NL8700927A - ANTI-INTERFERON GAMMA ANTIBODIES; AND THEIR THERAPEUTIC APPLICATION. - Google Patents

Description

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Annex to letter dated May 4, 1987 * P HP / AB / 19 - 1 -

Anti-interferon-γ antibodies; and their therapeutic application

Inflammatory reactions are part of many vertebrate diseases, such as humans. In the broadest sense, the term "ignition" refers to local as well as general reactions. Local inflammation is characterized by vasodilation, fluid transudation from the vessels, tissue infiltration by leukocytes and, in some severe cases, intravascular thrombosis, damage to blood vessels and blood extravasation. The general inflammatory reaction, also referred to as acute phase reaction, is characterized by 10 different reactions, including increased body temperature, changes in the level of the different blood components (cellular as well as dissolved elements) and changes in blood pressure. In severe cases, shock and death can occur.

Local and / or general inflammatory reactions occur as part of many diseases with endogenous or exogenous etiology, especially in (1) infections (local or general) with bacteria, viruses, fungi, parasites; (2) allergic reactions to certain environmental substances, medicines, faulty blood transfusions; (3) as part of auto-immune diseases in which the body's defense system mistakenly attacks its own tissues (eg, rheumatoid arthritis, lupus erythematosus, demye, linearization diseases, etc.); and (4) the rejection of tissue grafts.

In many cases the local inflammation is self-limiting. In some cases, the local inflammation may persist for a long time, in some cases lifelong, e.g. rheumatoid arthiritis. General inflammation is usually self-limiting in the case of certain acute viral infections. In other cases, however, the reaction can be so severe that vascular shock and death occur. This is '. 87 0 0927 - 2 - eg the case with Waterhouse-Friderichsen syndrome, a complication of a general infection with meningococci. Another example of a severe inflammatory response is the "Toxic Shock Syndrome" or "Tampon Disease" that occurs as a result of staphylococcal infection in women who use intravaginal tampons during menstruation.

The exact mechanism underlying the different inflammatory forms is only partially known. Several endogenous mediators are involved and initiate or regulate the changes observed in tissues, or in physiological parameters, of patients suffering from the inflammatory diseases. These mediators belong to a number of categories: (1) small molecules, such as histamine and prostaglandins; (2) serum proteins, such as the complement factors; (3) certain hormones, such as corticoids; and (4) cytokines, such as interleukin-1 (IL-1), tumor necrosis factor (TNF) and interferons.

The present invention is based on the observation that the administration of vertebrates to antibodies that bind and neutralize interferon-γ can suppress local as well as general inflammatory reactions.

The antibodies are proteins that belong to the class of immunoglobulins. They must be prepared in such a way that they have the ability to bind and neutralize interferon-γ molecules of a specific animal species in which inflammation must be suppressed. In the examples described, the mouse was used as a test animal and therefore interferon-γ antibodies directed against mice were raised. Several known techniques are available to obtain suitable antibodies, in particular: (1) polyclonal antibodies isolated from the serum of animals immunized with the desired interferon-γ; (2) monoclonal antibodies isolated from the supernatant of hybridoma cell cultures; (3) monoclonal antibodies isolated from serum - 87 0 0927 - 3 - or ascites fluid from animals inoculated with the hybridoma cells; and (4) monoclonal antibodies obtained by recombinant DNA technology.

Neutralizing interferon-γ with the help of the antibodies means that by adding these antibodies to interferon-γ, the latter must lose its biological activities. This biological activity can be measured with various techniques; the most suitable is based on the fact that interferon-γ shows anti-viral activity in cultured cells. Addition of antibodies must remove the antiviral activity of interferon-γ and other biological activities must also disappear.

Monoclonal or polyclonal antibodies can be administered by conventional routes. For example, the compositions are supplied in a freeze-dried, refrigerated state and may contain, in addition to a filler, a preservative, an antioxidant, etc. In the mouse, the preferred mode of administration is intraperitoneal injection. In other animal species, e.g., humans, other modes of administration may be more suitable, e.g., intramuscular or intravenous injection. For local administration through the surface of the body permeable to immunoglobulins, the composition containing the antibodies against interferon-γ may take the form of a gel, a cream or an ointment.

Vertebrates inoculated with monoclonal antibodies to homologous interferon-γ show a reduced response for various exogenous inflammatory stimuli. The extent and duration of the inhibition depends on the dose and epitope specificity of the antibody used. For a long duration of the inhibitory effect, it is essential that the administered antibody be slowly removed. Such removal depends inter alia on the differences that exist between the administered antibody and the body's own immunoglobulins. Ideally, the antibodies have a homologous origin.

Although the inflammatory reactions are usually a normal part of the body's defense against harmful agents, an excessive or continuous inflammation is often undesirable. In such cases it will be desirable to administer antibodies to γ-interferon, as this will reduce swelling, pain, bleeding, scarring and other unwanted effects of the inflammation.

Examples II-VI show the effect of anti-interferon-γ on local (examples II, III, IV and V) and general (example VI) inflammatory reactions elicited with bacterial lipopolysaccharides (LPS). LPS is a universal component from the wall of all Gram negative bacteria.

Although the polysaccharide part of the molecule differs from bacteria to bacteria, the lipoid core of the molecule has a constant structure. In vertebrate infections with Gram negative bacteria, the local as well as general inflammatory reactions are mainly due to the many biological effects of the lipoid core.

The local reaction consists of edema and cellular infiltration. The general reaction is formed by fever and the appearance of so-called acute phase proteins in the serum. In an extreme form, the reactions to LPS are known as the Shwartzman reactions, which can be local and / or general. They are characterized by intravascular thrombosis.

The general Shwartzman reaction is often lethal.

Although the milder forms of inflammation caused by LPS are common features of naturally occurring infections with Gram-negative bacteria, the more severe (Shwartzman-like) forms are quite rare in humans and animals. The classic example is Waterhouse-Friderichsen syndrome, which occurs as a complication of meningococcal infections. It is characterized by general intravascular coagulation. The fatal outcome is due to hypotension, which in turn is due to bleeding in the adrenal glands. Maternal infections with Gram -8700927 * - 5 - negative bacteria often lead to preterm labor and fetal death. A local Shwartzman-like reaction occurring in the placenta is believed to be the cause of this. Shwartzman-like lesions have been observed in the kidneys of children who have also died from E. coli enteritidis.

Example VII relates to an inflammatory reaction caused by a delayed hypersensitive hex response (DTH = Delayed type Hypersensitivity reaction). The DTH reactions occur as a result of repeated or continuous exposure of the body to certain proteins (antigens) that are foreign to the body. The pathogenesis is well known; the foreign antigens stimulate the multiplication and activity of T0g cells producing interferon-γ. This interferon-γ activates macro phages which become toxic to their environment. Inflammation is the result. DTH reactions are very frequent in the clinic. They occur in the form of inflammatory reactions in the skin (eczema) due to a wide range of environmental substances. A dramatic form of the DTH reaction is formed by the rejection of an organ or tissue in case the donor is not histocompatible with the acceptor. Certain organs such as the kidney and heart are relatively resistant to rejection reactions. However, the skin is particularly sensitive. Inhibition of skin graft rejection reactions (as shown in Example VI) is considered to be a very sensitive assay for an agent that suppresses the DTH reaction.

Example I Preparation of monoclonal antibodies against murine interferon-γ

An 8 week old female Lou / Ca rat was immunized intraperitoneally for 37 weeks with purified murin 35 interferon-γ (MuIPN-γ) emulsified in incomplete

Freund's adjuvant. A second set of intraperitoneal injections was given 17.5 weeks later. 21.5 weeks after the last .8700927-6 injection, the rat was injected intravenously with 300,000 units of pure MuIFN-γ. Four days after the last immunization, spleen cells in a 3: 1 ratio were mixed with non-secretor murine myeloma cells Sp2 / 0Agl4 and fused in the presence of 50% polyethylene glycol-4000 (Merck, Darmstadt, Federal Republic of Germany), one and the other according to standard methods. The fused cells were seeded in 96-well microtiter plates (50 μΐ / well; Costar 3596, Cambridge) in Eagle's Minimal Essential Medium (EMEM), which was supplied with 10% fetal calf serum (FBS), 2 mM glutamine, 2. 2 g / 1 sodium bicarbonate and HT (hypoxanthine, 0.1 mM, thymidine, 16 μΜ). The following day, an additional amount of EMEM was added to the wells, containing HAT medium (with aminopterin, 0.4 μΜ). The cultures showing hybrid-15 cell growth were examined for the presence of anti-MuFIN-γ antibodies using an ELISA method. A direct neutralization test was used to detect neutralization of the anti-viral activity. Positive hybridomas were subcloned into microtiter plates twice by the protein dilution method. Three stable hybridomas secreting IgG2a antibodies directed against MuIFN-γ were selected and designated Fi, F2 and F3.

Hybridoma cells (10 ^) were injected into the intra-peritoneal cavity of thymusless mice pre-treated with 0.5 ml Pristan mineral oil (2,6,10,14-tetra-methylpentadecane, 96%). Ascitic fluid and serum were harvested 7 to 14 days after cell inoculation.

Ascitic fluid and serum from the mice inoculated with hybridoma cells were examined for interferon-γ binding activity using an ELISA. Microtiter plates (Nunc, Immuno plate I-96F, Roskilde, Denmark) were coated with 0.1 µg of pure mouse interferon-γ and incubated at 4 ° C for at least 24 hours. After all nonspecific binding sites were saturated with 0.5% casein in phosphate buffered saline, the plates were washed three times with 0.15 M NaCl, 0.05% Tween-20. Next, sets of 0/5 log 10 dilutions of the ascites fluid and serum were added (100 µl) and the plates were incubated for 2 hours at room temperature. After washing, horseradish peroxidase conjugated rabbit anti-rat immunoglobulin (DakO / Denmark) was added at a dilution of 1/1000 in phosphate buffered saline (pH 7.4) containing 5% PBS, 5% mouse serum and 0. 05% Tween-20 contained. After incubation for 1 hour at room temperature, the plates were washed and 100 μl of color reagent (0.4 mg / ml 0-pheny-10-lendiamine-2-HCl in 0.1 M phosphate / citrate buffer, pH 6.0 containing 0.02% H2O2 sol (30%)). The reaction was stopped after 50 min by adding 50 μ 50 4 M H2SO4. The optical absorbance (OA) at 450 nm was used as a measure of the binding activity. Binding titers were expressed as the reciprocal of the dilution corresponding to 50% of the maximum OA reading.

The neutralization titer of the antiserum was determined by incubating series 0.5 log dilutions of the ascites fluid or serum with an equal volume of 30 units / ml of 20 mouse interferon-γ. The mixtures were incubated for 4 hours at 37 ° C and tested for antiviral activity on mouse L929 cells using a cytopathic effect inhibiting assay with Mengovirus as a challenge. The titers were expressed as the dilution of the antiserum yielding a 50% reduction in antiviral protection.

The results of the binding and neutralization tests with the obtained monoclonal antibodies are shown in Table I. The table shows that the preparations (Fi, F2 and F3) have an equivalent binding activity for interferon-γ.

F2 and F3 had greater neutralization potential for the antiviral activity of interferon-γ.

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Table I Neutralization and binding capacity of monoclonal antibodies against MuIFN-γ

Antibody Antibody Power Ratio 5 _ login units / ml A / B xlOOOa

Code Sample No. A. Neutrali- B. Binding _sation test test_

Fi 2574 serum 3.25 6.0 1.8 10 F2 2576 serum 5.5 4.9 3,980 2575 ascites 5.25 4.75 3,160 F3 2578 serum 5.75 5.7 1,120 15 2577 ascites 5.75 5. 7 1,120 a Calculated as the number of neutralization units per 1000 binding units 20

Example II Inhibition of local inflammation by anti-interferon-monoclonal antibodies

The footpad model was used in the mouse to demonstrate the inhibitory effect of anti-interferon-γ antibodies on local inflammation.

Female NMRI mice, 6 weeks old, were obtained from the test animals of the University of Leuven, Belgium. S. marcescens lipopolysaccharide B (LPS) was obtained from Difco (Detroit, Mich., United States of America).

The mice were injected in the right sole of the foot with 5 µg LPS (25 µ.). Phosphate buffered saline (PBS), pH 7.4 was injected into the left sole of the foot as a control. Footpad swelling was measured daily using a micrometer caliper. The footpad swelling was calculated as the percentage increase in the footpad thickness, by comparing the thickness of the LPS injected footpad .8700927-9 (li) with that of the footpad (C) of the other leg.

% increase = L - C x 100 C.

At least four mice were used for each experimental group. The percent inhibition of footpad swelling was calculated by the following formula;

Percentage inhibition = / l - avg. swelling in exp. group lx 100 10 V avg. swelling in control group /

Each of the three monoclonal antibodies was tested for its effect on the course of the local reaction in mice given a dose of 5 µg LPS in a footpad. The antibodies were administered as serum and ascites fluid in a single intraperitoneal injection 24 hours before the challenge with LPS. The dose was adjusted to inject equivalent amounts of interferon-γ binding units. Control mice were given a saline solution. The results are shown in Table II. This shows that the antibody preparations F2 and F3, which have neutralizing activity, inhibit footpad swelling, while Fi, which is a poorly neutralizing antibody, does not affect the swelling reaction in the footpad.

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Table II Effect of monoclonal antibodies against MuIFN-γ on LPS-induced footpad swelling reactions in mice: comparison of neutralizing (F2 and * 3> with poorly neutralizing (Ρχ) antibodies 5 __

Footpad swelling b

Injected antibodya (% + S.E? N = 4)

Code ^ Neutralization titre Day 2 Day 3 10 _ (- logi n) _

Fi serum 1.80 128 (9.79) 106 (15.28) F2 serum 5.40 14 (1.39) 11 (2.83) F2 ascitic fluid 5.25 12 (3.56) 7 ( 2.02) 15 F3 serum 4.50 31 (5.09) 23 (6.99) F3 ascitus fluid 5.50 13 (1.09) 11 (2.09)

Control (9% salt) - 135 (2.64) 121.5 (3.82) 20 a The antibodies were administered intraperitoneally 24 hours after a challenge with LPS at a dose of 0.1 ml containing 4.55 logio binding units / ml, b Swelling was measured 2 and 3 days after the LPS injection (5 µg).

Example III Inhibition of Local Inflammation with Monoclonal Anti-Interferon-γ Antibodies In this example, the same technique for measuring the local inflammation as in Example II was used. The design of this experiment was similar to that of the previous experiment except that the antibodies were given 56, 42, 28, 21, 10 and 5 days before the local challenge with LPS. Footpad swelling was recorded 2 and 3 days after the challenge. The results shown in Table III indicate that the inhibitory activity of the monoclonal antibodies on the initial phase of the footpad swelling reaction continued for 6 weeks.

Table III Effects of monoclonal antibodies to 5 MuIFN-γ on LPS-induced footpad swelling reaction in mice: duration of effect

Time of% Inhibition (+ SE) of Sole Swelling on Antibody Dosea Day 2 Day 3 10 (Days for LPS) _After LPS _After LPS_ 98-6 (+ 25) 5 (+ 22) 66 32 (+17) 8 (+ 23) 56 22 (+ 30) 26 (+ 27) 15 42 71 (+ 10) 61 (+ 20) 28 71 (+ 10) 85 (+ 2) 21 83 (+ 2) 91 (+ 2) 10 94 ( + 3) 89 (+ 2) 5 84 (+3) 84 (+ 3) 20 _ a Antibody dose (F3) s 1.0 ml i, p. (5.75 logio neutralizing units / ml). Each value represents the mean inhibition calculated from 4 treated mice compared to 4 control mice.

Example IV Inhibition of inflammation by monoclonal antibodies against interferon-γ: dose-response relationship 30

In this experiment, the same technique for measuring local inflammation as in Example II was used. The design of the experiment was also the same, except that the F3 monoclonal antibodies were given in three different doses. The footpad swelling reaction was examined on days 2 and 3. The results shown in Table IV indicate that the minimum dose of antibodies required to inhibit inflammation is 1 () 3.75 neutralizing units per mouse. Since the mice weighed an average of 25 g, the minimum dose can be expressed as 10 ^ 35 neutralizing units per kg body weight.

5

Table IV Effects of the monoclonal antibody (P3) against MuIFN-γ on the LPS-induced footpad swelling reaction in mice: dose-response relationship 10 Dose of antibody a% Inhibitory footpad swelling on logio neutralizing day 2 day 3 units / mouse_na LPS_na LPS_ 4.75 94.0 (1.2) 91.0 (1.6) 15 3.75 63.0 (9.5) 58.0 (13.8) 2.75 5.5 (13.0) -8 .5 (29.4) a Administered by intraperitoneal injection 0.1 ml, 24 hours before the LPS challenge.

b Each value represents the mean percent inhibition calculated from 4 treated mice and compared to the 4 control mice. By the way: standard deviation.

Example V Inhibition of inflammation with polyclonal antibodies to interferon-γ

The polyclonal antibodies were prepared from serum from rabbits immunized by repeat injections with mouse interferon-γ. The antiserum was found to contain 1q3.75 μ-u-γ neutralizing units per ml.

The ability of this serum to block inflammation was tested with an experimental protocol similar to that described in Example II. The results shown in Table V indicate that mice that received 4 intraperitoneal injections with 0.1 ml of antiserum (see the time scale in the footnote of Table V) have a notable. $ 700 927 - 13 - decrease in footpad response to LPS, showed.

Table V Effect of polyclonal antibodies to mouse IFN-γ on LPS-induced footpad swelling reaction in mice

Footpad swelling b

Treat in% £ S.E., N = 4_ _day 2_day 3_ 10

Polyclonal anti-ÏFN-γ antibody 57 (25/8) 55 (15.0)

Control (9% saline) 125 (6.8) 108 (2.8) 15 __ a The antibody or saline was injected i.p. given on days -1, 0, 1 and 2 relative to the LPS challenge and in a dose of 0.1 ml (= 2.75 logio neutralizing units-20 units per injection).

b The swelling was measured on days 2 and 3 after the LPS injection (5 µg).

Example IV Prevention of a lethal endotoxin shock by administering anti-interferon-y antibodies

A general inflammatory reaction known as the Shwartzman reaction or Sanarelli reaction was elicited in 30 mice. The technique consists of giving two consecutive injections with the endotoxin of Gram negative bacteria. The reaction is characterized by a general coagulopathy evidenced by the petechiae on the skin of the mouse ear. The general shock is evidenced by an inactivity of the mouse and a pilot erection. The shock reaction is lethal for a high percentage of the animals.

The experiment illustrated with the data • 8700927 - 14 - f from Table V used LPS from S. marcescens (see Example II). The mice received a local injection (5 µg) in the sole of the foot, followed 24 hours later by an intravenous dose of 100 µg. The results show that the occurrence of the disease could be completely prevented by treatment with monoclonal antibodies against interferon-γ.

Table VI Prevention of the lethal endotoxin shock by administration of anti-interferon-γ antibodies 10 _

Control (no Treatment with _anti body) _antibodies b_

Total number 12 12 15 Number of patients 0 12 0

Number of deaths 7 0 a. The shock was induced by a combination of two injections with LPS: a 5 µg dose preparation was administered in the sole of the foot, followed 24 hours later by an intravenous, provocative dose of 100 µg. b Intraperitoneal injection with 0.1 ml (4.55 log o binding units / ml) both 24 hours before the local injection and simultaneously with the intravenous, provocative dose.

° Inactivity, pilo-erection and petechiae.

Example VII Inhibition by anti-interferon-γ antibodies of skin graft rejection

It is known that skin grafts are usually shed faster than other organ transplants. Factors contributing to the rapid rejection of skin grafts include extensive nonspecific inflammation due to damage to the dermis and, to a significant extent, ischemic necrosis in the post-transplant period. • 87 0 0527 # - 15 - tation.

It. ability of monoclonal antibodies to MuIFN-γ to alter the survival time of a skin graft was investigated using skin-5 portions from donor C57BL / 6J mice transferred to recipient Balb / c mice. The mice received an intraperitoneal injection with anti-MuIFN-γ or phosphate buffered saline pH 7.4 1 hour before transplantation and then daily for 10 days. The grafts were examined daily and rejection was assumed if 100% of the transplanted area had undergone necrotic degeneration.

The results shown in Table VII indicate that control Balb / C mice receiving C57BL / 6J skin grafts shed their grafts from day 7. However, treatment with monoclonal antibodies to MuIFN-Y for 10 days delayed graft rejection.

Table VII The effect of monoclonal antibodies

HulFN-y on the survival of mouse skin graft a _ Viable grafts_ 25 Days after Treated with Control graft antibody b_ (no antibody) _N / total _% _ N / total_% 7 8/8 100 9/9 100 30 8 8/8 100 8/9 89 9 8/8 100 4/9 44 10 6/8 75 2/9 22 11 5/8 62.5 2/9 22 12 2/8 25 1/9 11 35 13 1/8 12, 5 0/9 0 14 1/8 12.5 0/9 0 15 _0 / 8_0_0 / 9_0 .8700927

, I

16 a Donors: C57BL / 6J mice; recipients: Balb / C mice b Intraperitoneal injection of 0.1 ml (5.0 logio neutralizing units / ml) 1 hour before transplant and daily for 10 days.

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Claims (5)

1. Immunoglobulin with neutralizing antibody activity against human or animal interferon-γ for use as an active therapeutic agent. 2. Immunoglobulin with neutralizing antibody activity against human or animal interferon-γ for use as an active agent to suppress inflammatory reactions. 3. Immunoglobulin with neutralizing antibody activity against human or animal interferon-γ for use as an active agent to suppress inflammatory responses due to infections, allergic reactions, autoimmune diseases, graft rejection reactions, DTH- reactions. 4. A medical or veterinary composition containing an immunoglobulin according to claims 1-3 and a pharmaceutically acceptable filler. 5. A medical or veterinary composition for suppressing inflammatory responses in man or an animal species, comprising immunoglobulin with neutralizing antibody activity against interferon-γ of human and animal species, respectively, and a pharmaceutically acceptable filler. 8700927