodies according to the invention are administered parenterally or enterally by any of a number of known routes. For example, administration may be subcutaneous, intravenous, intramuscular, intraperitoneal, transdermal, or intrathecal. Alternatively, or concurrently, administration may be by the oral, rectal or vaginal route. The antibodies may also be administered into the amniotic cavity for inutero treatment. The preferred routes are intravenous and intramuscular.

The dosage of antibody oligomer administered will be dependent upon the age, health, and weight of the recipient, kind of concurrent treatment, if any, frequency of treatment, and the nature of the effect desired. Effective amounts of the mAbs are from about 0.1 to about 500 mg per day, and preferably from about 3 to about 30 mg per day- Treatment may require infusion or injection or injection or the antibody over a period of days, weeks, months, or even years, as would be madily ascertained by one of skill in the art.

A typical treatment regimen comprises administration of an effective amount of antibody administered over between one and about six months. Duration of treatment required to achieve a therapeutic result will vary from patient to patient, depending upon the severity and stage of the illness and the individual characteristics of each patient.

The total dose required for each treatment may be administered by multiple doses or in a single dose. The mAb oligomers may be administered alone or in conjunction with other therapeutics directed to HIV-I infection, such as AZI, or directed to other disease symptoms.

The mAb oligomers of the present invention can be administered to HIV-infected expectant mothers. Since the antibodies of the present invention are of the IgG isotype, they can cross the placenta and reach the fetus. This may prevent infection of the fetus or, alternatively, provide effective therapy for an infected fetus.

Pharmaceutical compositions comprising the antibodies of the invention include all compositions wherein the antibody is contained in an amount effective to achieve its intended purpose. In addition to the antibody, the pharmaceutical compositions may contain suitable pharmaceutically acceptable carriers comprising excipients and auxiliaries which facilitate. processing of the active compounds into preparations which can be used phannaceutically. An additional pharmaceutical composition within the scope of the present invention is a combination of the antibody of the invention with an intravenous immunoglobulin preparation as is known in the art.

Pharmaceutical compositions include suitable solutions for administration by injection or orally, and contain from about 0.01 to 99 percent, preferably from about 20 to 75 percent of active component (i.e. the antibody) together with the excipient Pharmaceutical compositions for oral administration include tablets and capsules.

Compositions which can be administered rectally or vaginally include supposftories.

In the procedures that follow, "mAb-44T is the IgG1 recombinantly expressed version of the mAb 447-52D, which is a monoclonal antibody isolated from a heterohybridoma described in Gomy, M.K. et al., J. Vir. 66, 7518 (1992). Both mAb 447 and mAb 447-52D recognize the epitope GPXR of the V3 loop of the env gene of HIV-1. Clones coding for the recombinantly expressed version monoclonal antibody 447, also called mAb 447 herein, were deposited at the American Type Culture Collection on or about March 25, 1992, with accession numbers 68943 (light chain); 68944; and 68945 (heavy chain).

EXAMPLE 1 Dimerization of mAb 447 The procedure of IgG homodimer preparation is similar to that described previously by Wolff et al., J. linmunol. 148,2469 (1992). Separate 15 ml aliquots of mAb 447 at a concentration of 10 mgnnl (65 M) was incubated with either 200 'LM GMBS or 600 M 2iminothiolane HCl (2-IT or Traut's reagent) in order to introduce a maleimide or sulfhydryl group, respectively. The reaction was allowed to proceed for 1 h at ambient temperature.The reaction was perfonned in a phosphate buffered saline solution (PBS) containing 6.2 mM phosphate buffer, pH 7.2, with 150 mM Nail. The stock solution of GMBS was prepared in dimethylfomlamide (DMF); the protein and 2 IT stock solutions were prepared in PBS.

After the incubation period, both preparations were passed over a 5 ml Presto desalting column (Pierce) to remove reagents unreacted with the antibody. This gel filtration column was equilibrated with 100 mM sodium phosphate, 100 mM NaCl, pH 8. Fractions were monitored by absorbance at 280 nm and antibody fractions were pooled.

Both GMBS-and 2-IT-treated mAb 447 samples were then added together and allowed to react at nom temperature for 5 h.

Finally, 2-mercaptoethanol (fmal concentration 50 M) was added to quench the reaction, and 15 min later, N-ethylmaleimide was added to a final concentration of 50 M. The reaction mixture was dialyzed overnight against PBS at pH 7.2 and concentrated.

The concentrated conjugate material was loaded onto a Pharmacia Superose 6 column (HR5/5) equilibrated with 6.2 mM sodium phosphate, 0.15 M NaCl, pH 7A. The chromatogram was developed over 90 min at a flow rate of 0.2 mllmin. Elution was monitored by absorbance at 280 nm and antibody containing fractions were collected, concentrated and analyzed with non-reduced SDS-PAGE gel. Three peaks are obtained. The two leading peaks contain dimers and oligomers, respectively.

EXAMPLE 2 Assay for Measuring Binding Affinity (a) Immobilization of peptides on the sensor surface: The immobilization is performed at a flow rate of 5 L/min in HBS, pH 7A [10 mM Hepes, 0.15 M NaCl, 3.4 mM ethylenediaminetetraacetic acid disodium salt, 0.05% polyoxyethylene (20) sorbitan monolaurate]. The peptide is concentrated on the carboxylated dextran matrix by an ion exchange effect at pH below the isoelectric point of the peptide and covalently coupled via either primary amino or thiol groups. Primary amine coupling: The carboxylated dextran matrix is first activated with 35 'LL of an N-ethyl-N'-(3'-dimethylaminopropyl)carbodiimide hydrochloride (EDC) /N-hydroxysuccinimide (NHS) mixture.Twenty five-250 mcghnL of peptide (20-40 L) is injected in a buffer with pH lower than that of the peptide, e.g., 0.01 M acetic acid, pH 4. Unreacted groups on the sensor surface are blocked by 30 pL of ethanolamine HCl, pH 85. Thiol coupling: The carboxylated matrilarly activated with EDC/NHS mixture. A reactive disulfide, 2-(2- pyridinyldithio) ethaneamine (PDEA), 80 mM, pH 8.5, is introduced.

Twenty five-250 mcg/mL of a thiol containing peptide onto Ihe surface via the exchange of the disulfide tonds. The excess disulfides are deactivated with 50 mM cysteine in 1 M NaCI, pH 4.3.

(b) Measurement of kinetic rate constants: Recombinant 447 monoclonal HIV antibody, at concentrations ranging from 5 nM to 1 ,uM in HBS, is allowed to interact with sensor surfaces on which the different HIV gp 120 V3 loop-tip peptides have been immobilized. The runs are performed at 250C, at a flow rate of 5 L/min for 7 min (35 gL injection), taking report points every 5 s (for dRA/dt and RA values) [see Karlsson, R. et al., J. Immunol. Methods 145, 229 (1991) for mathematical definitions with analysis]. After the run, the surface is regenerated by injecting 5 L of a 10-20 mM HCl solution. The instrument software produces a table of dRA/dt and RA values that can be directly used in a plotting program.

(c) Three samples were analyzed for their off-rate constants on AL-I peptide(sequence provided in Gorny, cited above), gp120 (IIIB) and gp120 (MN), respectively. The results are shown in the tables below.

TABLET half life Fold improved (min) Monomer1/AL-1 265E-04 43 (Lot4479201B 6.95 mg/ml) Monomer2/AL-1 2.61E-04 44 (Lot 4479201B 23 mg/ml Dimer/AL-l 8.34E-05 138 3 (Lot 4479201B 2 mg/ml) Monomer1/gp120 7.62E-04 15 (IIIB) Monomer2/gp120 6.2IE-04 18 ("'B) Dimer/gpl20 9.68E-05 119 6.6-8 (IIIB) Monomerl/gpl20 4.50E-05 256 (MN) Monomer2/gp120 5.36E-05 215 (MN) Dimer/gp 120(MN) 1.86E-05 620 1.24-2.8 TABLE II SUMMARY OF SPR OFF-RATE MEASUREMENTS t 1/2 (min) MN AL-1 IIIB MAb447 89 44 12t (peptide) 240t - 18t (gp120) MAb447-DIMER 186 138 41 (peptide) 620 - 119t (pg120) MAb447-TRIMER 222 642 68 (peptide) - - TSTM(gp120) Css1#NB NB 136 (peptide)* NB NB 900 (gp120)* sCD4 35 -- 35 (gp120) *Correlate of Chimpanzee Protection t Average Value NB = No BINDING TSTM = Too Slow To Measure # C(31 is a mouse-human IgG1 chimeric monoclonal antibody. It binds the V3 loop domain of HIV-1 variant gpl20 and has potent hi IIIB-specific virus neutralizing activity.

EXAMPLE 3 Neutralization of HIV-1 Infectivity in-vitro by Human Monoclonal Antibody An assay was performed to quantitate the neutralization of cell-free HIV-1 virus imfection as well as the inhibition of cell-to-cell wad by measuring cell survival after exposure of culturesto antibody and virus for 7-8 days. Two-fold serial dilutions of the antibody under test were made in cell growth medium (RPMI1640+10% fetal calf serum), and 100 l volumes were placed in the wells of a 96-well dish.

Aliquots of 100 l of virus stock (prepared from chronically infected H9 cells or from newly established chronically infected FDA/H9 cells; three-day conditioned medium from the chronically infected cell population plated at a cell density of 2 x 105 cells/ml is clarified and 10 fold more than the last dilution of virus stock which kills all MT-4 cells in a 7 day assay is chosen as the challenge dose) was added to each well and the virus-antibody mixtures were incubated at 37 C for 1 hour.

MT-4 cells were added to each well (1 x 104 cells/well) in 50 1 of culture medium and the dish was incubated for 7 days at 370C at which time the endpoint was determined. The concentration of the last antibody dilution which prevented MT-4 cell killing is reported as the neutralization endpoint. Results are shown in Table N.

Table N: In vitro neutralization endpoints for MAb 447 preparations in the MT4 cell assay ( g/mL or serum dilution) TABLEN I. Batch 1: A. Std. Method B. Ab removed at 18 hr postinfection Ab preparation virus strain IIIB AL-1 IIIB AL-1 monomer 625 < 0.19 0.78 < 0.19 dimer 0.39 #0.19 0.39 #0.19 trimer 0.39 < 0.19 0.78 < 0.19 lot-005 447* 1.56 < 0.19 0.78 < 0.19 human 498 serum&num; 1:160 N.D. 1:80 N.D.

II. Batch 2: A. Std. Method B. Ab removed at 18 hr postinfection Ab preparation virus strain IIIB AL-1 IIIB AL-1 dimer 0.39 < 0.19 0.39 0.39 trimer 0.39 < 0.19 0.78 < 0.19 lot - 005 447 1.56 < 0.19 1.56 < 0.19 498 serum 1:80 1:1280 1:80 1:640 * Lot 005-447 is a sample of the nonrecombinant monoclonal antibody 447-52D, isolated from a heterohybridoma.

&num; Human 498 serum is a polyclonal anti-V3 loop antiserum.

While the foregoing specification teaches the principles of the present invention, with examples provided for the purpose of illustration, it will be understood that the practice of the invention encompasses all of the usual variations, adaptations, and modifications, as come within the scope of the following claims and its equivalents.

Claims (11)

WHAT IS CLAIMED IS:

1. An oligomer of a broadly neutralizing anti-HIV monoclonal antibody or monomer thereof, said oligomer having between 4 and 10 antibody combining sites.

2. The oligomer of claim 1 wherein the monoclonal antibody is specific for the V3 loop.

3. The oligomer Cf Ci8im 2 wherein the monoclonal antibody is 447-52D.

4. The oligomer of Claim 3 wherein the monoclonal antibody is IgG and wherein the oligomer is a dimer with 4 antibody combining sites.

5. The oligomer of Claim 3 wherein the monoclonal antibody is IgG and wherein the oligomer is a timer with 6 antibody combining sites.

6. The oligomer as in any of Claims 1-S, wherein said monoclonal antibody is constructed through recombinant DNA technology;

7. A dimer of 447 mA6.

8. The use of an oligomer of any of claims 1-5 or 7 in the manufacture of a medicament for neutralizing HIV.

9. The use of an oligomer of any of claims 15 or 7 in the manufacture of a medicament for the prevention of infection by HIV or the treatment of infection by HIV.

10. A phannaceirtical composition useful for neutralizing mV, comprising an effective amount of an oligomer of any of Claims 1-5, or 7 and a pharmaceutical carrier,

11. A pharmaceutical composition useful for preventing or treating infection of HIV or for treating AIDS or ARC, comprising an effective amount of an oligomer of any of Claims 1-5, or 7 and a pharmaceutically acceptable carrier.