NATURAL HUMAN ANTI-GAMMA INTERFERON ANTIBODIES

DETECTED AND PURIFIED BY SYNTHETIC PEPTIDES.

The invention relates to the use of human anti-gamma

interferon antibodies in human therapy and to the detection

and purification of said antibodies by means of ^• synthetic

peptides corresponding to regions of human gamma interferon.

Background Art

Interferons (IFNs) form an heterogeneous family of

proteins, defined according to their ability to prevent viral

replication. At present three major classes of human IFN have

been designed: human IFN-«( and IFN-β - which are 30% similar

at the primary amino acid sequence level - and IFN-y which is

similar to neither. IFN-y shares several characteristics and

activities with IFN-o( and IFN-β, but it also mediates various

immune functions. More recently, IFN-y has been established to

act as a potent immunomodulator (Table 1 ). Following

antigenic stimulation, T-cells and NK cells release IFN-y

which: influences the response of T-cells, B-ceils and

macrophages; enhances the proliferation of T-cells and the

functional maturation of cytotoxic T-cells; inhibits the

gereration of suppressor T-cells; enhances the immunoglobulin

secretion of B-cells when added late during an in vitro immune

response and switches on lgG2 a production. In addition to

these regulatory effects on the adaptative immune response,

IFN-y acts on the effector ceils of nonadaptive defence and

exerts proinfiammatory activity. Macrophages, primed with

IFN-y become able to kill bacteria, protozoa and tumor cells by

oxygen-dependent and-independent mechanisms. In ^• addition,

IFN-y induces endothelial cells and monocytes to release

chemiotactic factors, such as IL-1 and TNF and leads to

enhancement of synthesis and surface expression of class I and

class II antigens of the Major Histocompatibility Complex

(MHC), Fc receptor and leukocyte adhesion proteins (e.g.

integrins and integrin receptors). Induction of MHC antigens by

IFN-y may occur in several cell types that otherwise express

low or undetectable levels of these molecules. The

enhancement of MHC class II expression enables macrophages,

endothelial, epithelial and Langerhans ceils to present

antigens, but makes the same cells susceptible to the possible

cytotoxic effects of class II restricted T-lymphocytes. The

induction _of Fc receptors increases the capacity of

macrophages to phagocyte opsonized antigens. The

enhancement of the surface density of integrin receptors

promotes ' adhesion of monocytes to endothelial cells and to

immunological reactions.

While the general stimulatory activity of IFN- y in the

progression of immunologicai and inflammatory responses

indicates its potential therapeutical application as e.g.

immunomodulant, the agents that are able to neutralize its

activity may be beneficial when given to allotransplanted

patients and to patients affected by autoimmune disorders,

^• chronic inflammation, septic shocks or diabetes (Table 2) and

any clinical state where an enhancement of IFN-y production is

considered to be detrimental.

Antibodies are the most inherently specific natural

immunosuppressive agents. Billiau (1988), Immunol. Today 9_,

37, reported that murine anti-IFN-y antibodies experimentally

inhibited the Shwartzman reaction. His experiments opened up

interesting clinical applications for the treatment of

Shwartzman-related or-like inflammatory reactions. Other

data reported by Jacob et al. (1987), J. Exp. Med. 1 66. 798,

showed that anti-IFN-y antibodies protect NZB mice against

spontaneous development of autoimmune disease. Their

experiments indicated that anti-IFN-y antibodies, policlonal or

preferably monoclonal, may be candidated for trials in

connective-tissue diseases, such as Systemic Lupus

Erythematosus (SLE) and Rheumatoid Arthritis, Multiple

Sclerosis, and possibly of all those diseases where activated

4 -

cell-mediated immunity needs to be depressed. Antibody

immunosuppressive therapy is used in certain instances in

humans, for preventing Rh-related Erythoblastosis Fetalis.

This treatment, however, is limited to diseases when the

causative antigen is known, and a specific human antiserum is

available.

As substances self-recognized by human immune system,

IFNs should not elicit antibodies in man except in conjunction

with autoimmune disorders or when their structure and

antigenicity are modified. Antibodies to IFN-y were reported by

Caruso et al. (1989) J. Biol. Reg. Homeost. Agents 2., 8, in

patients infected with Human Immunodeficiency virus (HIV).

More recently, Caruso et al. (1990) J. Immunol. 1 44. 685,

reported the presence of natural antibodies to IFN-y in healthy

individuals ranging from newborn babies to adults and, at

higher levels, in patients suffering from different viral

infections. Those antibodies specific to IFN-y were

affinity-purified from sera taken from healthy individuals, and

viral-infected patients or other different biological sources

(See,- "Modes for carrying out the invention"), by using a

recombinant IFN-y -coupled CNBr-activated Sepharose 4B

column. The antibodies were found to be mainly of the IgG

class, and jmantained their ability ot bind recombinant IFN-y.

The did not neu ralize antiviral activit of IFN- while bein

capable of suppressing the IFN-y induction of class II MHC

antigens and of Fc receptor sites for immunoglobulins. In

addition, the natural human anti-IFN-y antibodies were found

to interfere, in a mixed lymphocyte culture (MLC), with the

proliferation and cytotoxic generation of lymphocytes, by

inhibiting endogenously produced IFN-y. The availability of

affinity purified human anti-IFN- y antibodies, capable of

neutralizing the immunomodulatory activity of IFN-y , opens up

interesting clinical applications, possibly for all those

diseases where activated cell-mediated immunity needs to be

regulated.

The ready availability of neutralizing human anti-IFN-y

antibodies may solve many of the problems associated with

the administration of heterologous immunoglobulins, both

polyclonal and/or monoclonal antibodies, which can induce

anti-lg antibodies.

The main objective of the present invention is to provide

techniques to detect anti-IFN-y antibodies and affinity-purify

them, using synthetic peptides corresponding to regions of

human IFN-y.

Disclosure of the Invention

The invention is based upon the discovery that synthetic

peptides corresponding to regions of human IFN-y can

6 -

substitute the natural or recombinant IFN-y proteins in detecting and purifying natural human anti-IFN-y antibodies. Accordingly, the first aspect of the invention is the use of

synthetic peptides in different immunological methods to detect human antibodies to IFN-y. The above mentioned methods are based on the use of synthetic peptides capable to

specifically bind anti-IFN-y antibodies allowing their, direct or

indirect detection. The second aspect is a method to affinity-purify human anti-IFN-y antibodies using synthetic peptides. The method is based on the binding of synthetic peptides to a support, or to other molecular carriers or on trapping the peptides into nitrocellulose sheets.

The third aspect of the invention is an immunotherapeutic treatment to control a disease associated with an activated cell-mediated immunity. The treatment consists in the administration of human antibodies directed against IFN-y to the individual, sufficient to control the clinical aspects of the disease. The fourth aspect of the invention is a "unit dosage"

for treatment of the above-described patients. The unit dosage form consists of human antibodies against IFN-y combined with a pharmaceutically acceptable vehicle. The amount of human antibodies in the dosage form has to be sufficient to substantially lessen manifestation of the disease.

symptoms associated with the disease, and by the presence of

auto antibodies associated with the disease while they are

absent, or at lower titer, in healthy individuals.

The fifth aspect of the invention are immunological

methods to detect the IFN-y by the use of purified human

anti-IFN-y antibodies. The method comprises the use of human

anti-IFN-y antibodies to specifically bind and detect (in a

direct or indirect manner) the IFN-y molecule(s).

The last aspect of the invention is a method to

affinity-purify the IFN-y molecule(s) recognized by the human

anti-IFN-y antibodies. The method comprises binding of human

anti-IFN-y to a support, or to other molecular carriers or

trapping them into nitrocellulose sheets.

Brief description of the Drawing

Figure 1 represents a graph showing the inhibition of the

expression of Fc-receptor sites and HLA-DR antigens on U937

cells stimulated with recombinant IFN-y by human anti-IFN-y

antibodies.

Figure 2 presents a graph showing the effect of human

anti-IFN-y antibodies, added at different times, on MLC

proliferation.

Figure 3 presents a graph showing the effect of human

anti-IFN-y antibodies on the development of cytotoxicity in

MLC.

Figure 4 presents the chromatography of human anti-IFN-y

antibodies on a IFN-y -relating peptide-sulfolinked agarose

column.

Figure 5 presents a Western blot analysis of the anti-IFN-y

antibodies affinity purified by an IFN- -relating peptide based

column showing their specific reactivity with IFN-y.

Figure 6 presents a graph showing the effect of human

anti-IFN-y antibodies purified by a peptide-based affinity

column of MLC proliferation.

Figure 7 presents a graph showing the correlation obtained

by an IFN- -relating peptide based ELISA and a recombinant

IFN-y based RIA in detecting and quantify human anti-IFN-y

antibodies in human serum.

Modes for Carrying Out the Invention

The practice of the present invention will employ, unless

otherwise indicated, conventional techniques of molecular

Q _

biology, microbiology, recombinant DNA, chemistry,

biochemistry, biotechnology and immunology, which are within

the skill of the art. Such techniques are explained fully in the

literature (See Literature, Appendix 1).

According to the invention, it is very useful and of

relevance for scientists and clinicians, to detect and quantify

natural antibodies to IFN-y in specimens by any of the known

assays.

For example, assays to detect human anti-IFN-y antibodies

may be useful:

a) in clinic, for a rapid diagnosis of viral diseases, and to

distinguish between a viral and bacterial infection; to monitor

a viral infection and, eventually, a specific antiviral therapy;

to study anti-IFN-y antibodies in patients with cancer, viral

infections, and/or immunological disorders; to monitor organ

transplantation, for a rapid diagnosis of viral infections in

transplanted patients, in order to prevent an eventual

allotransplant rejection; to monitor antiviral as well as

immunomodulating therapies;

b) in industry, for the quality control of immunoglobulin

preparations obtained by chromatography or other preparative

methodologies;

c) in blood bank, to detect viral infections in apparently

healthy blood donors.

Immunoloofcal assays

Assays for detection of antibodies to IFN-y may be

developed -using natural and recombinant IFN-y , or IFN-y-

-relating peptides, of various lenghts, which are

representative of the different epitopes of the original

molecule. These assays may be based on any of the classical

RIA and ELISA techniques.

Microtiter plates, strips, wells or other solid phase

supports may be coated with the antigen with any suitable

technique known in the art, like overnight incubation at

various temperatures in appropriate buffers. The antigen used

for coating may be a single protein/peptide or a mixture of

different antigens depending upon the antibodies which have to

be detected. Antibodies may be detected in several specimens

like plasma, serum, cerebro spinal fluid, urine, saliva, tissue

culture fluids etc.

After incubation of the specimens with the antigen, the

bound antibody can be detected by addition of an enzyme

conjugated antiserum or monoclonal antibody. All the

traditional enzyme tracers can be employed in this assay

(horse radish peroxidase, alkaline phosphatase, etc.) and the

sensibility of the assay may be improved by use of any

biotiπ-avjdin or streptavidin system for detection of the

anafyte. The final detection can be achived by addition of a

substrate solution which is variable depending upon the

enzyme used.

Examples of idoneous substrate are o-phenylendiamine,

tetramethylbenzidine, paranitrophenylphosphate and others.

Results can be read by eye or by a spectrdphotometer and the

assay can be both qualitative or quantitative. Quantification

can be obtained by end point dilution, standard curve system or

by any other suitable quantification technique.

Any principle of assay can be used for the development of

an ELISA assay for detection of antibodies and particularly

they could be homogeneous assays or eterogeneous assays like

classical or modified competition assays, titration assays,

direct and indirect sandwich-assays, Ig capture assays and the

like.

The same principles used for detection of antibodies by

ELISA may be used by RIA or time resolved fluoroimmunoassay

or other assays based on the use of different tracers.

At the state of the art, antisera and monoclonal antibodies

obtained using natural and recombinant IFN-y can be used to

develop assays for detection of IFN-y in tissue culture fluids

and in biological specimens. Purified antibodies to IFN-y

obtained by affinity chromatography or other purification

procedures, may be used as well as antisera and monoclonal

antibodies and/or together with these reagents, for

U

development of antigen detection assays. All the conventional

thchniques may be applied to develop antigen detection assays

which may. be, for example, capture or competition

immunoassays (RIA, ELISA, TR-FIA etc.). To perform

competition immunoassays natural recombinant IFN-y, or IFN-y

-relating peptides can be used. They may be conjugated to

various tracer molecules. For example, they may be conjugated

to enzymes suitable for ELISA techniques, radioactive

materials for RIAs, fluorescent molecules for TRFIA and other

immunofljuorescent techniques, and others tracers. Detection

of antigen may also be obtained by immunofluorescence, flow

cytometry or other techniques known in the art.

Peptides

The general methodologies for obtaining peptides are well

known. Peptides of IFN-y may be obtained using different

methods:

a) Enzyme digestion and chemical cleavage of natural or

recombinant proteins. See, e.g., Arakawa et al. (1986), J. Biol.

Chem. 261. 8534; and Seeling et al. (1988), Biochem. Z∑, 1981.

b) Enzyme catalized synthesis in vitro. See, e.g., Mitin and

Zapevalova (1990), Int. J. Peptide Protein Res. &5_ 352.

c) Enzime modification of analogues.

d S nthesis b recombinant techni ues. See e. . Charbit

et al. (1987), J. Immunol. 139. 1658.

e) Site-specific and regionally directed mutagenesis of

protein-encoding sequences. See, e.g., Kunkel (1985), Proc.

Natl. Acad. Sci. U.S.A., fi£, 488.

f) Solution peptide synthesis. See, e.g., Bodansky (1984) in

Principles of Peptide synthesis, Springer-Verlag, Heidelberg;

Bodansky (1984), The practice of peptide synthesis,

Springer-Verlag, Heidelberg.

g) Solid-phase peptide synthesis. See, e.g., Sheppard

(1989), Solid Phase Peptide Synthesis, IRL Press, Oxford.

h) Segment condensation. See, e.g., Pettit (1976), in

Synthetic Peptides, Vol. 4, p22, Elsevier, Amsterdam.

i) And, in general, all the appropriate combinations of the

above mentioned methods.

The 146-amino acid sequence of mature human IFN-y,

deduced from the nucleotide sequence of a cloned cDNA is well

known. See, e.g., Gray et al. (1982) Nature, 295. 503, Grey and

Goeddel (1982), Nature 2 8, 859.

IFN-y -relating peptides of different lenghts may be

synthesized. For example, we have obtained IFN- -relating

peptides, spanning 14 residues which, at 7 amino acids

intervals, which cover all the native IFN-y primary sequence

from amino acid 1 (Cys) to amino acid 146 (Gin) (Table 3). This

list is not meant to be exhaustive, and shorter or longer

peptides within the sequence from peptide 1 to 23 (see table

3), may be obtained, combined, and used to detect or purify

natural human anti-IFN-y antibodies.

Provided that at least 5 aminoacids are present whereas

the upper limit is not critical but practical reasons limit the

maximum number of aminoacids to about 20. Particularly

preferred peptides are n° 1 , 2, 3, 9, 10, 11 ,13, 14, 15, 21, 22

in Table 3. IFN-y -relating peptides may have C-terminal

functions as free acid, alcohol, amide, ester, hydrazide, etc;

and N-terminat functisn modified. A number of techniques to

modify the N-terminal function are known in the art, the most

common are being acylation, aikylation, etc.

Some amino acid residues forming IFN-y-relating peptides

may be repeated, deleted or substituted in a conservative or

nonconservative manner. For example, amino acids from the

native structure of IFN-y may be substituted by other

uncorrelated amino acids that mimic the tertiary structure of

the epitope, forming a mimotope; amino acid residues may also

be replaced by pseudoisosteric aminoacids. Morover Methionine

can be replaced by N-Leucine or N-ethyl-Norleucine. Relevant

sequences may be also inserted into peptide skeleton to

increase its affinity to antibodies and its stability.

Peptides can also be conveniently giycosilated by

different su ars or modified su ar molecules. A number of

techniques for obtaining glycosilation of peptides are known in

the art. Peptides of IFN-y may be also modified in a way that

alter their backbone conformation. For example,

conformational constraints may be obtained inserting in the

peptide chain D-amino acids, alpha methyl amino acids, Proline

and other amino acids, even modified. See, e.g., Marshall and

Bassbard (1972), Circ. Res., Suppl., Suppl. II to 30, - 41 , 143;

Manavalan and Momany (1980), Biopolymers JJ 1943; Madison

and Kopple (1980), J. Am. Chem. Soc. 102. 4855.

Other possibilities of backbone modification may include

the C7 turn mimics of Huffman & Callahan, the β-turn mimics

of Freidingen, Kahn, Kemp and others. See, e.g., Huffman et al.

(1988). Peptides: Chemistry and Biology. Proc. 10th. American

Peptide Symp. (Marshall, G.R. ed.) pp 105-108, ESCOM, Leiden;

Freidingen, (1981) Peptides: Synthesis, Structure and Function,

Proc 774 American Peptide Symposium (Rich, D.H. & Gross, E.,

eds), pp. 673-783, Pierce Chemical Co., Rockford, IL; Kahn

(1988) Peptides, Chemistry and Biology, Proc. 10th American

Peptide Symp. (Marshall, G.R., ed.) pp. 109-111, ESCOM, Leiden;

Kemp & Sun (1982) Tetrahedron Lett. 22., 3759.

Peptide bonds may be modified so as to induce

conformational restriction or to increase stability to

enzymatic attack.

Peptide bonds modification can be either -C H< and

-CO-NH- without altering the N-C-C backbone atomic

sequence, for example C > - disubstituted peptides,

-β-dehydropeptides, thiated peptides, N-aikylated peptides,

N-hydroxylated peptides, nitrono peptides etc. Further

alterations may be obtained by N-C-C backbone modification

for example to form compounds containing d -hydroxy- or

it/ -amino acids; [yCH ₂ 0]; [y-CH (OH) CH ₂ ]; [y-CH ₂ -NH]; [ CH=CH].

These peptide surrogates or others can be used. See e.g.,

Spatola (1983) Chemistry and Biochemistry of Amino Acids

Peptides and proteins (Weinstein, B. ed.) Vol 7, pp 267-357,

Dekker, NY, McQuade et al (1990), Science, 247, 454, Miller et

al, (1989). For nomenclature see, e.g., IUPAC-1UB Commission

in Biochemical Nomenclature (1984) European J. Biochem., 138.

9. On the other hand IFN-y -relating peptides can be

conformationally restricted by either short-, medium- and

long-range cyclization to form homodetic cyclic peptides,

heterodetic cyclic peptides, bicyclic systems for instance

through N**-C ^[ , C *→C , C — C, N«C , C'^C ¹ , N-N, or

spiro-system formation, or even by other techniques known in

the art. See, e.g., Toniolo, 1990, Int. J. Peptide Protein Res. 35.

287.

Nucleotides, nucieosides deossinucleotides oligo- and

polinucleotides, lipides, glycolipids, terpens and their analogs

Lipids, glycolipids, terpens, and appropriate derivates of

these compounds may be bound to the IFN-y-relating peptides.

INF-y -relating peptides may also be formed by various

combinations of modified or unmodified peptides, obtainable

by different techniques known in the art.

Carriers

Peptides may be linked, for different purposes (for

example, to facilitate their binding to a support), to a suitable

carrier to form a conjugate. Any carrier may be used, such as

the various serum albumins, tetanus toxoids, or keyhole limpet

hemocyanin (KLH) as in the state of the art.

Peptide-protein conjugates can be obtained basically

using simmetrical or asymmetrical bifunctional reagents. They

may be incorporated into the final conjugate or may activate

certain reactive sites on one molecule for the subsequent

linkage with the other one.

A number of techniques for obtaining such linkage are

known in the art, including glutaraldehyde, bis imido esters,

carbodiimides, imido esters, toluene diisocyanate,

p-nitrobenzoyl chloride, hystamine dihydrochloride, MBS

(Maleimidobenzoyl-N-hydroxysuccimimide) and many others.

Point of attachment of carrier molecules on a peptide can be

different, depending on the peptide structure and the steric

requirements. If the peptide lacks a sulphydryl, this can be

provided by addition of a cysteine residue. These reagents

create a disulfide linkage between themselves and peptide

cysteine residues on one side, and an amide linkage through the

£ -amino on a Lysin or other free amino group on the other. A

variety of such disulfide/amide-forming agents are known.

See, e.g., Immun. Rev. (1982) 62, 185. Other bifunctional

coupling agents form a thioether rather than a disulfide

linkage.

Binding of INF-y-relating peptides to the carrier molecule

may be achived by non covalent bonds. For example, a peptide

may be linked to a carrier protein through a hydrophobic

domain in a hydrophobic poket.

Purification

Human natural antibodies directed against IFN-y, can be

purified from different specimens, including plasma, serum,

urine, saliva, or already purified human immunogiobulin

preparations. Human immunoglobulins may include different

classes (IgM, IgG, IgA, IgD or IgE). They may be in the native

structure or denaturated under a variety of experimental

conditions knows in the art., or may include antigen binding

fragments (F(Ab') ₂ , Fab, Fab ¹ , FV) of immunoglobulins. The

antigen binding fragments of immunoglobulins may be obtained

chemically, enzymatically, or by recombinant DNA techniques,

e.g. miniantibodies.

Human natural antibodies to IFN-y can be purified by any of

the known affinity techniques, using IFN-y or IFN-y-relating

peptides as specific binding molecules linked to a support.

One of the many techniques known in the art. is the

affinity chromatography.

IFN-y-relating peptides may be linked to many of the

known matrices including Agarose, silica gel, polyacrylamide,

and the like.

Binding of peptides to the matrix may be achieved

cross-linking the functional groups on the matrix and on the

peptide by interposition of a spacer arm. Spacer arms most

often are linear aliphatic C6 - C8 chain with functional groups

able to form a bridge between matrix and peptide. Both

hydrophilic compounds (i.e., alcohols) and hydrophobic

compounds (i.e. spacer arms including a benzene ring or others

groups) can be used. Carrier molecules can be used instead of

spacer arms.

In order to avoid denaturation of the peptides following

their attachement to a matrix and to facilitate this process, it

may be useful to preactivate the matrix so that subsequent

binding of the iigand may be achieved under mild conditions.

Activation is a chemical reaction between the matrix and the

2 «• ϋ -

activating compound, thus resulting in the formation, at the

surface of the matrix itself, of reactive groups (usually

electrophiltc) which readily combine with groups of the Iigand

(usually nucleophiiic, e.g. amino groups). Reactive groups such

as imidocarbonate, oxirane (epoxy-activation) ,

trichlorotriazine, O-imidazolylcarbonyl, and many others are

known in the art. By reacting with an N-hydroxysuccinamide

ester of ^' bromoacetic acid, a spacer arm with a terminal amino

group may be activated to give rice to a highly reactive

alkylating agent.

Ready to use matrices with activated spacer arms may be

used, such as, for example, SulphoLink coupling gel, in which

the active site is a iodoacetil group that readely react whith a

free sulphydryl group.

IFN-y-relating peptides may be also bound to a matrix by

non covalent bonds using, for example, a triazine dye resin.

Moreover, peptides of IFN-y may also be bound to a matrix by

reversible covalent bonds.

On the other hand, purified human antibodies to IFN-y may

also be linked to a support to affinity-purify the recognized

form(s) of IFN-y.

Affinity linkage between antibodies and IFN-y to the

affinity matrix can be broken either directly, by creating

conditions which are unfavourable for biospecific interactions

or by means of competitive affinity elution.

Therapy

Murine anti-IFN-y antibodies were shown to prevent the

rejection of allogeneic tumor cells, to inhibit the

Shwartzman-related or-like reaction, and to protect NZB mice

against spontaneous development of autoimmune diseases. See,

e.g., Landolfo et al. (1985), Science 229. 176, Billiau (1988),

Immunol. Today 9_. 37, Jacob et al. (1987), J. Exp. Med. 166. 798.

Purified human antibodies to IFN-y may be used in clinic as

specific antagonists of IFN-y to selective immunosuppress the

physiological response(s) induced by IFN-y , and more

specifically, those responses which are involved in the

up-regulation of the immune or autoimmune process. Being

proteins, the antibodies will be administered parentally,

preferably . intravenously. Since they may react with white

blood cells, they will preferably be administered slowly,

either from a conventional IV administration set or from a

subcutaneous depot. The dose for individuals, and for different

diseases, is determined by measuring the effect of the

anti-IFN-y antibody on the lessening of those parameters which

are indicative of the disease being treated. Based on the

experience of Jacob et al. (1988), J. Exp. Med. 166. 798, and

considering the natural clearance of antibodies, the dose of

human anti-IFN-y antibodies may have to be repeated

periodically depending upon the particular disease. When used

as prophylaxis, it may be possible to administer short courses

of human anti-IFN-y antibodies bimonthly, semiannually or

annually. In treating an existing disease it is expected a most

frequent antibody administration, also using infusion devices.

For autoimmune diseases that are known to be triggered or

aggravated by particular environmental factors which increase

the level of IFN-y, the dosage regimen will be scheduled

accordingly.

- When administered parentally, the human anti-IFN-

antibodies will be formulated in a unit dosage injectable form

(solution, suspension, emulsion) in association with a nontoxic

and nontherapeutic acceptable parental vehicle. Nonacqueous

vehicles may also be used. The vehicle may contain substances

that enhance isotonicity and chemical stability.- The antibody

is preferably formulated in purified form, substantially free of

aggregates and other proteins, at various concentrations

ranging approximately from 0.5 mg/ml to 20 mg/ml.

Examples

The ^following examples further illustrate the invention.

These examples are not intended to limit the scope of the

invention. In light of the present disclosure, numerous

embodiments within the scope of the claims will be apparent

to those of ordinary skill in the art.

I. Inhibition of natural and recombinant IFN-v-induced Fr.

receptor and HLA-DR antigens bv affinitv-purified human

anti-IFN-y antibodies.

U937 cells, when incubated in a medium containing

natural or recombinant IFN-y, increase both Fc receptor

and HLA-DR antigens on their surface. The maximal

effect is usually reached at 24h, using IFN-y at a

concentration of 200 U/ml.

As shown in figure 1 , when affinity-purified human

anti-IFN-y antibodies were present in U937 cell cultures

stimulated with IFN-y , they dramatically inhibited the

expected increase in Fc receptor and HLA-DR antigens.

The inhibition was .found to be dose-dependent (Table 4)

maximally effective with human anti-IFN-y antibodies at

a concentration of 4.8 At the same time no

inhibitory effect was observed when unrelated purified

human immunoglobulins were added, as a control, to IFN-y

treated U937 cell cultures.

Expression of Fc receptor sites (A, B, C), and HLA-DR

antigens (A ¹ , B ¹ , C ¹ ) on U937 was evaluated by flow

cytometric analysis.

Continuous line: cultures set up in the absence of IFN-y.

Dotted line: cultures set up in the presence of IFN-y (A,

A ¹ ), in the presence of IFN-y and human anti-IFN-y

antibodies (B, B ¹ ), and in the presence of IFN-y and human

unrelated immunoglobulins (C, C).

I.A.1. Effect of human anti-IFN-y antibodies on the Ivmphocvte

proliferation induced bv irradiated allooeneic peripheral

blood leukocy es (PBL).

On day 7, mixed lymphocyte cultures result in marked

lymphocyte proliferation. The addition of human

anti-IFN-y antibodies at the beginning of the culture

resulted in a markedly reduced uptake of [ ³ H]thymidine.

Tfte inhibiting ability of human anti-IFN-y antibodies

was gradually lost with decreasing concentrations of

antibodies (Table 5). Figure 2 shows that lymphocyte

proliferation was inhibited when human antibodies

specific to IFN-y were added on days 0 and 1. When the

same antibodies were added later (days 2, 3, and 4), no

inhibition in proiiferative response was observed.

Purified human anti-IFN-y antibodies were added at the

concentration of 4.8 μg/well at the initiation of mixed

lymphocyte culture or on day 1 , 2, 3, 4. [ ³ H]thymidine

pulse for 18h on day 6.

I.A.2. Effect of human anti-IFN-y antibodies on allooeneic

induced Ivmphocvte cvtotoxicitv.

Figure 3 shows a representative experiment on the

cytotoxic response of mixed lymphocyte cultures,

evaluated on PHA-stimulated PBL or on K562 cells. The

cytotoxicity of effector cells, recovered from cultures

set up in the presence of human anti-IFN-y antibodies,

was strongly reduced against PHA-stimulated PBL. On

the other hand, the human anti-IFN-y antibodies had only

a moderate influence on the development of cytotoxic

lymphocytes to K562 cells. In other experiments, the

cytotoxic activity to K562 cells was reduced to only

10-20% as compared to control cultures. PBL were activated in vitro with irradiated allogeneic

PBL. Purified human antibodies to IFN-y (•) or unrelated

purified human immunoglobulins (o) were added, at the

concentration of 4.8 /jg/well, at the initiation of mixed

lymphocyte culture. (■ ) PBL kept with medium only.

Target cells were PHA stimulated lymphocytes (A) and

K562 (B).

I I. Chromatography of human anti-IFN-y on an agarose

matrix cross-linked through a spacer arm to peptide n°

22 (See Table 3_.

Two mg of peptide n° 22, provided with an additional

L-cysteine at its N terminal, was linked to 2 mi of

Sulfolink coupling gel (PIERCE) following a standard

protocol • recommended by PIERCE. The affinity column

was connected to an FPLC apparatus (PHARMACIA) and

equilibrated with phosphate buffered saline (PBS).

Purified human antibodies (50 mg) were applied to the

column at a flow rate of 0.1 ml/min., and the bound

antibodies were eluted at the same flow rate with 0.1 M

glycine, pH 3.0.

Figure 4 shows a typical pattern of elution of antibodies

bound to the affinity matrix (See, peak 2).

I II. Western blot analysis of the affinity-purified human

anti-IFN-y antibodies.

Antibodies specifically reacting to IFN-y, and purified on

a IFN-y -relating peptide based affinity column, were

, confirmed to be immunoglobulins and to react to

recgmbinant IFN-y by Western blot analysis. Figure 5

shows that by using ¹²⁵ I-conjugated goat anti-human Ig

as a tracer, the anti-IFN-y antibodies, in a denaturated

form, were recognized as two reactive bands of 25,000

jand -50,000 molecular weight, being the light and heavy

chain of immunoglobulins respectively (a). At the same

time, these antibodies were capable of reacting with

- recombinant IFN-y proteins of 16,000 and 32,000

molecular - weight (Hoffmann-La Roche) (b). The

specificity _% of the recombinant IFN-y reactivity of

purified human antibodies was confirmed by comparing

it with the reactivity of a commercially available

anti-IFN-y monoclonal antibody (Boehringer) (g).

Human anti-IFN-y antibodies did not react with natural

IFN- (gift of Dr. Kari Kantell) (b), natural IFN-β (Serono)

(c), recombinant IFN-c( (Hoffmann-La Roche) (e), and

recombinant interleukin-2 (Boehringer) (f).

IV. ELISA for detection of anti-IFN-y antibodies in human

serum.

Wells of polystyrene microtitration plates were coated

with IFN-y-relating peptides or recombinant IFN-y . In

order to quantify the amount of specific anti-IFN-y

antibodies in the specimens, a negative control and a

series of positive controls were included in each assay.

The positive controls were given a value of 1 , 25, 30,

50, 60 and 100 arbitrary antibody units (AU). A standard

curve was plotted for each test run referring to the

adsorbance of these controls and each test specimen

was given a value in AU based on such standard curve.

A panel of 88 serum specimens was used to study the

correlation of the results obtained from an ELISA based

on an IFN- -relating peptide (peptide n° 21 in this

example; See, Table 3) and a RIA based on recombinant

IFN- as antigen on the solid phase. As shown in figure 6,

the correlation between ELISA and RIA was very high,

with a correlation coefficient of 0,94, and 80,5% of the

results foiling within ± 1 SD from the theoretical values

indicated by the linear regression line.

Ut i l ity

The various embodiments of the invention are useful for

the detection, quantitation, purification of human anti-IFN-y

antibodies, and for treatment of individuals susceptible to

autoimmune diseases or, more in general, of individuals

suffering from all those diseases where activated

cell-mediated immunity needs to be depressed.

APPENDIX 1

Literature

Maπiatis, Fritsch and Sambrook, MOLECULAR CLONING: A

LABORATORY MANUAL (1982); DNA CLONING, Volumes I e II (D.N.

Glover ed. 1985); OLIGONUCLEOTIDE SYNTHESIS (M.J. Gait ed.

1984); NUCLEIC ACID HYBRIDIZATION (B.D. Hames and S.J.

Higgins; eds. 1984); ANIMAL CELL CULTURE (R.K. Freshney, ed.

1986); IMMOBILIZED CELLS AND ENZYMES (IRL Press, 1986); B.

Perbal, A P ACTICAL GUIDE TO MOLECULAR CLONING (1984);

- The series, METHODS IN ENZYMOLOGY (S. Clowick and N. Kaplan,

eds., Academic Press, Inc.), and HANDBOOK OF EXPERIMENTAL

IMMUNOLOGY, Volumes l-IV (D.M. Weir and C.C. Blackwell, eds.,

1986, Blackwell Scientific Publications); M. Bodanszky,

PRINCIPLES OF PEPTIDE SYNTHESIS (1984); K. March, ADVANCED

ORGANIC CHEMISTRY (1988); THE PEPTIDES, ANALYSIS,

SYNTHESIS, BIOLOGY (E. Gross and J. Meienhofer, eds., 1980,

Acxademic Press, Inc.); P. Tijssen, PRACTICE AND THERAPY OF

ENZYME IMMUNOASSAYS (R.H. Burdon and P.H. Van Knippenberg,

eds., 1988, Elsevier); LA. Osterman, METHODS OF PROTEIN AND

NUCLEIC ACID RESEARCH, Volumes 1-3 (Springer-Verlag);

PROTEIN ENGINEERING (D.L. Oxender and C.F. Fox, eds., 1988,

Alan R. Liss, Inc.).

Table 1. The IFN-γ multiple activities.

T-lymphocytes Promotes T lymphocytes proliferation. Induces maturation of cytotoxic T lymphocytes. Inhibits maturation of suppressor T lymphocytes

B-lymphocytes Promotes Ig synthesis and switch to IgG2a Inhibits IgE synthesis

Macrophages Induces or increases the expression of MHC class II, Fc receptor, Integrin receptors Mac-1, LFA-1, CR3; induces synthesis of IL-1, TNF, chemotactic factor. Increases release of proteolytic enzymes. Activates the oxidative burst and killing of microorganisms and tumor cells.

Endothelial cells Induces the expression of MHC class II and ICAM-1 and release of chemotactic factors.

Epithelial cells Induces the expression of MHC class II and ICAM-1.

PMN Enhances the release of proteolytic enzymes and oxidative radicals.

3 1

Table 2. Disease known to benefit of a therapy with IFN-γ antagonists.

Type I Diabetes Delayed by immunosuppressive agents. Exacerbated by IFN-y.

Multiple Sclerosis Exacerbated by IFN-γ.

Lupus-Erythematosus Delayed by anti-IFN- ₇ MAbs.

Development of nephritis exacerbated by IFN-γ.

Adjuvant Arthritis Exacerbated by IFN-γ (early phase of the disease) and delayed by anti-IFN-γ MAbs.

Shwartzman Reaction Lethal effects of endotoxin, thrombosis and hemorrhagia prevented by anti-IFN-γ MAbs.

Delayed hypersensitivity Local recruitment of T cell inhibited by anti-IFN-γ MAbs.

Allotransplant Rejection Rejection of tumor skin and heart allografts delayed o blocked by anti-IFN-γ MAbs.

Table 3. Sequences of peptides of the invention

1 - Cys-Tyr-Cys-Gln-Asp-Pro-Tyr-Val-Lys-Glu-Ala-Glu-Λsn-Leu. (aa. 1-14).

2 - Tyr-Val-Lys-Glu-Ala-Glu-Asn-Leu-Lys-Lys-Tyr-Phe-Asn-Ala. (aa.7-20).

3 - Asn-Leu-Lys-Lys-Tyr-Phe-Asn-Ala-Gly-His-Ser-Asp-Val-Ala. (aa. 13-28).

4 - Asn-Ala-Gly-His-Ser-Asp-Val-Ala-Asp- sn-Gly-Thr-Leu-Phe. (aa. 19-32).

5 - Val-Ala-Asp-Asn-Gly-Thr-Leu-Phe-Leu-Gly-Ile-Leu-Lys-Asn. (aa.25-38).

6 - Leu-Phe-Leu-Gly-Ile-Leu-Lys-Asn-Trp-Lys-Glu-Glu-Ser-Asp. (aa.31-44).

7 - Lys-Asn-Trp-Lys-GIu-Glu-Ser-Asp-Arg-Lys-Ile-Met-Glπ-Ser. ^' (aa.37-50).

8 - Ser-Asp-Arg-Lys-Ile-Met-Gln-Ser-Gln-Ile-Val-Ser-Phe-Tyr. (aa.43-56).

9 - Gln-Ser-Gln-Ile-Val-Ser-Phe-Tyr-Phe-Lys-Leu-Phe-Lys-Asn. (aa.49-62). 0 - Phe-Tyr-Phe-Lys-Leu-Phe-Lys-Asn-Phe-Lys-Asp-Asp-GIπ-Ser. (aa.55-68) 1 - Lys-Asn-Phe-Lys-Asp-Asp-Gln-Ser-Ile-Gln-Lys-Ser-Val-Glu. (aa. 61-74). 2 - Glα-Ser-Ile-Gln-Lys-Ser-Val-Glu-Thr-Ile-Lys-Glu-Asp-Met. (aa. 67-80). 3 - Val-Glu-Thr-Ile-Lys-Glu-Asp-Met-Asn-Val-Lys-Phe-Phe-Asn. (aa.73-86). 4 - Asp-Met-Asn-Val-Lys-Phe-Phe-Asn-Ser-Asn-Lys-Lys-Lys-Arg. (aa.79-92). 5 - Phe-Asn-Ser-Asn-Lys-Lys-Lys-Arg-Asp-Asp-Phe-Glu-Lys-Leu. (aa. 85-98). 6 - Lys-Arg-Asp-Asp-Phe-Glu-Lys-Leu-Thr-Asn-Tyr-Ser-Val-Thr. (aa. 91-104). 7 - Lys-Leu-Thr-Asn-Tyr-Ser-Val-Thr-Asp-Leu-Asn-Val-Gln-Arg. (aa. 97-110). 8 - Val-Thr-Asp-Leu-Asn-Val-Gln-Arg-Lys-Ala-Ile-His-Glu-Leu. (aa. 103-116). 9 - Gln-Arg-Lys-Ala-Ile-His-Glu-Leu-Ile-Gln-Val-Met-Ala-Glu. (aa. 109-122). 0 - Glu-Leu-Ile-Gln-Val-Met-Ala-Glu-Leu-Ser-Pro-Ala-Ala-Lys. (aa. 115-128). 1 - Ala-Glu-Leu-Ser-Pro-Ala-Ala-Lys-Thr-Gly-Lys-Arg-Lys-Arg. (aa. 121-134). 2 - Ala-Lys-Thr-GIy-Lys-Arg-Lys-Arg-Ser-Gln-Met-Leu-Phe-Gln. (aa. 127-140). 3 - Lys-Arg-Ser-Gln-Met-Leu-Phe-GIn-GIy-Arg-Arg-Ala-Ser-Gln. (aa. 133-146).

Table 4. Effect of natural human antibodies to IFN-y, added at differe concentrations on the expression of Fc receptor and HLA-D antigens on U937 cells stimulated with rlFN-y (a).

4.80 83.6 73.5 0.7

a. U937 cell cultures were stimulated for 24 hr with 200 U of rlFN-y a at the same time, they were treated or not with differe concentrations of human anti-IFN-y antibodies, or with purified hum unrelated Ig.

Table 5. Effect of human anti-IFN-y antibodies added at different concentrations on allogeneic induced lymphocytic proliferation (a).

Addition to [ ³ H] thymidine Inh ibition culture incorporation (%) (cpm + SD)

Exp. n. 1

97 32.9

0

0

Exp. n. 2

94 16

0

3.2

a. Lymphocytes admixed with irradiated allogeneic PBL, at a responder: stimulator ratio of 1 :1 , were incubated with medium alone or with medium containing different ratios of human antibodies to IFN-y , or of unrelated human antibodies. Proliferative response was measured on day 7 after 18 hr pulse with [ ³ H] thymidine.