WO1998015577A1 - METHODS OF ACTIVATING OR ENHANCING Fc RECEPTORS - Google Patents

Abstract

This invention relates to a method of activating FcR or of improving the function thereof, by contacting FcR with an effective amount of a molecule, preferably an antibody. It also relates to a more sensitive assay for HITTS, diagnostic kit therefor and to reagents, compositions and kits for use in diagnosis, prophylaxis or treatment of diseases where activation or improvement of FcR activity play a role.

Description

METHODS OF ACTIVATING OR ENHANCING FC RECEPTORS

The present invention provides methods of activating or improving the activity of Fc Receptors (FcR) , a reagent for use in the methods and methods of diagnosis, treatment or prophylaxis of disease where activation or improvement of FcR activity assists in diagnosing or combating the disease. It also relates to improvement in immune responses, and to improvement of the efficacy of antibodies used in treating diseases.

BACKGROUND

Immune complexes that form as a result of the interaction between antibody and antigen, or aggregation of antibodies, are able to bind to Fc Receptors which are specific cell surface molecules present on many different cell types. Such receptors include FcγRI , FcγRII, and FcγRIII which are receptors for IgG, and receptors for other immunoglobulin types such as FcαRI, the IgA receptor, FcεRI, the IgE receptor and so on. However in certain circumstances immune complexes cannot bind to, or fail to activate, Fc receptors. One example is the binding of certain types of immunoglobulins to FcγRII. It is known that mouse IgGl will not bind to a form of human FcγRII called the "non-responder" . Similarly, human IgG2 will not bind to a form of human FcγRII called the "responder" . However, human IgG2 will bind to the "non-responder" form. Thus, it is clear that in a number of situations, certain Fc receptors are either not able to bind immunoglobulins or bind them poorly and therefore, cannot be activated. This has significance in assays involving FcR function; such assays will not be reliable as certain immune complexes will be unable to bind to or activate their receptor. It is also clear that the inability of Fc receptors to bind immunoglobulins may predispose individuals to diseases either where immune complexes cannot be removed or where the binding would otherwise activate the cells. In addition, antibody mediated therapy of a disease assumes that such antibodies, and the immune complexes that they form, will be able to bind to Fc receptors and activate appropriate responses. However, this will not always be the case and clearly, such antibody mediated therapy would be ineffective or have a profoundly reduced efficacy. Thus, any method that could improve FcR based assays, improve therapy of a disease by antibody or improve FcR function in vivo would be useful . In work leading to the invention, the inventors have surprisingly found that antibodies which recognise epitopes on human Fc receptors are able to convert nonfunctional Fc receptors into functional receptors which are able to bind to immune complexes and/or activate cellular responses. They have also found that these antibodies are able to enhance activity m already functional FcR. Without wishing to be bound by any proposed mechanisms, the conversion from non-functional receptors to functional receptors appears to be associated w th a change in the shape of the FcR molecule and/or multimeπsation of the receptor .

SUMMARY OF THE INVENTION

Accordingly, m a first aspect the invention provides a method of activating or improving the function of FcR, comprising the step of contacting FcR with an effective amount of a molecule capable of inducing said activation or improvement, for a time and under conditions sufficient to allow said activation or improvement to occur.

Preferably the molecule is an antibody or fragment thereof capable of inducing said activation or improvement. More preferably the antibody is capable of inducing a change m configuration of the FcR, such as multimeπsation, and/or a shape change m FcR. Such antibodies may be polyclonal or monoclonal antibodies or fragments of antibodies, such as scantibodies , humanised antibodies, antibody mimetics (Smyth and Von Itzstein) and the like. Preferably the antibodies are monoclonal antibodies. More preferably the monoclonal antibodies have an activity comparable to that of monoclonal antibodies 8.2, 8.26 and CIKM5 as disclosed herein, or are biological equivalents thereof.

In a second aspect, the invention provides a method of activating or improving the function of FcR in an assay which utilises FcR as a reagent, comprising the step of contacting an effective amount of a molecule with said FcR prior to or during the assay, wherein said molecule is capable of inducing said activation or improvement.

The term "an assay which utilises FcR as a reagent" refers to any assay in which FcR is used. Generally, such assays will utilise the ability of FcR to bind antibody or immune complexes. However, the FcR may be used because of other properties of the receptor, such as phagocytosis, cell activation, platelet aggregation or induction of killing of unwanted cells. Such assays may be used in determining the amount of Ig in a sample, detecting immune complexes in a sample, removing Ig from a sample (such as during plasmapheresis) , diagnosis of diseases and the like, such as those disclosed in PCT/AU95/00606. An example is a diagnostic assay, wherein the assay comprises use of FcR the function of which is activated or improved in accordance with the method of the invention. In a preferred aspect, the assay is a diagnostic assay for heparin-induced thrombocytopaenia syndrome (HITTS) .

HITTS is a condition in which heparin-antibody complexes ( IgG-platelet factor 4 immune complexes) activate platelets by binding to the platelet FcγRII. This results in loss of platelets from circulation. The diagnostic assay for HITTS involves taking a third party's platelets and mixing these with heparin and serum of a patient. If the platelets aggregate in the presence of the serum and heparin, this indicates that HITTS is the likely diagnosis. However, the source of donor platelets is a problem as the platelets of many people (up to 20%) do not aggregate in the assay. This is because their FcR is unable to be activated. However, the platelets of these individuals are susceptible to activation by the molecules used in the present invention. Thus, the invention ensures that heparin-antibody complexes bind to platelet Fc receptors and activate platelets when the molecule is added; patients previously shown to be negative for HITTS using less sensitive, conventional assays can be positvely identified. Furthermore, equivocal results obtained using such assays can be resolved by using the method of the invention.

In addition to the poor specificity and sensitivity, conventional methods of diagnosis of HITTS are also expensive. For example, a commercially available ELISA for the condition costs about $3000 per assay. In a third aspect, the invention provides a diagnostic kit for HITTS, comprising FcR and an effective amount of a molecule which activates or improves FcR function, thereby to enhance the binding of the FcR to the heparin-antibody complexes. Preferably, the specificity and/or sensitivity of the FcR for the immune complexes is also enhanced. The invention thus enables a cheaper and more sensitive way to detect HITTS.

In a fourth aspect, the invention relates to a method of diagnosing HITTS, comprising the step of contacting serum of a subject suspected of having HITTS with an FcR activated in accordance with the invention. Preferably, the method comprises steps taken in conventional assays with the exception that the donor platelets used are treated in accordance with the method of the invention to activate platelet FcR.

In a fifth aspect, the invention provides a reagent comprising FcR and an effective amount of a molecule capable of inducing activation or improvement in function of FcR, optionally together with appropriate carriers, buffers or the like. In a preferred embodiment, the reagent is provided as part of a a kit comprising the FcR and the molecule which are contacted with each other to thereby activate the FcR.

The FcR may be any FcR such as those described herein below. The molecule may be any molecule capable of effecting the required changes in FcR. Preferably, the molecule is an antibody, more preferably a monoclonal antibody. Still more preferably the antibody has an activity equivalent to monoclonal antibodies 8.2, 8.26 or CIKM5 as described herein. In a sixth aspect the invention relates to a method of treatment or prophylaxis of a disease in a subject wherein activation or improvement of the activity of FcR in the subject assists in combating the disease, comprising the step of administering to said subject an effective amount of a molecule capable of activation or improvement of the function of FcR.

The disease may be any disease in which activated or enhanced endogenous or exogenous FcR may be of assistance in fighting the disease. FcR is involved in activating cells which include phagocytes, killer cells and platelets which remove or kill unwanted cells, pathogens or aetiological agents from the body. The diseases may include, but are not limited to, autoimmune diseases (such as HITTS), parasitic, yeast, bacterial and viral infections, and cancers.

In one situation contemplated, the subject may be able to mount a normal antibody response to the pathogen but the FcR is not able to clear immune complexes containing the pathogen from the body for example, by phagocytosis, or killing by FcR-dependent mechanism. In such a case, the molecule used in accordance with the invention enhances the antibody-dependent immune functions in the patient.

In another situation, the subject is administered an exogenous antibody specific for an aetiological or pathogenic agent or an unwanted cell or tissue (such as a cancer cell, parasite or a virus-infected cell) . When the molecule used in the method is administered to the subject or brought into contact with FcR, this is able to stimulate the FcR and remove immune complexes formed between the aetiological agent, pathogen or unwanted cell and the exogenous antibody. The immune complex may be an antibody- coated pathogen or cell which is the target for Fc receptors on cells such as phagocytes or killer cells. Thus, in this aspect of the invention, an exogenous antibody specific for an aetiological agent and/or unwanted cell is administered to the subject prior to, or simultaneously with said molecule. Such a method may be important in the treatment of tumours and other cancers, protozoan, bacterial, yeast or other fungal or viral infections such as candidiasis, tuberculosis, AIDS, hepatitis, malaria, plague and the like. The exogenous antibody is preferably a therapeutic antibody, eg. antiviral, anti-bacterial, anti-parasite, anti-yeast, or anti- tumour antibodies.

In a seventh aspect, the invention therefore provides a composition comprising a therapeutic agent such as an antibody and an FcR activated in accordance with the method of the invention. Also contemplated is a kit comprising a therapeutic agent and an FcR activated in accordance with the invention. Preferably, the kit comprises at least one therapeutic agent, FcR and an amount of a molecule which activates or improves the function of the FcR when said molecule is contacted with the FcR thereby to enhance the efficacy of the therapeuctic agent eg. an antibody. The subject may be any subject where FcR is of assistance in combating the disease. Preferably, the subject is a vertebrate, more preferably a mammal. The subject may be any animal which would benefit from the method including humans, domestic pets, farm animals and the like. In an eighth aspect, the invention relates to a method of activating or improving the function of monocytes/macrophages since FcR are found on these cells. In a particularly preferred embodiment, macrophages cultured m vi tro are contacted with immune complexes and anti-FcR mAb prior to infusion into a subject. In this way, presentation of the antigens m the immune complexes is enhanced by virtue of the improved binding of the activated macrophages. Thus, the method of the invention provides a method of enhancing or improving antigen presentation by combination of phagocytosis and endocytosis by the anti-FcR activated macrophages. Monocytes and platelets may also be activated m a similar manner.

The term "activating or improving the function of FcR" means that the FcR is changed from a non-active state to an active state, or the ability of an already active FcR is improved or enhanced above levels prior to contact with said molecule. The function of FcR includes, but is not limited to, the ability to bind immune complexes, the ability to activate cellular responses and the ability to to cause aggregation of platelets.

The term "FcR" refers to any protein with an ability to bind immunoglobulm. The FcR may be specific for any immunoglobulm class, including IgG, IgE, IgA, IgM or IgD. The FcR may be from any species, preferably a vertebrate species, more preferably a mammalian species. Still more preferably the FcR is of human or other mammalian origin such as rat, mouse, rabbit and primate origin. Depending on the use contemplated, the FcR may be native FcR, including that m si tu on cells, (such as m plasmapheresis or m vivo treatment of a patient), isolated or purified FcR, recombmant FcR, synthetic FcR (for example those made by peptide synthesis), a functional fragment of FcR, or soluble FcR which may be used in vi tro such as m kits or assays. Alternatively the FcR may be bound to a solid support. In addition, the FcR may also be labelled with a suitable marker or reporter molecule. The term "an effective amount" of the molecule refers to an amount sufficient to bring about the required activation or improvement FcR. The phrase "for a time and under conditions sufficient to allow said activation or improvement to occur" refers to the amount of time and the conditions necessary to allow the activation or improvement in FcR function to take place. Such time and conditions may vary depending on the environment in which the method is conducted, such as in vivo or in vi tro, and may easily be determined by a person skilled in the art.

The term "a molecule capable of inducing said activation or improvement" refers to any molecule capable of inducing such an activation or improvement . The inventors have discovered that this activation or improvement appears to be associated with multimerisation and/or a change in shape of the FcR which increases the affinity of the receptor for antibodies. Multimerisation may be dimerisation, or aggregation of a larger number of monomers such as tetramers, etc. and may result in clustering of FcR on the surface of platelets or other cell types .

Description of Preferred Embodiments.

The invention will now be described with reference to the following non-limiting figures and examples . Figure 1 is a histogram of results of anti-Leu4- induced T cell proliferation assays using intact anti- hFcγRII mAbs . Anti-Leu4 was used at a final dilution of 1:400 (A) and 1:4000 (B) using PBMC from a HR individual, and 1:400 (C) and 1:4000 (D) using PBMC from a LR individual. The anti-hFcγRII mAbs (IV.3, 8.7, 7.30, 8.2 and 8.26) and the control mAb (1841) were used as ascites at a final dilution of 1:1600. cpm=counts per minute of incorporated [³H] -Thymidine .

Figure 2 is a histogram of results of OKT3- induced T cell proliferation assays using intact anti- hFcγRII mAbs. (A) PBMC from a HR individual were incubated with OKT3 ascites at a final dilution of 1:4000 and 1:4 x 10⁵, using mAbs 1841 (control), IV.3, 8.7, 7.30, 8.2 and 8.26. (B) PBMC from a LR individual were incubated with OKT3 ascites at a final dilution of 1:4000 and 1:4 x 10°, using mAbs 1841 (control), IV.3, 8.7, 7.30, 8.2 and 8.26. cpm=counts per minute of incorporated [³H] -Thymidine .

Figure 3 is a histogram of results of anti-Leu4- induced T cell proliferation assays using F (ab' ) ₂/F (ab' ) fragments of the anti-hFcγRII mAbs. Anti-Leu4 was used at a final dilution of 1:400 (A) and 1:4000 (B) using PBMC from a LR individual. The anti-hFcγRII mAbs used were IV.3 F(ab'), 8.7 F(ab')₂, 8.2 F(ab')₂, 8.26 F(ab') and CIKM5 F(ab')₂ all used at a final concentration of 1.25μg/ml. cpm=counts per minute of incorporated [³H] - Thymidine . Figure 4a is a graph of binding of F(ab') fragments of IV.3 mAb changes affinity of receptor for immobilised F(ab')2 fragments of 8.2 mAb. F(ab')2 fragments of the anti-FcγRII mAb, 8.2, were immobilised on the surface of a CM5 biosensor chip using standard a ine chemistry. A constant flow rate of 5 μl/min of HBS (0.005% P20 detergent added) was maintained across the surface of the chip between injections. The diagram is an overlay of five BIAcore sensorgrams where the response (in RU) is a measure of change in the mass of protein bound to the sensor surface. In each sensorgram a 20 μl injection of HSA-FcγRII (48 μg/ml in HBS) was injected followed immediately by a 50 μl injection of one of the following: HBS alone, 15 μg/ml 8.2 F(ab')2 fragments (rebinding control), 15 μg/ml 1302 F(ab')2 fragments (irrelevant antibody control), 15 μg/ml IV.3 Fab' fragments, or 25 μg/ml intact IV.3 mAb.

Figure 4b is a graph of binding of F(ab'2) fragments of 8.2 mAb changes affinity of receptor for immobilised F(ab') fragments of IV.3 mAb. F(ab') fragments of the anti-FcγRII mAb, IV.3, were immobilised on the surface of a CM5 biosensor chip using standard amine chemistry. A constant flow rate of 5 μl/min of HBS (0.005% P20 detergent added) was maintained across the surface of the chip between injections. In each sensorgram a 20 μl injection of HSA-FcγRII (48 μg/ml in HBS) was injected followed immediately by a 50 μl injection of one of the following: HBS alone, 50 μg/ml IV.3 F(ab) fragments (rebinding control), 50 μg/ml 1302 F(ab')2 fragments (irrelevant antibody control) or 50 μg/ml 8.2 F(ab')₂ fragments.

Figure 5 is a graph showing binding of 8.2 mAb to immobilised FcγRII alters affinity of 8.7 mAb for its epitope on FcγRII. HSA-FcγRII was immobilised on the surface of a CM5 biosensor chip using standard amine chemistry. A constant flow rate of 5 μl/min of HBS (0.005% P20 detergent added) was maintained across the surface of the chip between injections. In sensorgrams a and b 8.2 mAb (200 μg/ml) was injected followed immediately by HBS alone (a) or 8.7 (200 μg/ml)) . Sensorgram c shows the binding of 8.7 mAb to immobilised FcγRII without prior treatment with 8.2 mAB. Figure 6 is a graph showing abolition of the mAb

IV.3 inhibition of Fc dependent platelet aggregation by preincubation with the anti FcγRII mAb 8.2 F(ab')₂. Washed platelets were preincubated with irrelevant mouse mAb MOPC131 (lmg/ml) - A or mAb 8.2 F(ab')₂ (7μg/ml) -B for 5 minutes. Subsequently 50μl of the mAb IV.3 (Iμg/ml) was added to both sets of platelets and further incubated for 5 minutes. The degree of FcγRII dependent platelet aggregation was then assessed by adding 50μl of mAb VM58 (2.3μg/ml). Platelet aggregation was only seen in tracing A, with the irrelevant mAb, compared to no aggregation in tracing B, with mAb 8.2 F(ab')₂.

Figure 7 is a histogram showing the effect on thromboxane generation in high responders to VM58 (2.3μg/ml), CD9 (2μg/ml) and 8.26 (5μg/ml) after preincubation with IgGl (control), 8.2 F(ab')2, 8.26 F(ab'), IV.3 or saline control. The mean value of 5 separate experiments are shown with standard error bars . Figure 8 is a graph showing marked enhancement in a low responder by mAb 8.2 F(ab')₂ in VM58 induced platelet aggregation. Washed platelets were preincubated either with 50μl of mAb 8.2 F(ab')₂ (7μg/ml) - A or an irrelevant IgGi monoclonal antibody MOPC131 (lOμg/ml) - B for 5 minutes.

Figure 9 is a histogram showing generation of thromboxane B₂ in response to heat-aggregated IgG and plasma from heparin induced thrombocytopaenia following preincubation with 8.2 F(ab')₂, 8.26 F(ab'), IV.3 or saline control. The mean value of 5 separate experiments are shown with standard error bars .

Figure 10 is a graph showing enhancement of HITTS plasma-induced platelet aggregation by monoclonal antibody 8.2 F(ab')₂. Washed platelets were preincubated either with 50μl of A - an irrelevant IgGi mAb MOPC131 (lOμg/ml) or B - mAb 8.2 F(ab')₂ (7μg/ml) for 5 minutes. 50μl of the same HITTS plasma was simultaneously added to each cuvette with irreversible platelet aggregation at 10 minutes only occurring in the platelets pre-incubated with mAb 8.2 F ( ab ' ) ₂.

Figure 11 is a graph showing inhibition by soluble recombinant human FcγRII of HITTS plasma-induced platelet aggregation. Plasma was incubated for 5 minutes prior to the addition of soluble FcγRII - B compared to the effect of saline - A. Heparin (0.5U/ml) was added to both after 5 minutes to cause heparin-dependent platelet aggregation only in A.

Figure 12 shows the enhancement of release of thromboxane B₂ (TxB₂) and aggregation (slope) of platelets by 8.2F(ab)₂/ and inhibition by soluble rFcγRII, in plasma with weak HITTS antibodies.

Figure 13 represents the inhibition of platelet dense granule release and prolongation of lag phase by soluble rFcγRII in a plasma with weak HITTS antibodies, and enhancement by 8.2F(ab)₂.

Figure 14 shows the inhibition of HITTS-antibody induced platelet aggregation by soluble rFcγRII . Figure 15 shows the enhancement of HITTS-antibody induced platelet aggregation.

Example 1 : Antireceptor Antibodies can Alter FcR Function.

This example demonstrates that anti-receptor antibodies can alter Fc receptor function. This is demonstrated in a system using whole cells and anti-CD3 antibodies. Anti-CD3 induced T-cell proliferation using peripheral blood cells involves a set of interactions between: -

(a) the anti-CD3 monoclonal antibody and the T-cell receptor CD3 complex;

(b) the Fc portion of anti-CD3 monoclonal antibody; and (c) the cell surface human Fcγ receptor on monocytes.

The activation of resting T-cells by soluble anti-CD3 monoclonal antibodies requires the cross-linking of the T-cell receptor CD3 complex to achieve optimal stimulation. This requirement can be fulfilled by monocytes which bind the Fc portion of the anti-CD3 antibody via the Fc receptor. In this assay the CD3 positive T-cells that are coated with anti-CD3 antibody form an immune complex.

This complex binds to Fc receptors of monocytes . The binding to the Fc receptors can be indirectly indicated, for example, by proliferation of the T-cells as anti-CD3 antibody that is attached to CD3 on the surface of the T-cell is cross linked by the monocyte Fc receptors. The cross linking (aggregation) of CD3 stimulates the T- cells to proliferate.

It has been clearly shown however, that certain forms of FcγRII are unable to bind the anti-CD3 immune complex, and individuals bearing this particular receptor can be identified by the failure of their cells to induce T-cell proliferation. The individuals bearing this particular receptor are called non responder individuals. Materials and Methods :

MAbs and reagents .

The anti-hFcγRII murine mAbs used, 8.2 IgGi, 8.7 IgGi, 8.26 IgG2b, 7.30 IgGi, IV.3 IgG2b and CIKM5 IgGi are described elsewhere (47, 49, 50). Control murine mAbs used included, 1841 IgGi (anti-2-4-6-trinitrobenzene sulphoric acid), 1459 IgGi (anti-Ly 3.1), 1305 IgGi (anti- Ly 2.1) and 1302 IgG2a (anti-Ly 2.1) (51, 52; Dr V. Sutton, Austin Research Institute, Austin Hospital, Heidelberg, Victoria, Australia) . Pepsin fragments of mAbs 8.2 [F(ab')₂], 8.7 [F(ab')₂], 8.26 [F(ab')], IV.3 [F(ab')], CIKM5 [F(ab')₂] and 1302 [F(ab')₂] were produced (50) . Two anti-CD3 mAbs were used in the T cell proliferation assays; OKT3 (murine IgG2a) (53) and anti- Leu4 (murine IgGi) (54) (Becton Dickinson, Mountain View CA) .

Anti-CD3 -induced T cell proliferation assays.

PBMC were isolated using a Ficoll density gradient (Pharmacia, Uppsala, Sweden) , harvested, washed, resuspended in RPMI 1640 (Flow Laboratories, Australia) supplemented with 10% heat-inactivated foetal calf serum (Commonwealth Serum Laboratories, Australia), 100 U/ml penicillin, lOOμg/ml streptomycin, 2mM glutamine (Commonwealth Serum Laboratories, Australia) and 0.05mM 2- ME (Koch-Light Ltd, Suffolk, England) , counted and 5x10"' cells (lOOμl volume) were aliquoted in triplicate into U- bottomed sterile 96 well microtitre plates (Nunc, Roskilde, Denmark) . The cells were incubated with either (a) 1:1600 final dilution of whole anti-hFcγRII mAb ascites or control mAb (1841) ascites, and (b) 1.25μg/ml final concentration of F(ab')₂ or F(ab') fragments of the anti-hFcγRII mAbs or medium alone (control); cells and anti-hFcγRII mAbs were then incubated with either OKT3 or anti-Leu4 in a final volume of 200μl. The anti-CD3 mAbs were used at two dilutions (determined using a HR individual); saturating (1:400 for anti-Leu4 and 1:4000 for OKT3 ) and subsaturating (1:4000 for anti-Leu4 and 1:4 x 10⁶ for OKT3 ) . The cells and antibodies were incubated for 72 hours at 37°C and over the last 14 hours, pulsed with lμCi of [³H] - thymidine per well (3000 Ci/mol, Amersham, Buckinghamshire, England) . Cells were harvested onto glass paper discs and incorporation of [³H] - thymidine measured by liquid scintillation counting, expressed as counts per minute (cpm) .

Anti-CD3 -induced T cell proliferation assays were performed in triplicate using PBMC from two HR and two LR individuals. Representative results from each experiment are shown below and all values are expressed as the mean ± SE.

Murine IgGi EA rosette inhibition assays

Blocking anti-hFcγRII mAbs (50μl of intact mAb ascites at 1:400 dilution or purified pepsin fragments at 2μg) were incubated with 50μl of hFcγRII⁺ K562 cells (5 x 10⁶ cells/ml) for 45 minuted on ice. EA complexes were prepared by coating sheep red blood cells with 2, 4, 6- trinitrobenzene sulphonic acid (TNBS) (Research Organics Inc., Ohio, USA) and sensitising these cells with a mouse IgGi anti-TNBS mAb (52) . Fifty microlitres of 2% EA complexes was then added to the K562 cells with the anti- hFcγRII mAbs, incubated for 5 minutes at 37^υC and centrifuged at 200g for 3 minutes. Pelleted cells and EAs were incubated for 30 minutes on ice, stained with ethyl violet and the number of rosette forming cells (at least 5 SRBC or 50% of the cell covered) were counted in a total of 100 cells. All dilutions and incubations were performed in L-15 medium modified with glutamine (Flow Laboratories, Australia) and control mAbs used were intact 1305 and 1302 F(ab' )₂.

IL-1 and IL-6 assays

The release of IL-1 and IL-6 from monocytes into the tissue culture supernatant was assayed in the presence of the anti-hFcγRII mAbs. Monocytes from a LR individual were purified by counter-current centrifugal elutriation (55), resuspended in RPMI and 10% foetal calf serum, penicillin, glutamine and 2-ME (described above) and aliquoted at 10⁵ cells/well. The cells were incubated with; (a) medium only (b) intact anti-hFcγRII mAbs (ascites 1:400 final dilution) and (c) F(ab') or F(ab')₂ fragments of anti-hFcγRII mAbs ( 0.5-1. Oμg/ml final concentration). The incubations were performed either in the presence, or absence, of anti-Leu4 (1:1000 final dilution, known to induce T cell proliferation in the presence of the stimulating anti-hFcγRII mAbs) for 24 hours at 37°C, in a volume of 200ml. IL-1 was then assayed in cell free supernatants using the cell line, NOB-1, and IL-6 using 7TD1 cells (56, 57) . Monocytes were 95% pure as determined by non-specific esterase (58) .

Results

The anti-Leu4-induced T cell proliferation assay defines two functionally distinct groups of anti-hFcγRII mAbs.

T cell proliferation assays were performed with HR and LR individuals using anti-Leu4 and OKT3 at two dilutions. The addition of intact anti-hFcγRII mAbs to these assays resulted in division of the mAbs into two groups; (a) stimulating anti-hFcγRII mAbs, 8.2, 8.26 and

CIKM5, and (b) non-stimulating anti-hFcγRII mAbs, 8.7, 7.30 and IV.3 (Figure 1) . Using PBMC from a HR individual, mAbs 8.7 and 7.30 were shown to partially inhibit T cell proliferation, whereas mAb IV.3 completely blocked T cell mitogenesis at both dilutions of anti-Leu4 (Figure 1A and IB) . This inhibitory phenomenon has been described in previous studies (26, 34, 35, 45, 47, 48), and is attributed to the inhibition of Fc (anti-Leu4) binding to monocyte hFcγRII, preventing the cross-linking of the TcR/CD3 complex, which is essential for optimal T cell proliferation. In contrast, intact mAbs 8.26 and 8.2 enhanced T cell proliferation compared to the control mAb 1841, thus classifying these antibodies as "stimulators" (Figure 1A) . This stimulating effect was less evident at higher dilutions of anti-Leu4 (Figure IB) and was completely lost in the absence of anti-Leu4. Using anti- Leu4 and PBMC from a LR individual, T cell stimulation was also evident with intact mAb 8.2 and 8.26 (Figure 1C) , demonstrating the capacity of the anti-hFcγRII mAbs to activate non-proliferating T cells; this effect was lost as higher dilutions of anti-Leu4 (Figure ID) . Similarly, intact CIKM5 in a separate experiment and F(ab')₂ fragment of CIKM5 (later in this study) were shown to stimulate T cell proliferation in HR and LR individuals only in the presence of anti-Leu4. Therefore, the stimulatory signal provided by mAbs 8.2, 8.26 and CIKM5 can be categorised as an "accessory signal " , since they alone do not activate T cells, but do so in combination with anti-Leu4.

The stimulating effect with the anti-hFcγRII mAbs was not observed with OKT3 in both HR and LR individuals (Figure 2). Since saturating amounts of OKT3 (1:4000 dilution) could induce maximal T cell proliferation, which would mask any possible accessory signal, assays using subsaturating dilutions of OKT3 (1:4 x 10⁶) were also performed. No additional T cell stimulation was induced by the anti-hFcγRII mAbs at the higher dilution of OKT3 (Figure 2A and 2B) . Similar results were shown for CIKM5 in a separate experiment .

T cell stimulation is induced by the antigen binding region of mAbs 8.2, 8.26 and CIKM5 To determine the activity of the antigen binding region of the anti-hFcγRII mAbs independent of their Fc portions, F(ab) (8.26 and IV.3) and F(ab')₂ (8.2, 8.7 and CIKM5) fragments were tested in the T cell proliferation assay. F(ab')₂ fragments of mAbs 8.2 and CIKM5 profoundly enhanced T cell stimulation in the LR individual, in contrast to F(ab') fragments of 8.26, which induced minimal or no T cell stimulation (Figure 3A and 3B) . T cell stimulation induced by intact and F(ab')₂ fragments of 8.2 and CIKM5 indicates that this effect results from the interaction of hFcγRII with the antigen binding sites of the mAbs. F(ab') fragments of mAb 8.26 lose the capacity to induce T cell stimulation when compared to intact 8.26 (Figure 1C and 3A) , demonstrating that hFcγRII cross- linking (aggregation) is necessary for optimal T cell stimulation. It should be noted that the production of the F(ab') fragments of 8.26 resulted in minimal loss of antigen binding activity , and therefore, the lack of stimulation by 8.26 F(ab') is not due to inactive mAb fragments (50). In the HR individual, F(ab')₂ fragments of 8.2 and CIKM5 also resulted in enhanced T cell stimulation (not shown). MAb 7.30 F(ab')₂ were not available; however, this mAb is functionally identical to mAb 8.7 (50).

The T cell stimulatory effect of mAb 8.26 was inhibited by pre-incubating 8.26 with a recombinant soluble form of hFcγRII (recombinant soluble hFcγRII would block the antigen binding site of 8.26) before the addition of

8.26 to the anti-Leu4-induced T cell stimulation assay (59, 60, 61). This demonstrates that the stimulation was dependent on the interaction of the antigen binding region of 8.26 with cell-surface hFcγRII. Several conclusions can be drawn from these results. An accessory signal leading to enhanced T cell activation and proliferation is observed with the stimulating anti-hFcγRII mAbs (8.2, 8.26 and CIKM5) and is dependent on the presence of anti-Leu4. This effect is not evident with mAbs 8.7, 7.30 and IV.3. The synergism between the stimulating anti-hFcγRII mAbs and anti-Leu4 is seen in both HR and LR individuals. Furthermore, the accessory signal induced by mAb 8.26 required hFcγRII cross-linking (dimerisation) for the induction of maximal T cell proliferation.

On the basis of these observations it is clear that the anti-receptor antibodies modify the ability of Fc receptors to interact with immune complexes. There are a number of possible mechanisms.

One possibility is that the anti-receptor antibody dimerises the cell surface FcγRII and as a result enables it to interact more efficiently with immune complexes. This would have the effect of being able to provide a more effective interaction of the receptor with immune complexes which would in turn deliver a more effective cross linking signal to the T-cells. It is also possible in this setting that the interaction of the antibody with the receptor induces a shape change in the molecule which enables it to interact with immune complexes more effectively. This too, would have the effect of delivering a more effective cross-linking signal to the T- cells and thereby inducing proliferation.

In models of cell-to-cell interaction, hFcγRII can act as an anchoring molecule, which stabilises interactions between cells, and/or a signalling cell- surface molecule, which transmits specific cell signals (62). Thus the binding of the stimulating anti-hFcγRII mAbs to cell-surface hFcγRII could mediate the accessory signal by several possible mechanisms. Human FcγRII may act predominantly as an anchoring molecule, so that the stimulating anti-hFcγRII mAbs bind simultaneously to hFcγRII on monocytes and T cells, enabling a stable cell- to-cell interaction between monocytes and T cells, which would enhance TcR/CD3 cross-linking by anti-Leu4 and hFcγRII on monocytes (CD8⁺ T cells have been shown to express hFcγRII; H. Zola, personal communication, Department of Immunology, Flinders Medical Centre, South Australia, Australia) . Secondly hFcγRII may act as a signalling molecule. Since monocytes can secrete important soluble mediators (eg. IL-1 and IL-6), which provide accessory signals for T cell activation (17, 19), it is possible that the stimulating anti-hFcγRII mAbs preferentially release T cell-stimulatory cytokines from monocytes, B cells or T cells, or induce the expression of a cell-surface antigen on monocytes or B cells (eg. B7 ) which is capable of providing a costimulatory signal to enhance T cell proliferation (63, 64). Alternatively, the stimulating anti-hFcγRII mAbs could interact with the T cell hFcγRII to provide a direct accessory signal to the T cell. The next part of the study examines several of these possible mechanisms of stimulation.

MAbs 8.2, 8.26 and CIKM5 inhibit the binding of mouse IgGi EA complexes to cell-surface hFcγRII

If hFcγRII and the stimulating anti-hFcγRII mAbs had solely an anchoring role in the stimulation of T cell proliferation, then enhanced TcR/CD3 cross-linking by anti- Leu4 and hFcγRII on monocytes would mediate the accessory signal. This model would provide an efficient accessory signal only if the anti-hFcγRII mAbs did not block anti- Leu4 (murine IgGi) Fc binding to hFcγRII, i.e., if the anti-hFcγRII mAbs inhibit the binding of mouse IgGi complexes to hFcγRII, then cross-linking of TcR/CD3 complex by hFcγRII on monocytes and anti-Leu4 would be inefficient. The epitope specificities of the anti-hFcγRII mAbs in relation to the Fc-binding region of murine IgGi on hFcγRII were studied by examining the ability of the mAbs to inhibit the binding of murine IgGi EA complexes to cell- surface hFcγRII. Intact 8.7, 7.30 and IV.3, and 8.26

F(ab'), 8.7 F(ab')₂ and IV.3 F(ab') all inhibited murine IgGi EA rosette formation, indicating that these mAbs bind at or near the Fc-binding region. The 8.26 antibody only partly inhibited EA rosetting indicating close location to the binding site but distinct from the epitope of IV.3. Intact mAb 8.2 completely inhibited the binding of mouse IgGi EA complexes, whereas F(ab')₂ fragments of 8.2 failed to inhibit the binding of EA complexes, similar to CIKM5 F(ab')₂ in other studies (65) . This indicates that mAbs 8.2 and CIKM5 bind to a site distant from the Fc-binding region of mouse IgGi, and the Fc portion of the intact mAb (8.2 and CIKM5) interacts with hFcγRII to inhibit the binding of EA complexes. Results are summarised m Table

I.

Table I Effect of Anti-hFcγRII mAbs on Mouse IgGi EA

Rosette Formation

mAb Intact mAb F(ab') or F(ab')₂

8.26 20 (78) 20 (78)

8.2 0 (100) 90 (0)

8.7 0 (100) 0 (100)

7.30 60 (33) NT^b

IV.3 0 (100) 0 (100)

Control mAb 90 (0) 90 (0)

a) . Percentage of EA rosettes using FcγRII⁺ K562 cells as the target cells and the mAbs as blocking antibodies. Ascites of all intact mAbs were used at a dilution of 1:400, and F(ab') (IV.3 and 8.26) or F(ab')_ (8.2, 8.7) fragments were all used at 2μg. Control mAbs used were 1305 ascites and 1302 F(ab')₂ fragments. EA rosettes the absence of antibody was 90%. The data is represented as percentage of EA rosettes, with percentage of inhibition of EA rosettes relative to the control mAbs, m parentheses. The results are representative of at least 2 experiments.

b) NT = Not tested.

These results have important implications with respect to the mechanism of T cell stimulation by mAbs 8.2, CIKM5 and 8.26. TcR/CD3 cross-lmkmg via hFcγRII is considered to be a prerequisite for the monocytes to provide their accessory function m the induction of T cell proliferation (35). The requirement of monocyte hFcγRII m the induction of T cell proliferation by mouse IgGi anti- CD3 can be demonstrated by the inhibition of T cell proliferation using anti-hFcγRII mAbs which inhibit IgG Fc- binding (35; also described herein). MAbs 8.7, 7.30 and IV.3 inhibit both the binding of murine IgGi EA complexes and the anti-Leu4 (murine IgGi ) -induced T cell proliferation, as expected. In contrast, intact 8.2, CIKM5 and 8.26 inhibit the Fc binding of murine IgGi EA complexes to hFcγRII; however, they stimulate T cell mitogenesis. Thus, the anchoring model with enhanced anti- Leu4 Fc binding to monocyte hFcγRII and TcR/CD3 cross- linking is not the major mechanism for the accessory signal provided by mAbs 8.2, CIKM5 and 8.26. In addition, the data also demonstrate that extensive TcR/CD3 cross-linking is not an essential requirement for efficient T cell activation.

IL-1 or IL-6 does not mediate 8.2, 8.26 and CIKM5-induced T cell proliferation

Under optimal conditions, TcR/CD3 cross-linking by anti-CD3 mAbs via Fc receptors or immobilised anti-CD3 mAbs provides all the necessary signals to mediate T cell proliferation. When TcR/CD3 signalling is suboptimal, the accessory signals provided by monocytes may be essential for T cell activation (13); incubation with intact mAb 8.2, 8.26 and CIKM5, which block IgG Fc binding, would provide suboptimal conditions for TcR/CD3 cross-linking and signalling. Both IL-1 and IL-6 are soluble cytokines secreted from monocytes, which are able to provide the accessory signal requirements for T cell proliferation (17, 18, 38-42). Therefore, preferential release of IL-1 or IL-6 induced by the stimulating anti-hFcγRII mAbs (8.2, 8.26 and CIKM5), compared to the non-stimulating anti- hFcγRII mAbs (8.7, 7.30 and IV.3), may account for the differences in T cell proliferation observed with anti- Leu4. This hypothesis was investigated by determining the level of IL-1 and IL-6 release following the incubation of the anti-hFcγRII mAbs with purified monocytes from a LR individual in the presence, or absence, of anti-Leu4. Purified monocytes from a LR individual were incubated with intact and F(ab') or F(ab')₂ fragments of the anti-hFcγRII mAbs with, or without, anti-Leu4 (1:2000 final dilution); this dilution of anti-Leu4 is known to induce T cell proliferation. The results are summarised in Table II.

Table II IL-1 and IL-6 Release from Purified Monocytes Induced by Anti-hFcγRII mAbs and Anti-Leu4

IL -1 IL- 6

No anti- -Leu Anti-Leu4 No Anti- -Leu4 Anti-Leu4

Medium 3.6±0.4 5±1 37+1 43±1

Intact mAb

8.2 6. l±O.4 7.3±0.4 42±3 42±2

8.26 lO±l 11.3±0.1 55±3 58±2

8.7 5.8±0.3 6. l±O .1 43±2 51±1

7.30 9. l±O .6 8.2±0.3 47±2 50±3

IV.3 5. O±O .3 5.4±0.1 36±2 47±1

Fragments

8.2 F(ab' )₂ 6.5±0.3 7.3±0.3 49±2 56±2

8.26 F ( ab ' ) 4.2±0.1 5.2±0.1 47±3 50±3

8.7 F(ab' )₂ 6.2±0.1 6.9±0.3 45±2 52±2

IV.3 F(ab' ) 5. l±O .3 5.2±0.3 37±2 43±1

IL-1 and IL-6 assays were performed as described herein. Values are expressed as the mean ±SE (n=3) , in U/ml .

The results demonstrate that there was no preferential cytokine release by the stimulating anti- hFcγRII mAbs, compared to the non-stimulating mAbs. The only notable difference was observed with intact 7.30 (non- stimulator) and 8.26 (stimulator), which released higher levels of IL-1 compared to the remaining anti-hFcγRII mAbs. The addition of the anti-Leu4, which is an absolute requirement for T cell stimulation with the anti-hFcγRII mAbs, did not significantly alter the pattern of cytokine release from purified monocytes. Therefore, the increased proliferation of T cells induced by mAb 8.2, 8.26 and CIKM5 with anti-Leu4 is not a direct result of IL-1 or IL-6 release from monocytes induced by these antibodies.

The results from this study lead to new findings in two areas: (i) T cell activation with anti-Leu4 and the role of hFcγRII and (ii) biological function and triggering of hFcγRII. Stimulating mAbs (8.2, 8.26 and CIKM5) were identified which can mediate an accessory signal to enhance T cell proliferation induced by anti-Leu4 with PBMC from HR or LR individuals. Incubation of PBMC with mAbs 8.2, 8.26 or CIKM5 alone had no effect on T cell mitogenesis; enhanced T cell stimulation required the presence of anti- Leu4 and the stimulating anti-hFcγRII mAbs. The accessory signal was a direct result of the interaction between the anti-hFcγRII mAbs and cell-surface hFcγRII, and studies with mAb 8.26 demonstrated that hFcγRII cross-linking was necessary for optimal T cell stimulation. A number of mechanisms may be involved in the anti-hFcγRII mAb-induced stimulation. It seems likely that the mechanism involved results in a change in the capacity of Fc receptors to bind immune complexes. In this way the so-called non responder FcγRII that is normally unable to bind the IgG¹ immune complexes is now able to do so and to induce T-cell proliferation.

This Example extends our knowledge of the functional aspects of hFcγRII, including the possible mechanisms of hFcγRII triggering. Human FcγRII can mediate diverse biological effects, including phagocytosis, ADCC, release of inflammatory mediators and regulation of Ig production (67, 68, 69). Many of these biological events can be triggered by cross-linking hFcγRII with anti- receptor mAbs alone, or in some cases more extensive cross- linking with a bridging antibody (69). The general requirement of hFcγ receptor clustering to trigger the biological response is also observed with the T cell- stimulating effect of mAb 8.26, which loses its capacity to efficiently induce T cell proliferation when F(ab') fragments, rather than intact mAb, are used (Figure 1 and 3) .

It is now clear that hFcγ receptor cross-linking is not sufficient to induce all the biological effects observed and additional factors are involved. Functional studies have shown that superoxide generation from neutrophils can be induced by immune complexes (IgG-Latex), but not by mAb IV.3 cross-linked by an anti-mouse Ig (70) . In addition, it has been demonstrated that mAb CIKM5 alone, but not IV.3 or 2E1, was able to trigger Ca²⁺ mobilisation in U937 cells (65, 71) . This functional difference was attributed to more efficient cross-linking by CIKM5, compared to IV.3 and 2E1. Although hFcγRII cross-linking is an important requirement for hFcγRII triggering, functional differences in epitopes recognised by these mAbs need to be considered. Indeed, this concept is supported by the data in our study using the anti-Leu4-induced T cell proliferation assay as a model to study hFcγRII triggering. Monoclonal antibodies 8.2, 8.7, 8.26, 7.30, IV.3 and CIKM5 have been previously divided into four clusters, each cluster defining a different structural epitope in the extracellular domains of hFcγRII (50). Cluster 1 (mAbs 8.2 and CIKM5) defines a combinatorial epitope with determinants in both extracellular domains of hFcγRII, and Cluster 2 (mAb 8.26), Cluster 3 (mAb IV. ) and Cluster 4 (mAbs 8.7 and 7.30) have determinants in the second domain only. Using these anti-hFcγRII mAbs in the anti-Leu4- induced T cell stimulation assay, the clusters of mAbs can now be classified into functional epitopes, and these can be related to structural regions in the extracellular domains of hFcγRII. Clusters 1 and 2 define activational epitopes, compared to Clusters 3 and 4, which define non- stimulating epitopes.

These data indicate that hFcγRII can be triggered in two ways; (a) hFcγRII cross-linking or aggregation and (b) perturbation of discrete stimulatory or non-stimulatory epitopes. The concept of stimulatory and non-stimulatory epitopes may explain functional differences noted with anti-hFcγRII mAbs used in the studies described above (65, 70, 71) eg. intact CIKM5, which defines a stimulatory epitope herein, was shown in another study (65) to stimulate Ca²⁺ mobilisation in U937 cells. In contrast, intact IV.3, which was classified as a non-stimulatory mAb in our study, failed to induce Ca²⁺ mobilisation unless cross-linked with anti-mouse Ig. The presence of activational and non-stimulating epitopes defined by the anti-hFcγRII mAbs broadens the diversity of hFcγRII function and triggering, and when considered in the context of structural differences of the isoforms in their membrane-anchoring and cytoplasmic domains one can now begin to explain why the interaction of identical or similar ligands with hFcγRII is able to stimulate a variety of biological functions .

Further diversity in ligand binding and signal transduction can be generated by the influence of the conformation of the Fc portion of the IgG molecule, particularly after binding antigen, on receptor-ligand interaction (72).

These data imply the existence of at least two distinct functional sites - one for binding of immune complexes and one for activation of receptor.

The findings herein may also have biological implications in autoimmune disease in that the antibodies may be able to assist patients in removing immune complexes from their bodies. The production of autoantibodies is a common feature of autoimmune diseases in both humans and mice (73, 74), and in particular, anti-murine FcγRII autoantibodies can be detected in the serum of mice with systemic lupus erythematosus (75) . In many autoimmune diseases, the autoantibodies are directed against intracellular components, and it is not clear how these antigens are involved in the pathogenic process unless they are released during cellular degradation and form circulating immune complexes. Autoantibodies directed against cell-surface molecules are often capable of altering the functional activity of the cell-surface antigens and have been directly implicated in the pathogenesis of various autoimmune diseases (75-77). The functional importance of these anti-murine FcγRII autoantibodies was demonstrated by the correlation of the level of the anti-murine FcγRII Ig in the serum with impaired phagocytosis of immune complexes, and the ability of the sera containing anti-murine FcγRII Ig to inhibit the binding of immune complexes to cell-surface murine FcγRII.

In addition, the activation of monocytes, macrophages or other FcR expressing cells has implications in infecious disease where antibodies are produced by patients or given to patients for therapy. Clearly FcR activation or enhancement can lead to improved biological consequences, for example, phagocytosis or antibody dependent killing, both well known FcR dependent functions. (67-69) . Other molecules which can activate FcR in accordance with the invention may be identified using assays such as those used for detecting HITTS. For example, putative molecules which activate the receptors may be incubated with FcR. The treated FcR may then be used in an assay for HITTS as described in Example 3 below. Example 2 : Binding Kinetics of Antibodies to Recombinant Form of FcγRII

This example examined the binding kinetics of some antibodies useful in the present invention. The anti-FcγRII monoclonal antibodies 8.2 and 8.7 were described by Ierino et al (1993) while IV.3 was described by Looney et al 1986. Monoclonal antibodies were purified by protein A affinity chromatography . F(ab')2 fragments of 8.2 and 8.7 (both IgGi) were produced by pepsin digestion. Pepsin treatment of the IV.3 IgG2b mAb produced monovalent F(ab') fragments. All antibodies and antibody fragments were diluted in or dialysed into Hepes buffered saline (HBS; 150 mM NaCl, 10 mM Hepes pH 7.4) . Mapping of the epitopes of the monoclonal antibodies, 8.2 and IV.3, to the recombinant receptors, sFcγRII and HSA-FcγRII, was carried out using a surface plasmon resonance (SPR) biosensor (BIAcore 2000) . In the first experiment (Fig. 4a) 8.2 F(ab')2 fragments were immobilised to the surface of a BIAcore CM5 sensor chip using a standard amine coupling procedure. Receptor (HSA- FcγRII or sFcγRII) was then injected across the surface of the sensor chip and captured by the immobilised 8.2 as indicated by the increase in biosensor response between 0 and 240 s (Fig. 4a). Antibody F(ab') fragments of IV.3 or buffer alone were then injected. The presence of IV.3 caused a decrease in the response signal when compared to buffer alone. This decrease in signal was not observed when an irrelevant mAb (1302, anti-Ly-2.1) was injected in place of the IV.3 mAb or when 8.2 F(ab')2 fragments were injected to test for rebinding of the dissociated receptor to the immobilised 8.2 antibody. The second experiment (Fig.4b) used immobilised IV.3 F(ab') fragments in place of 8.2 F(ab')2 fragments. In this reverse experiment, injection of 8.2 F(ab')2 fragments also caused a decrease in response signal compared to injection of buffer alone, irrelevant antibody or IV.3 F(ab') fragments.

These results differ from conventional epitope mapping results m which the signal either increases or remains the same following the addition of the second antibody. An increased signal would have indicated that the two antibodies had separate epitopes and that the binding of one antibody did not influence the binding of the second. If there were no change m response signal, the antibodies must share at least part of their epitopes or the binding of one antibody causes a conformational change which completely prevents the binding of the second. The results actually obtained are consistent with 8.2 and IV.3 binding to separate epitopes and that the binding of either one of the antibodies to the receptor causes a decrease affinity of the receptor for the other antibody.

Results from a different type of epitope mapping experiment are shown Fig. 5. In this experiment, HSA- FcγRII was immobilised to the surface of a CM5 sensor chip using standard amme coupling procedures . The monoclonal antibody, 8.2 (200 ug/ml) was injected across the surface. This concentration of antibody gave a near-maximal response indicating that all of the available 8.2 epitopes on the immobilised FcγRII were occupied. Subsequent injection of 8.7 monoclonal antibody (200 μg/ml) resulted a further increase response signal indicating that 8.2 and 8.7 probably have different epitopes on FcγRII. In addition both the association and dissociation kinetics of 8.7 from FcγRII were altered if the FcγRII had been pretreated

8.2 antibody. In particular 8.7 dissociated from FcγRII-8.2 complexes more slowly than from FcγRII alone. This result suggests that the binding of 8.2 to FcγRII resulted m a conformational change the FcγRII molecule which was reflected m the altered binding characteristics of the 8.7 epitope .

Example 3 : Effect of Antibodies in HITTS Diagnosis Assays

This Example relates to studies conducted at Royal Perth Hospital. Specifically, 110 assays for HITTS have been conducted without any false positive or any increase in false negative results compared to the conventional assay (101) . A number of patients who would previously have been classified as not suffering from HITTS were positive in the assay described below. This means that the antibody confers improved sensitivity on the assay.

1. Platelet Preparation. Venous blood was collected by clean venipuncture into 4.5 ml evacuated tubes (81) and anticoagulated with 0.129 mol/1 tri-socium citrate at a ratio of 9:1. It was centrifuged at 300g for 5 min and the platelet-rich plasma was separated and recentrifuged at 800g for 10 min to produce a loose platelet plug. Plasma was decanted and the platelets were washed twice with Tyrode's buffer (0.15 mol/1 NaCl, 2.7 mmol/1 KC1, 0.36 mmol/1 NaH₂P0₄, 0.1% dextrose, 30 mmol/1 sodium citrate, 1 mmol/1 Mg₂Cl₂6H₂0) and resuspended in Tyrode' s/Hepes buffer (0.15 mol/1 NaCl, 2.7 mmol/1 KC1, 0.36 mmol/1 NaH₂P0₄, 0.1% dexrose, 5mmol/l Hepes, 1 mmol/1 Mg₂Cl₂6H₂0, 2 mmol/1 CaCl₂) to a final platelet count of 150 x 10⁹/1. For flow cytometry the platelets were resuspended in pH 7.4 phosphate buffered saline (PBS) containing 1% bovine serum albumin and 2 mmol/1 potassium EDTA (PBS/EDTA/BSA) .

2. Monoclonal Antibodies

Two mAb known to cause platelet activation were obtained. mAb VM58 (IgGi) was a gift from Dr M Berndt , Baker Institute, Melbourne Australia. It is an anti- glycoprotein IV antibody, and has been described to have a variable platelet aggregation response amongst individuals associated with the known high/low responder polymorphism of platelet FcγRII (82; 83; 84). The mAb against CD9 (ALB-6 clone-IgGi) was purchased from Dako Corporation, California and reproducibly causes FcγRII-dependent platelet activation (85; 86). IgG was extensively dialysed against phosphate buffered saline (PBS pH 7.4) before platelet aggregation studies.

The anti-human FcγRII mAb 8.2 and 8.26 was produced from hybridoma cell lines as previously described (87) . mAb 8.2 (IgGi) defines a combinatorial epitope distant from the IgG binding site with determinants in both domain one (Dlγ) and domain two (D2γ) of the extracellular portion of FcγRII. The mAb 8.26 (IgG_2b) binds to a epitope located entirely in D2γ which is involved in the IgG Fc binding by inhibiting EA rosette formation. F(ab') and F(ab')₂ fragments were made as previously described (87) . Briefly mAb were purified by protein A affinity chromatography, eluted at pH 3.0 to 3.5 and pepsin (Boehringer - Mannheim, Germany) digests with an enzyme to substrate ratio of 1:50 were carried out at 37° C for 1 hour. Undigested IgG or Fc fragments were removed by absorption to protein A and purified mAb 8.2 F(ab')₂, and mAb 8.26 F(ab') fragments were shown to be free of whole Ig and Fc fragments by SDS-PAGE analysis. Anti -human FcγRII mAb IV.3 (IgG_2b) was produced from a hybridoma cell line obtained from the American Type Culture Collection (ATCC accession no. HB217) . mAb 41H.16 was a gift from Dr Jan van de Winkel from the University Hospital, Utrecht, The Netherlands. The mAb 41H.16 binds strongly to the high responder form of the FcγRII polymorphism, FcγRII^Hls131 (88) and the ratio of binding of mAb 41H.16 to IV.3 is useful to classify patients' platelet FcγRII phenotype as being homozygous low responder, homozygous high responder and high/ low responder heterozygotes (89; 90) .

3. Heat Aggregated Human Immunoglobulm.

Human immunoglobulm was purified from a commercial source of immunoglobulm (Intragam - CSL Ltd, Melbourne, Australia) . After extensive dialysis against PBS pH 7.4, the immunoglobulm was further purified by protein A chromatography with the bound material eluted with glycine pH 3.0. After immediate neutralisation with 3M Tris and dialysed overnight in PBS pH 7.4, the purity of the IgG was confirmed by SDS-PAGE analysis. Aggregated IgG (HAGG) was produced at a concentration of 20mg/ml by heat treatment at 63°C for 30 minutes.

4. Plasma from patients with heparin induced thrombocytopenia .

Two patients with heparin induced thrombocytopenia were studied. Each satisfied the following diagnostic criteria: (1) the thrombocytopaenia (platelet count less than 100 x 10⁹/L) occurred when the patients were receiving heparin; (2) the thrombocytopaenia resolved when the heparin was withdrawn; (3) a heparin- dependent platelet antibody was detected in the patients' plasma by platelet aggregometry as previously described (91), and was inhibited by the mAb IV.3 (lμg/ml); and (4) exclusion of other causes of thrombocytopaenia. Patients' plasma was preheated at 60°C for 30 minutes to inactivate complement and traces of thrombin.

5. Production of Human Soluble Recombinant FcγRII . The soluble form of human FcγRII was produced and characterised as previously described (92). Briefly, a truncated form of human FcγRII was produced by inserting a translation termination codon into the FcγRIIa cDNA segment encoding the 5' of the transmembrane domain. The Hfc3.0 cDNA encodes the FcγRIIa allelic variant (low responder) expressing glutamine and histamine at amino acid position 27 and 131 respectively (93). Quantitation of soluble recombinant human FcγRII (rhFcγRII) was performed using a capture : tag ELISA assay using two anti-FcγRII mAbs, 8.26 and 8.7. The cell culture supernatant containing soluble rhFcγRII was affinity purified using a HAGG Sepharose 4B column, with bound rhFcγRII eluted with 0.1 M acetate buffer pH 4.0 containing 0.5 M NaCl. The eluent was neutralised with Tris and dialysed/concentrated against PBS pH 7.4. The protein content of the purified samples was quantified in the capture : tag ELISA assay by comparison with a standard curve of rhFcγRII of known concentration. Purity and homogeneity of the rhFcγRII were confirmed by SDS - PAGE analysis. The soluble rhFcγRII was stored at - 80°C and thawed immediately prior to platelet studies at the final concentration of 50μg/ml.

6. Platelet studies and measurement of thromboxane B₂ generation.

Three subjects who were used routinely in our laboratory for their responsiveness in the routine HITTS platelet aggregation testing were chosen. Two were heterozygote HR/LR and one LR homozygote, as defined by the binding of mAb 41H16 and IV.3 by flow cytometry as previously described. (89; 90) . The aim of these experiments in these platelets of known phenotype was; (1) to determine the effect of the anti FcγRII mAb 8.2 and 8.26 to the activating mAb, HAGG and plasma from patients with HITTS and (2) to examine the extent of inhibition by soluble rhFcγRII on the platelet activating mAb, HAGG and plasma from patients with HITTS.

Platelet aggregation was performed on washed platelets using a Chronolog 560 whole blood aggregometer . Tracings were recorded on a Chronology Chart-Strip recorder with a chart speed setting of 2 cm/min. 100% light transmission was set using Tyrodes buffer. 400 μl of washed platelets were added to a stirred cuvette, warmed for 1 minute at 37°C and the recorder was set to 0% light transmission. The intensity of the platelet activation was measured by the lag phase after the addition of the agonist prior to aggregation, the gradient of the aggregation slope and the amount of TxB₂ generated. Where no aggregation occurred (less than 3% change in light transmission) the lag phase was recorded as greater than 100mm and the slope less than 3mm. TxB₂ was extracted after 5 minutes following the addition of the agonists by 0.5ml of 100% ethanol . The samples were then centrifuged at 10,000 g for 5 minutes and the supernatant assayed for TxB₂ as previously described (94) . Washed platelets were preincubated for 5 minutes with 50μl of the mAb 8.2 F(ab')₂ (7μg/ml) , mAb 8.26 F(ab') (9μg/ml) and mAb IV.3 (lμg/ml) . We and others have shown that mAb IV.3 at this concentration completely saturates the FcγRII and completely inhibits Fcγ dependent platelet activation (91) . The incubating concentrations of mAb 8.2 F(ab')₂ and mAb 8.26 F9ab') were shown repeatedly to completely abolish the mAb IV.3 inhibition of Fc dependent platelet activation (see figure 6) . This suggests that the conditions used the experiments concerning the concentration and incubation time of the mAb 8.2 F(ab')₂ and mAb 8.26 F(ab') completely saturates the platelet Fc receptor. To examine the effect of soluble rhFcγRII on platelet activation, 50μl of the soluble rhFcγRII (50μg/ml, final concentration) was preincubated for 5 minutes prior to the addition of the agonist and compared to the addition of 50μl of PBS. After the preincubation with the above anti

FcγRII mAb, 50μuL of each the platelet activating antibodies mAb VM58 (2.3 μg/ml), mAb CD9 (2μg/ml), HAGG (250μg/ml) and 2 patients' plasma with HITTS were compared to the response to a control irrelevant mAb M0PC131 (lmg/ml) . The concentrations of the mAb and HAGG were found by previous experiments to cause maximal platelet aggregation in responding subjects. To our surprise, during our preliminary experiments we found intact mAb 8.26 to be a strong and mAb 8.2 a weak direct platelet activating antibody. We therefore compared mAb 8.26

(5μg/ml) to the other tested platelet activating antibodies to determine whether this mAb activated platelets by directly binding via the F(ab') region or whether, like the other antibodies, via its Fc portion. All concentrations shown are final concentrations in the cuvette.

The anti FcγRII antibody 8.26 (intact Ig) was able to induce both intense platelet aggregation and thromboxane release (Table 3 and Figure 7) . The platelet activation was dependent upon the Fc portion rather than occurring by direct antigen binding to FcγRII by mAb 8.26, because there was no response to mAb 8.26 F(ab') fragments. It was surprising to find that the mAb 8.2 F(ab')₂, which detects an entirely separate epitope, was able to completely inhibit the mAb 8.26 intact antibody-induced platelet activation (Table 3 and Figure 7) despite its previously being shown not to directly inhibit mAb 8.26 binding (92) . As expected the mAb 8.26 F(ab') completely inhibited the mAb 8.26 Ig induced platelet response. These results suggest that the Fc-FcγRIIa platelet interaction is complex.

To further analyse this interaction, two monoclonal antibodies against platelet antigens

(glycoprotein IV (VM58) and CD9 , which are known potent inducers of aggregation and thromboxane release) were used. In addition, platelets with the different polymorphism of FcγRIIa (either being a high responder, ie. able to bind murine IgGi, or low responder ie. unable to bind murine

IgGi, were also used. As expected with the high responder platelets, the IgGi VM58 antibody induced rapid aggregation and thromboxane release (Table 3 and Figure 7) . This response was Fc dependent, as demonstrated by the anti- FcγRII antibody IV.3, which blocks Ig : FcγRIIa interaction, and which completely inhibited the activation. Surprisingly, the other anti FcγRII antibodies 8.2 and 8.26 did not inhibit VM58 antibody induced aggregation, but both enhanced platelet aggregation to a similar extent (increased slope to 110 and 118%, reduced lag phase by 67 and 73% and increased thromboxane release 156 and 147% respectively when compared to 100% baseline (Table 3 and Figure 7 ) .

Since the 8.2 and 8.26 antibodies enhanced the platelet reaction from the high responding individuals, their effect on low responding platelets was investigated. The VM58 antibody was unable to aggregate low responder platelets on its own because it is an IgGi murine antibody. However prior incubation of the 8.2 F(ab')₂ antibody to platelets allowed the VM58 antibody to intensely activate platelets measured by platelet aggregation (Table 3) and thromboxane release (increase from 292 ng/ml to 3654 ng/ml) . That is, it converted the non-responding platelets to the high responding phenotype, as demonstrated graphically in Figure 8.

Similar marked enhancement of VM58 platelet activation was obtained with pretreatment of the low responder platelets with the mAb 8.26 F(ab') antibody (Table 3), increasing thromboxane generation from 292 ng/ml to 3654ng/ml.

In order to establish that the enhancement of platelet activation by these anti FcγRII antibodies was seen with other anti-platelet antibodies, the activation of platelets by an anti-CD9 antibody was investigated. Results similar to those obtained with VM58 were seen with both 8.2 and 8.26 fragments, as shown in Table 3 and Figure 7) . In view of the fact that heat aggregated IgG appeared to activate platelets independently of FcγRIIa phenotype, studies were designed to examine whether the same phenomena as observed with the anti - FcγRII antibodies existed with human heat-aggregated IgG. The results showed a similar enhancement of platelet activation by preincubation with 8.2 fragments. There was an increase in slope to 133% decrease in lag phase to 39% and increased thromboxane release to 160%, compared to a baseline of 100% (Table 3 and Figure 9) . However 8.26 fragments instead of augmenting the reaction as was seen with the VM58 and CD9 antibodies, caused a decline in intensity of platelet activation by decreasing the scope to 42%, increasing the lag time to 165% and decreasing thromboxane release to 49% of baseline of 100% (Table 3 and Figure 9) . As the clinical syndrome of heparin-induced thrombocytopaenia is caused by Fc-dependent platelet activation, we then examined the platelet activation pattern with the anti-FcγRII antibodies in plasma samples from these patients. Similar contrasting results were seen with the HITTS plasma as with heat-aggregated IgG (Table 3 and Figure 9), with comparable enhancement in platelet activation with mAb 8.2 and partial inhibition with 8.26 fragments. In one example, mAb 8.2 preincubation converted a non-response or negative test to a brisk response or positive test to HITTS plasma as graphically shown in Figure 5. Preincubation of the platelets with either soluble rhFcγRII (50 μg/ml) abolished all aggregation to mAb 8.26, VM58, CD9 and HAGG and reduced to less than 9% TxB₂ generation. It appeared that the degree of inhibition by soluble rhFcγRII to HITTS plasma depended on the initial intensity of aggregation response. The weakest reacting

HITTS plasma showed complete inhibition to rhFcγRII in the high responding platelets as shown graphically in Figure 11. There was a reduction in platelet reactivity, but not complete inhibition, in the low responding platelets to both HITTS plasmas and in the high responding platelets with the stronger HITTS plasma. The mean gradient in these reacting platelets decreased to 72%, the average lag phase increased to 148% and TxB₂ release reduced to 31% of the 100% baseline value. The results show that there were differences in the FcγRII binding regions between anti-platelet monoclonal antibodies (VM58 and CD9) and human immune complex induced platelet activation (HAGG and HITTS plasma) . The binding of anti-FcγRII mAb 8.2 F(ab')₂ caused substantial enhancement of all measures of Fc dependent platelet activation, and even converted the phenotype of the mAb VM58 non-responder to a brisk responder.

The anti-FcγRII mAb 8.26 F(ab'), which was expected to abolish Fcγ mediated platelet activation by preventing Ig binding, surprisingly caused the same enhancement phenomena as mAb 8.2 F(ab')₂ to the platelet activating monoclonal antibodies (VM58 and CD9), and even produced brisk aggregation in the non-responder platelets . In contrast to mAb 8.2 F(ab')₂, platelet activation by human immune complexes (HAGG and HITTS plasma) was partially inhibited by mAb 8.26 F(ab') . This evidence suggests that after binding of these anti-FcγRII mAb there is a conformational alteration in the three-dimensional structure of the receptor that exposes previously unrecognised Ig binding sites that are different for the Fc portion of anti-platelet monoclonal antibodies and human immune complexes (HAGG and HITTS plasma) . These changes in FcγRII structure can not only markedly affect the rate of response of platelets to immune complexes but also the amount of metabolites (TxB₂) produced in response to the same stimulus. We have also demonstrated that the LR soluble form of rhFcγRII completely inhibits both mAb- and HAGG-induced platelet activation. The effectiveness of inhibition of the soluble rhFcγRII to HITTS-induced platelet activation appeared to depend on the initial platelet reactivity of the particular HITTS antibody to the platelet donor.

The attachment of mAb 8.2 F(ab')₂ increases the rate of association of the complexed IgG to the receptor, as measured by decreased lag time to commence platelet aggregation (Table III) . The effect is also to increase the stimulus response coupling of the IgG binding to the FcγRII as demonstrated by enhancement of the slope of aggregation and TxB₂ synthesis to the identical stimulus (Table III) .

PLATELET ACTIVATING ANTIBODIES

Murine Human

MOPC 131 8.26 VM 58 CD 9 HAGG HITTS (1) ^* HITTS (2) ^** HR LR

Monoclonal antibody preincubation Saline 0 + + + + + 0 + + + ++ _ 8.2 F(ab)₂ 0 0 + ++ + + + ++ + + + + +++ + ++ 8.26 F (ab) 0 0 + + + + + + + + + + + NT IV.3 0 0 0 0 0 0 0 0

Table I I I :

Platelet aggregation response to platelet activating monoclonal antibodies after 5 minutes incubation with anti

FcyRII monoclonal antibodies. The final antibody concentrations were 8.26 (5μg/ml), VM 58 (2.3 μg/ml), CD9

(2μ/ml), HAGG (250 μg/ml), plasma from patients with HITTS, control MOPC 131 (lOμg/ml), 8.2 F(ab)₂ (9μg/ml),

8.26 F(ab) (9μg/ml) and IV.3 (lμg/ml).

The grading of the response was judged using the lag phase before aggregation commenced and the slope of aggregation as follows:

+++ lag<30mm; slope>100mm; ++ lag 30 - 40mm; slope>60mm

10 + lag>30mιrι; slope<60mm; 0 no aggregation

Abbreviations: HR - high responder; LR - low responder; HAGG - heat aggregated IgG; HITTS - heparin induced thrombocytopenia .

* Platelets from high responder individual

** Platelets from non-responder individual.

An alternative explanation of this phenomenon is the direct priming of the receptor upon binding of a mAb 8.2 F(ab'h/ but without the Fc portion of mAb 8.2, we could not demonstrate platelet aggregation or TxB₂ synthesis to any degree (Table III) . The conversion of the low responder phenotype to a brisk responder by mAb 8.2 F(ab')₂ has important implications for improving the diagnosis of HITTS by platelet aggregation. A negative result with diluted positive weak HITTS antibody was converted into an unequivocal strong positive response, as shown by the platelet aggregation pattern in Figure 10. The binding of mAb 8.2 F(ab')₂ prior to HITTS testing will substantially enhance FcγRII dependent response, increase the rate of aggregation and diminish the variation in reactivity amongst donor platelets from different subjects. This preincubation step may improve the sensitivity and turnaround time of HITTS testing by platelet aggregation for the clinical laboratory.

The only Fc dependent mAb induced platelet activation that was inhibited by 8.2 F(ab')₂ was the intact anti FcγRII mAb 8.26. As expected, the mAb 8.26 F(ab') fragments could inhibit the whole mAb 8.26 activation and that mAb 8.26 F(ab') FcγRII binding did not itself cause signal transduction (Table 3). These findings suggest that the platelet activation by mAb 8.26 is not direct F(ab') binding to FcγRII causing activation, but, like other mAb, through the Fc portion of the intact IgG. The region on the Fc receptor to which mAb 8.26 causes Fc dependent platelet activation is unique, because unlike the other platelet activating mAb, 8.26 was completely inhibited instead of enhanced with the 8.2 antibody. There are two possible explanations for this observation. Firstly, unlike most mAb previously described, this is one of the few mAb of the IgG_2D rather than the IgGi subclass that has been shown to cause Fc dependent platelet activation (95) . It is possible murine IgG_2b binds to a different FcγRII region than murine IgGi. However, inhibition by mAb 8.2 F(ab')₂ using heat aggregated mouse IgG_2b, and which produced the pattern found with human HAGG was not observed. The more likely explanation is that mAb 8.26 binds to the main IgG binding region on D2γ and this exposes a unique secondary Fc binding region blocked by mAb 8.2 F(ab')₂- Perhaps the physical limitations of the mAb 8.26 F(ab') binding to the FcγRII, then the same molecule subsequent Fc binding on the platelet Fc receptor, causes this specific inhibition pattern of 8.26 FcγRII dependent platelet activation by mAb 8.2.

It is apparent that the binding of mAb 8.26 F(ab') does not inhibit IgG binding near residue 131 of FcγRIIa because platelet activation by mAb VM58, which is dependent on this polymorphism, is enhanced by this antibody. It is likely that HAGG and HITTS plasma have IgG complexes of the IgGi subclass (96) and the partial inhibition of platelet activation by mAb 8.26 reflects blocking of the IgGi binding on FcγRIIa (87). The remaining platelet activation is generated from other Ig binding sites particularly near FcγRIIa . Human IgG₂ binds with higher affinity to FcγRIIA^Hls131 (97; 98) and this subclass of IgG has been reported to be the major component of the HITTS antibody in the platelet factor 4/heparin ELISA assay (99) . Another study has not confirmed this finding in HITTS with the IgGi and IgG₃ subclasses predominating (96) . The IgG subclass repertoire produced in the immune response in HITTS probably is heterogeneous, depending on the clinical circumstances . It is likely that platelet activation after mAb 8.26 (Fab') binding in response to HITTS plasma indicates binding of IgG near

FcγRIIA^Hls131. The functional platelet response after mAb 8.26 (Fab') binding may readily identify those patients who have developed an IgG response that would bind to the FcγRIIA^Hls131 genotype causing the platelet activation and the clinical expression of thrombocytopaenia and thrombosis that is found in HITTS. This information will be important, because the FcγRIIA^HlΞl31 allele has been shown by several groups to be over-represented in HITTS patients (34.4% compared with 19%) (91; 100) and may help explain the heterogenous platelet reactions to HITTS antibodies.

Example 4 : Further Studies of the Effect of mAb8.2 in

HITTS Diagnosis Assays.

The anti-FcγRII monoclonal antibody, mAb 8.2 was used in the following studies. a) . Firstly, platelets from the same healthy donors were pre-incubated either with 8.2 F(ab)₂ or with mouse

IgG. Plasma from each of 12 patients with a positive diagnosis of HITTS were tested for reactivity with these platelets in the presence of 0.5 units/ml heparin and 100.0 units/ml heparin. The intensity of the reaction was assessed by the gradient of aggregation, the lag phase before aggregation, thromboxane B₂ release and mepacrine staining of platelet dense granules.

The sensitivity and specificity of the HITTS assay system in the presence and absence of 8.2 F(ab)₂ was also studied in 131 patients using a variety of donor platelets .

In the initial study, it was found that with weak

HITTS antibodies, platelet reactivity by all measures were markedly enhanced by preincubation with 8.2F(ab)₂. In the second study, 31/131 patients had a positive response, of which 11 were detected only after 8.2

F(ab)₂ preincubation. The number of non-specific reactions did not increase with 8.2 F(ab)₂ (8/131).

These results suggest that incubation of donor platelets with mAb 8.2 F(ab)₂ improves the sensitivity of the HITTS assay by more than 50% without loss of specificity .

b) . 1) A retrospective study was undertaken on heat inactivated plasma from 11 patients with the positive diagnosis of HITTS by clinical evaluation and FcγRII dependent platelet aggregometry . The plasma reactivities were studied in platelet rich plasma (PRP) using platelets from the same donor. Aggregation was quantitated by measuring the lag phase before aggregation and the gradient. The release of thromboxane B₂ (TxB₂) was assayed by an RIA technique. Mepacrine staining of dense granules, to assess platelet activation and release, was measured by flow cytometry. Anti-FcγRII mAb IV.3, which is known to completely inhibit FcγRII induced platelet activation, was used as a control. The plasmas were divided into weak (5) and strong

(6) antibodies according to the reaction strength in the aggregometry test. Those which had a lag phase of greater than 80 mm or a gradient of less than 50 mm/min were defined as weak antibodies. A saturating concentration of mAb 8.2 F(ab)₂ [8 μg/ml], rsFcγRII [50 μg/ml] or IV.3 [1 μg/ml] was preincubated at 37°C with 200 μl HITTS plasma and 250 μl donor platelet rich plasma for 5 min prior to the addition of heparin (0.5 units/ml). 2) A further prospective study was performed on

102 consecutive patients with a putative diagnosis of HITTS. Pre-incubation of mAb 8.2 F(ab)₂ with donor platelets prior to aggregometry was compared with the standard aggregation test for HITTS. All samples were tested with a standard (0.5 units/ml) and high (100 units/ml) concentration of heparin to determine the heparin specificity of the reactions.

The results showed that with the weak HITTS antibodies, reactions by all measures were markedly enhanced by the mAb 8.2 F(ab)₂ as shown in Figures 12 and

13) .

Inhibition by soluble rFcγRII was variable, but the reaction was reduced in 8 out of 11 patients. In 4 of these, reduction was more than 50% of the baseline. Figure 14 demonstrates a typical example of the inhibitory effect of soluble rFcγRII. Overall, pre-mcubation with 8.2 F(ab)₂ increased the rate of platelet aggregation, with

shortened lag phase increased gradient enhanced TxB₂ release reduced mepacrine staining

Conversely, after incubation with soluble rFcγRII, aggregation was reduced with:

increased lag phase reduced gradient reduced TxB₂ release increased mepacrine staining

These results are summarised in Table IV.

Table IV

Aggregation Aggregation Mepacrine Thromboxane Slope Lag phase labelling B₂ Release

Pre-incujation

Saline 100 100 100 100

8.2 F(ab)₂ 127 27 54 137 soluble 70 132 160 69 rFcγRII

IV.3 <3 >300 339 3

Percentage change in HITTS plasma induced platelet activation markers after pre-incubation with saline, FcγRII mAbs (8.2 and IV.3) and soluble rFcγRII (mean of 11 different experiments) .

From the further 102 samples tested, 24 had HITTS. Of these, 8 were positive only after prior incubation with 8.2 F(ab)₂. Figure 15 shows an example of 8.2 F(ab)₂ conversion of a negative reaction to a positive reaction. High dose heparin (100 units/ml) inhibited all positive HITTS reactions, even those enhanced by treatment with 8.2 F(ab)₂. Reactions were judged not to be heparin dependent if aggregation still occurred at the high heparin concentration. The number of non-specific reactions (7/102) did not increase with 8.2 F(ab)₂ pre-incubation .

Anti-FcγRII 8.2 F (ab) ₂ Enhancement The ¹⁴C-serotonin release assay (SRA) is commonly regarded as the most sensitive and specific laboratory test for the diagnosis of HITTS. Disadvantages of this test are:

it is technically difficult and time-consuming the use of radioactive material is required

Platelet aggregation testing is commonly used because it is :

widely available technically simple rapidly performed facilitates testing for LMWH/heparinoid cross- reactivities

Sensitivity and specificity approaching that of the SRA can be achieved by:

careful platelet donor selection two-point heparin testing.

In this study, pre-incubation of the donor platelets with the F(ab)₂ fragments of the anti-FcγRII mAb 8.2 improved the sensitivity of heparin-dependent platelet aggregation for the diagnosis of HITTS by 50% without increasing false positive results. The application of this reagent leads to a routine test which is easily performed, and which is equivalent to, or better than, the SRA for the diagnosis of HITTS in the clinical laboratory.

Soluble rFcγRII Inhibition Circulating soluble FcγRII is raised in HITTS and plays a protective role in vivo by neutralising immune complexes that would otherwise cause platelet activation.

When this mechanism is overwhelmed, thrombocytopenia and thrombosis may result . The addition of soluble rFcγRII to the platelet aggregation test has an inhibitory effect on HITTS antibody induced platelet activation.

The reaction in most patients was substantially inhibited. However, the in vitro test conditions were contrived in favour of aggregation. Without wishing to be bound by any proposed theory, it is believed that soluble rFcγRII would be more potent in vivo and has therapeutic potential .

Example 5 : Improvements in the Functions of Activated

FCR.

Enhancement of Fc receptor function by monoclonal antibodies can result in improved Fc receptor function. Included in these functions, but not limited to these, are improved antibody-dependent killing, also known as antibody-dependent cell mediated cytotoxicity (ADCC) , phagocytosis, antigen presentation, immune complex-mediated platelet activation, and monocyte activation and regulation of antibody production.

Antibody Dependent Cytotoxicity

Fc receptor positive cells are able to kill antibody-coated cells or viruses, bacteria, parasites, yeasts or other pathogens. Such killing is mediated when the target cell or pathogen is coated with antibodies, either as a result of a natural immune response or after administration of antibodies in a therapeutic setting. The coating of these pathogens and their killing can be measured m vi tro a number of ways, including antibody- dependent killing of chromium-labelled red blood cells. This is a standard and widely accepted model for antibody- dependent killing by Fc receptor positive cells. In these experiments, red blood cells are labelled with a radioactive isotope of chromium, exposed to antibody and then Fc receptor positive cells such as macrophages, and over time the killing of these antibody cells is measured by the release of chromium. Indeed, such readouts are possible with other isotopes or other methodologies, but the principle of antibody dependent killing is still the same. The inclusion m this system of anti-Fc receptor antibodies such as antibody 8.2, or 8.26 results m the increased killing of these red blood cells, either measured as greater chromium release or more sensitive killing of the red cells at a given antibody dose (101) .

Phagocytosis Another essential Fc receptor function is the phagocytosis or the mgestion of antibody-coated particles by Fc receptors. Antibody-coated particles such as red blood cells, bacteria, viruses, yeasts, cancer cells etc can be ingested by Fc receptor positive cells such as macrophages, neutrophils or other like cells. The uptake of these coated particles is a standard measurement for phagocytosis, and this process leads to the elimination of such antibody-coated cells or pathogens. Assays using adherent macrophages or neutrophils and the presence of anti-receptor antibodies detected by the uptake of labelled particles either with radio-activity, fluorescence or other like indication. The uptake of the labelled particles into Fc receptor positive phagocytes is easily measured either by directly counting the ingested particles or by indirectly counting the degree of radio-activity or label uptake (102) . In such assays the uptake of antibody-coated particles is enhanced by the anti-FcgR antibody, such as 8 . 2 or 8 . 26 .

Antigen Presentation

Antigen presentation is performed by antigen- presenting cells and leads to the stimulation of lymphocytes . Fc receptors are able to take up immune complexes. Having taken up these immune complexes, entry is gained into the antigen presenting pathways inside the cell, and ultimately leads to presentation of antigen to lymphocytes and the induction of immunity. In such assays, antigen-presenting cells are co-cultured with leukocytes in appropriate media, and in the presence or absence of the monoclonal anti-Fc receptor antibodies, the degree of stimulation of T-cells is measured, either by cytokine secretion or T-cell proliferation by any standard technique (103) .

In all of the above in vi tro experiments, similar in vivo experiments are conducted to measure the killing and/or disposal of antibody-coated particles such as cells or pathogens. Other immune complexes are also measured in vivo by using the presence of the anti-Fc receptor antibodies. Such in vivo experiments are conducted in transgenic animals expressing human Fc receptors or in humans. In addition, improved antigen presentation leading to greater immune responses in vivo is achieved by administration of the anti-receptor antibodies to transgenic animals expressing human Fc receptors or patients . Included in the concept of disposal of immune complexes is the killing and subsequent phagocytosis, or killing only, of cells expressing viruses, or of cells which are cancerous, or of other cell types which are altered and to which antibodies have bound.

Our studies with anti-FcγRII mAb indicate that multiple IgG binding sites exist on the platelet FcγRII, which are different for platelet activation induced by monoclonal antibodies and human immune complexes. These different anti-FcγRII mAb are useful to enhance Fc dependent platelet activation (mAb 8.2 F(ab')₂), and define IgG binding regions, particularly near to the FcγRIIa¹³¹ polymorphism that has been associated with HITTS and other immune complex disease (mAb 8.26 F(ab') . These observations increase the sensitivity of HITTS testing in the clinical laboratory and improve the definition of the antibody FcγRII binding response in patients with HITTS. We have shown that soluble recombinant FcγRII can inhibit FcγRII dependent platelet activation, and it is possible that plasma soluble forms of FcγRII may similarly modify immune complex mediated platelet activation such as it occurs in patients with heparin induced thrombocytopenia.

It will be apparent to the person skilled in the art that while the invention has been described in some detail for the purposes of clarity and understanding, various modifications and alterations to the embodiments and methods described herein may be made without departing from the scope of the inventive concept disclosed in the specification .

References cited herein are listed on the following pages, and are incorporated herein by this reference.

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Claims

1. A method of activating or improving the function of FcR, comprising the step of contacting FcR with an effective amount of a molecule capable of inducing said activation or improvement, for a time and under conditions sufficient to allow said activation or improvement to occur.

2. A method according to claim 1, wherein said molecule activates or improves the function of the FcR by inducing a change in configuration of the FcR and/or a change in shape of the FcR.

3. A method according to claim 2 , wherein the change in configuration of the FcR is characterised by multimerisation.

4. A method according to any one of claims 1 to 3 , wherein said molecule is an antibody selected from the group consisting of a monoclonal antibody, a polyclonal antibody, a fragment thereof, a humanised antibody, a scantibody and an antibody mimetic.

5. A method according to claim 4, wherein said monoclonal antibody is selected from the group consisting of mAb 8.2, 8.26, CIKM 5 and biological equivalents thereof .

6. A method according to any one of claims 1 to 5, wherein the FcR is a reagent in an assay, and wherein the molecule is contacted with the FcR prior to, or during the assay.

7. A diagnostic assay, wherein the assay comprises use of FcR the function of which is activated or improved in accordance with the method according to any one of claims 1 to 6.

8. A method according to claim 6 or an assay according to claim 7, wherein the assay is for HITTS.

9. A diagnostic kit for HITTS, comprising FcR and an effective amount of a molecule which activates or improves FcR function thereby to enhance the binding of the FcR to heparin-antibody complexes.

10. A diagnostic kit according to claim 9, wherein the specificity and/or sensitivity of the FcR for the heparin-antibody complexes is enhanced.

11. A diagnostic kit according to claim 9 or claim

10, wherein the molecule is selected from the group consisting of mAb 8.2, 8.26, CIKM5 and biologically equivalents thereof.

12. A method of diagnosing HITTS, comprising the step of contacting serum of a subject suspected of having HITTS with an FcR activated in accordance with the method of any one of claims 1 to 6.

13. A reagent comprising FcR and an effective amount of a molecule capable of inducing activation or improvement in function of FcR, optionally together with appropriate carriers, buffers or the like.

14. A kit comprising FcR and a molecule, which molecule is provided in an amount effective to activate or improve the function of the FcR when said molecule is contacted with the FcR.

15. A method of treatment or prophylaxis of a disease in a subject, wherein activation or improvement of the activity of FcR in the subject assists in combating the disease, comprising the step of administering to said subject an effective amount of a molecule capable of activation or improvement of the function of FcR.

16. A method according to claim 15, wherein the disease is selected from the group consisting of autoimmune diseases, parasitic infections, yeast infections, bacterial infections, viral infections and cancer.

17. A composition comprising a therapeutic agent and an activated FcR, which FcR is activated by a method according to any one of claims 1 to 6.

18. A kit comprising at least one therapeutic agent, an FcR, and a molecule which is provided in an amount effective to activate or improve the function of the FcR when said molecule is contacted with the FcR thereby to enhance the efficacy of said therapeutic agent.

19. A composition or kit according to claim 17 or claim 18, wherein the therapeutic agent comprises one or more of an agent selected from the group consisting of anti-venom, anti-toxin antisera, antibacterial, anti-viral, anti-parasite, anti-yeast and anti-tumour agents.

20. A method of activating or improving the function of monocytes and/or macrophages, comprising the step of contacting said monocytes or macrophages with an effective amount of a molecule capable of inducing said activation or improvement, for a time and under conditions sufficient to allow said activation or improvement to occur.

21. A method according to claim 20, wherein the macrophages or monocytes are further contacted with an immune complex.

22. A method of improving antigen presentation, comprising the step of activating or improving the function of monocytes and/or macrophages according to the method of claim 20 or claim 21.

23. A method, kit, reagent or composition according to any one of claims 1 to 22, wherein the FcR binds to an immunoglobulm selected from the group consisting of IgG, IgE, IgA, IgM and IgD.

24. A method, kit, reagent or composition according to claim 23, wherein the FcR is from a mammal.