JP2009524664A - Anti-FCRN antibodies for the treatment of auto / alloimmune diseases - Google Patents

Abstract

  Antibodies against the heavy chain of human FcRn are provided that function as non-competitive inhibitors of IgG binding to FcRn. Such antibodies may be polyclonal antibodies, monoclonal antibodies, chimeric antibodies or humanized antibodies, or antigen-binding fragments thereof. These antibodies are useful for reducing the concentration of pathogenic IgGs in an individual and are therefore used as therapeutic tools in autoimmune and alloimmune diseases.

Description

This work was supported by registrations HL067347 and AI60687 from the National Institutes of Health. The institution has certain rights in this invention.
The present invention relates generally to the field of autoimmune and alloimmune diseases.

Humoral autoimmune and alloimmune diseases are mediated by pathogenic antibodies. Some examples of autoimmune diseases include immune neutropenia, myasthenia gravis, multiple sclerosis, lupus and immune thrombocytopenia (ITP).
ITP is a disease of increased peripheral platelet destruction, and most patients develop antibodies that bind to specific platelet membrane glycoproteins. Antiplatelet antibodies effectively opsonize platelets, leading to rapid platelet destruction by cells of the reticuloendothelial system (eg, macrophages). Since studies have shown that most patients have normal or reduced platelet production, relative marrow failure may be involved in this disease. In general, attempts to treat ITP include suppressing the immune system and consequently causing an increase in platelet levels.

ITP occurs more frequently in women than in men, and ITP is considered to be an autoimmune disease common to children and adults. Onset is 1 in 10,000. In the United States, the incidence of ITP in adults is approximately 66 cases per 1,000,000 people per year. The estimated average incidence in children is 50 cases per 1,000,000 people per year. Internationally, childhood ITP occurs in approximately 10-40 cases per 1,000,000 people per year.
This problem is significant because chronic ITP is one of the major blood disorders in both adults and children. It is responsible for the considerable hospitalization and treatment costs of specialized hematology departments in the United States and around the world. Each year about 20,000 new cases occur in the United States, and the costs for ITP and special treatment are extremely high.

Most children with ITP have a very low platelet count, which causes sudden bleeding and is accompanied by typical symptoms including contusions, small red spots on the skin, nosebleeds and gum bleeding . Children can sometimes recover without treatment, but many physicians recommend careful observation, reduction of bleeding, and treatment with intravenous gamma globulin.
Large intravenous doses of human immunoglobulin (IVIG) have been shown to increase platelet counts in children afflicted with immune ITP, making IVIG useful as a treatment for several other autoimmune diseases It is shown that there is.

  Many studies have investigated the mechanism by which IVIG is effective in treating autoimmune diseases. With respect to ITP, early investigations led to the conclusion that the effect of IVIG was mainly due to the blockade of the Fc receptor responsible for the phagocytosis of antibody-opsonized platelets. Later studies have shown that Fc-depleted IVIG preparations increase platelet counts in some patients with ITP, and in recent years the effect of IVIG has been due to stimulation of FcγRIIb expression in macrophage cells It has been reported to lead to inhibition of platelet phagocytosis. Such IVIG treatments, however, have considerable side effects and are very expensive to develop and administer. In addition, other therapies used for the treatment of autoimmune / alloimmune diseases other than IVIG include polyclonal anti-D immunoglobulins, corticosteroids, immunosuppressants (including chemotherapeutic agents), cytokines, plasma exchange , In vitro antibody adsorption (eg, using a Prosorba column), surgical procedures such as splenectomy and others. However, like IVIG, these treatments are complicated by incomplete efficacy and high costs.

  Recently, it has been proposed to produce anti-human FcRn antibodies in knock-out mice lacking the FcRn gene (Roopenian, 2002, US Publication No. 2002/128863). The author argues that high affinity antibodies that bind to the same epitope of FcRn as IgG competitively inhibit the binding of pathogenic IgG to FcRn and therefore increase clearance. However, none of such antibodies have been demonstrated and therefore the efficiency of such antibodies is still a question. Furthermore, due to the high affinity of endogenous IgG for FcRn and the high concentration of endogenous IgG in the blood, competitive inhibition of FcRn requires very high doses, and therefore, with current IVIG therapy. It may be accompanied by similar side effects.

  Based on the prior art description, there is a substantial need to develop new therapies for autoimmune and alloimmune diseases that do not have the low potency and high cost of IVIG. It is therefore desirable to identify safer and more effective alternatives to IVIG for the treatment of autoimmune and alloimmune diseases.

Summary of the Invention The present invention provides compositions and methods for the treatment of autoimmune and alloimmune diseases. The composition of the invention comprises an agent that functions non-competitively to inhibit the transport of IgG by the FcRn receptor. Non-competitive receptor inhibitors, by definition, exhibit inhibitory activity that is independent of the concentration of receptor ligand (eg, IgG). With respect to FcRn-mediated transport of IgG, non-competitive inhibition is due to non-competitive binding of inhibitors to FcRn at physiological pH, and the inhibitor-FcRn complex during the time course of endosomal sorting and transit. It can be achieved by incomplete dissociation. Alternatively or additionally, non-competitive inhibition may be achieved by binding to a site that is remote from and / or not identical to the ligand binding site. The antibodies of the invention bind to FcRn, resulting in inhibition of pathogenic antibody binding to FcRn, thereby improving clearance of the pathogenic antibody from the individual's body. In one aspect, the agent that binds to FcRn is a polyclonal or monoclonal antibody directed against the heavy or light chain of FcRn. In one aspect, the invention provides polyclonal and monoclonal antibodies against the human FcRn receptor. In other embodiments, the antibody is chimeric or humanized.

According to the present invention, a method for alleviating an autoimmune or alloimmune disease, which functions as a non-competitive inhibitor of IgG to bind to FcRn, binds to FcRn and binds pathogenic antibodies to FcRn Also provided is a method comprising administering to an individual a composition comprising an agent that inhibits. In another embodiment, such agent is a polyclonal, monoclonal, chimeric or humanized antibody directed against FcRn, particularly the human FcRn receptor. In another embodiment, the antibody is directed against the heavy chain of the FcRn receptor.
As used herein, the term “pathogenic antibody” refers to an antibody that causes a pathological condition or disease. Such antibodies include antiplatelet antibodies.

  The present invention provides compositions and methods for increasing the clearance of pathogenic antibodies. These compositions and methods are useful for the treatment of autoimmune and alloimmune diseases. The compositions and methods of the invention are also known as FcRn (newborn Fc-receptor, FcRP, RcRB and Brambell Receptor) in a manner sufficient to prevent pathogenic antibodies from binding to FcRn. Is directed to combine).

  In the present invention, specific anti-FcRn treatment is provided. Most of the inhibitors of enzymes or receptors act as competitive inhibitors of substrate or ligand binding, so that they bind to the same site on the receptor as the ligand, and therefore the extent of inhibition , A direct function of the relative affinity and concentration of the inhibitor and ligand. US Patent Application No. 2002/0138863 to Roopenian (see paragraph 0031) shows that an antibody to FcRn binds to FcRn at the same site important for binding of IgG to Fc, so that when the antibody binds to FcRn, It is stressed that the binding of IgG to the site of FcRn is inhibited. While this prior art argument has been directed against competitive inhibitors, it has surprisingly been found in the present invention that non-competitive inhibitors of IgG for binding to FnRn have therapeutic value. It was done.

  Furthermore, Roopenian et al. Recognize that it is difficult to generate highly specific anti-FcRn antisera (see paragraph 0085). This cited document aims to overcome this problem by generating anti-FcRn antibodies in FcRn-deficient mice in which FcRn is recognized as an extracorporeal molecule. However, in the present invention, we repeatedly immunize animals with human FcRn light chain and with a novel immunoconjugate consisting of a human FcRn heavy chain peptide fragment covalently linked to mussel hemocyanin. Was able to generate high affinity and specific antibodies to both FcRn light and heavy chains.

  In a preferred embodiment, the antibody or fragment thereof is a non-competitive inhibitor of IgG binding to or transport by human FcRn. An antibody or fragment consists of any isoform (eg, IgA, IgD, IgE, IgG, IgM, etc.), and such antibodies may be made in any species (eg, mouse, rat, etc.). Depending on the type of origin (see Ober et al., 2001, Int Immunol 13: 1551-9), IgG isoform antibodies can competitively inhibit IgG binding to human FcRn. Such antibodies can be used as long as they also act as non-competitive inhibitors of IgG binding to FcRn. That is, one antibody that is both a non-competitive inhibitor of IgG binding to FcRn and a competitive inhibitor may be used.

  FcRn binds to its ligand (ie, IgG) with a pH dependent affinity. It exhibits virtually no affinity for IgG at physiological pH. Thus, anti-FcRn antibodies that bind to FcRn at physiological pH (7.0-7.4) can act as non-competitive inhibitors, and as a result, the binding of anti-FcRn antibodies to FcRn is not affected by the presence of IgG. Concentrations of antibodies of the invention in a pH-independent manner in the pH range 6-8 and in a non-competitive manner, less than required for competitive inhibitors due to their ability to bind FcRn Allows functional inhibition of FcRn-mediated transport of IgG at Without intending to be bound by any particular theory, due to pH independence in the range of pH 6-8, such inhibitors bind to (physiological pH) FcRn on the cell surface, and It is hypothesized that it is possible to remain bound to FcRn during the course of intracellular transport, thereby inhibiting FcRn binding to IgG within acidic pH (˜6) endosomes. The non-competitive mode of binding allows these inhibitors to be used at much lower concentrations than competitive inhibitors, making them more attractive for therapeutic purposes. Without intending to be bound by any particular theory, it is believed that as a result, FcRn-mediated protection of IgG from intracellular catabolism is inhibited, thereby increasing IgG clearance.

  As shown in the Examples herein, IVIG mediates a dose-dependent increase in the elimination of pathogenic antibodies in an animal model of ITP, and this effect is mediated by IVIG interaction with FcRn. . However, in part, due to the putative mechanism of IVIG inhibition of FcRn binding with pathogenic antibodies (i.e. competitive inhibition) and in part, IgG is at physiological pH (i.e. pH 7.2-7.4). ), A very high dose of IVIG is required to produce a substantial increase in clearance of pathogenic antibodies (i.e., typical of IVIG). Clinical dose is 2 g / kg).

  The present invention provides for non-competitive inhibition of FcRn binding to pathogenic antibodies at physiological pH and for non-competitive inhibition of FcRn binding to pathogenic antibodies for specific anti-FcRn therapy It is. Thus, the present invention provides a method for preventing pathogenic antibodies from binding to FcRn as a treatment for autoimmune and alloimmune diseases. The methods of the invention also provide compositions useful for specifically inhibiting FcRn in a manner sufficient to prevent pathogenic antibodies from binding to FcRn. The compositions and methods of the present invention rather act both in increasing the rate of pathogenic antibody excretion and in alleviating the morbidity and disease caused by pathogenic antibodies in those who receive the treatment.

  In one aspect of the invention, anti-FcRn (heavy chain) antibodies are provided. These antibodies can be prepared by using a whole heavy chain or a peptide corresponding to the heavy chain sequence. Examples of such antibodies are shown in Examples 12-15. The antibody was observed not to bind to b2 microglobulin. However, because they bind to the FcRn complex (including both heavy chain and b2 microglobulin) and were generated against the heavy chain sequence of FcRn, it is possible that these antibodies bind to the heavy chain of human FcRn. Most likely.

  The compositions and methods of the present invention are therefore not limited and include immunocytopenia, immune neutropenia, myasthenia gravis, multiple sclerosis, lupus and antibodies that are Suitable for use in autoimmune diseases, including other diseases that cause the disease. In addition to humans, the antibodies of the present invention can also be used in other species.

The composition of the invention comprises an agent that can inhibit FcRn binding to pathogenic antibodies such as anti-platelet antibodies. Such compositions include, but are not limited to, monoclonal antibodies, polyclonal antibodies and fragments thereof. The antibodies can be chimeric or humanized, antibody fragments, peptides, small molecules, or combinations thereof that can prevent pathogenic antibodies from binding to FcRn receptors. Antibody fragments that contain antibody binding sites may also be used. Such fragments include, but are not limited to, Fab, F (ab) ′ ₂ , Fv and single chain Fv (ie, ScFv). Such fragments include all or part of the antigen binding site, and such fragments retain the specific binding properties of the parent antibody.

  The antibody of the present invention may be a chimeric antibody or a humanized antibody. “Chimeric” antibodies are those that are encoded by immunoglobulin genes that have been genetically engineered so that the light and heavy chain genes are composed of immunoglobulin gene segments belonging to different species. For example, a variable (V) segment of a gene from a mouse monoclonal antibody may be linked to a human constant (C) segment. Such a chimeric antibody is considered to be less antigenic to humans than an antibody having a mouse constant region and a mouse variable region.

A “humanized” antibody is an immunoglobulin, an immunoglobulin chain, or a fragment (Fv, Fab, Fab ′, F (ab ′) ₂ or a fragment thereof containing minimal sequence derived from a non-humanized immunoglobulin, or Other antigen-binding sequences of antibodies). Humanized antibodies include human immunoglobulin residues from the complementarity determining region (CDR) of non-human species such as mice, rats or rabbits that have the desired specificity, affinity and capacity. Includes those replaced by residues from CDRs. In some examples, the Fv framework residues of the human immunoglobulin are replaced with corresponding non-human residues. Humanized antibodies may contain residues that are found neither in human antibodies nor in non-human transducible CDRs or framework sequences. In general, a humanized antibody will contain at least one and typically substantially all of the two variable domains, all or substantially all of the CDR regions corresponding to those of a non-human immunoglobulin. And all or substantially all of the FR region is of human immunoglobulin consensus sequence.

  Methods for humanizing non-human antibodies are well known in the art. Generally, a humanized antibody has one or more amino acid residues introduced therein from a non-human source. These non-human amino acid residues are referred to as “import” residues and are typically derived from an “import” variable domain. Humanization is performed by the method of winter and co-workers (Jones et al., Nature, 321: 522 525 (1986); Riechmann et al., Nature, 332: 323 327 (1988); Verhoeyen et al., Science, 239: 1534 1536 (1988). ), Essentially by replacing the rodent CDRs or CDR sequences with the corresponding sequences of a human antibody. In practice, humanized antibodies typically replace some CDR residues and potentially some FR residues with residues from similar sites in rodent antibodies. It is a human antibody.

  Polyclonal antibodies directed against FcRn or fragments thereof, light chains or heavy chains, etc. immunize appropriate subjects with FcRn or portions thereof, light chains, heavy chains, peptide sections contained within the molecule, etc. Can be prepared. The anti-FcRn or fragment antibody titer in the immunized subject can be monitored gradually by standard methods such as ELISA using FcRn or fragments thereof. If desired, antibody molecules directed against FcRn or fragments thereof can be isolated from mammals (eg, from blood) and further purified by well-known methods, such as protein A chromatography to obtain IgG fractions be able to.

  Monoclonal antibodies directed against FcRn or fragments thereof can be produced by standard methods, the hybridoma method first described by Kohler and Milstein (1975, Nature 256: 495-497). Briefly, an immortal cell line (typically myeloma) is fused to lymphocytes (typically splenocytes) from a mammal immunized with FcRn or a fragment thereof and the resulting hybridoma cells Are searched for hybridomas that produce monoclonal antibodies that bind to FcRn. Typically, an immortal cell line (eg, a myeloma cell line) is derived from the same mammalian species, lymphocytes, and the like. Hybridoma cells producing the monoclonal antibodies of the invention are detected by probing the hybridoma culture supernatant for antibodies that bind FcRn using a standard ELISA assay. Human hybridomas can be prepared in a similar manner.

An alternative to preparing monoclonal antibody-secreting hybridomas is to identify and isolate monoclonal antibodies by searching a recombinant combinatorial immunoglobulin library (antibody phage display library) using FcRn or a fragment thereof. It is.
Administration of the composition of the present invention can be carried out by methods known to those skilled in the art. Where the particular inhibitor of FcRn comprises an antibody, administration can be accomplished, for example, by intravenous, intramuscular or subcutaneous injection, cannula or other methods known to those skilled in the art. Similarly, administration of small molecules effective to prevent binding of anti-platelet antibodies to FcRn receptors can be performed by methods well known to those skilled in the art.

  The inhibitor antibody or antibodies of the invention can be administered for the elimination of pathogenic antibodies in the treatment of autoimmune and alloimmune diseases. The effect of an inhibitor antibody on the elimination of pathogenic antibodies in a particular individual is the method of administration, inhibitor antibody pharmacokinetics (i.e., inhibitor distribution and excretion rate and degree), inhibitor antibody affinity for FcRn, FcRn transport capacity, and Potentially, it is recognized that there is a tendency due to FcRn receptor turnover. Animal studies presented herein have demonstrated that model inhibitor antibodies result in a dose-dependent transient increase in IgG excretion in rats. Due to the temporary nature of the effect, it is possible to control the duration of FcRn blockade and to minimize any risk associated with FcRn blockade (eg, risk of infection).

  The pH 6-8 independent and non-competitive inhibitors of the present invention result in corresponding reductions in endogenous pathogenic and non-pathogenic IgG antibody concentrations. As such, the effect of high affinity, non-competitive inhibitors of FcRn on pathogenic antibody concentrations can be assessed based on the effects of the inhibitors on total serum concentrations of endogenous IgG. FcRn inhibitors may be administered as single and / or multiple doses. In general, 1 to 2000 mg / lg, preferably 1 to 200 mg / kg and more preferably 1 to 40 mg / kg can be administered to patients suffering from autoimmune or alloimmune diseases, and these therapies are Preferably, it is designed to reduce the serum endogenous IgG concentration to less than 75% of the pre-treatment value. Intermittent and / or chronic (continuous) administration methods may also be applied.

The present invention is illustrated by the following examples, which are intended only to illustrate certain embodiments of the invention and are not intended to be limiting in any way.
[Example 1]
In this example, the general method used is described. 200-225 g female Sprague-Dawley rats were used for in vivo analysis. Rats were fitted with a jugular vein catheter 2 days prior to treatment. 7E3, a murine anti-glycoprotein IIb / IIIa (GPIIb / IIIa) monoclonal antibody was generated from hybridoma cells obtained from the American Type Culture Collection (Manassas, Va.). Hybridoma cells were grown in serum-free medium (Life Technologies®, Rockville, MD) and then antibodies were purified from the medium using protein G chromatography. IVIG preparations were obtained from Baxter Healthcare® (Hyland Division, Glendale, Calif.) And Bayer® (Pharmaceutical Division, Elkhart, Ind.). Both IVIG preparations were made by solvent / surfactant-treated and cold ethanol fractionation of human plasma. Old human platelets were obtained from American Red Cross (Buffalo, NY and Salt Lake City, UT). Murine anti-methotrexate IgG1 monoclonal antibody (AMI) was made in our laboratory and then purified. Goat anti-human IgG (no cross-reactivity to goat and mouse serum proteins) and alkaline phosphatase-conjugated goat anti-mouse IgG (no cross-reactivity to goat and human serum proteins) were both from Rockland (Gilbertsville, PA). obtained. Mouse anti-human IgG, fluorescent isothiocyanate (FITC) -labeled anti-mouse IgG and p-nitrophenyl phosphate were from Pierce® (Rockford, Illinois). Calf serum albumin (BSA) and buffer reagents were obtained from Sigma® (St Louis, MO). The buffers were phosphate buffered saline (PBS, pH 7.4), 0.02M Na ₂ HPO ₄ (PB), and PB + 0.05% Tween-20 (PB-Tween).

  Examples 2-5 illustrate the effect of IVIG on antiplatelet antibodies. In these examples, IVIG can attenuate the effects of antiplatelet antibodies in a dose-dependent manner in a rat model of ITP, and IVIG is dramatic and clearly nonspecific in antiplatelet antibody clearance. It has a good effect.

[Example 2]
This example demonstrates that IVIG removes anti-platelet antibodies in a rat model of IPT. Rats were administered IVIG (0.4, 1, or 2 g / kg) via a jugular vein catheter. After IVIG administration, a blood sample (0.15 mL) was collected for baseline measurement of platelet count. Rats are then dosed with anti-platelet antibody, 7E3, 8 mg / kg, and platelet counts are then measured using a Cellu-Dyne1700 multiparameter hematology analyzer (Abbott Laboratories®, Abbott Park, IL). Obtained over time. Control animals received saline and then 7E3. The platelet nadir of each animal was the lowest platelet count observed. Platelet count data was normalized by the initial platelet count because of the large inter-animal variability in the initial platelet count. By normalizing the data, the effects of 7E3 and IVIG can be better compared between animals. Blood samples (0.15 mL) were taken for pharmacokinetic analysis at 1, 3, 6, 12, 24, 48, 96 and 168 hours after administration of 7E3. 7E3 plasma concentration was measured using an enzyme linked immunosorbent assay (ELISA) as follows. Human GPIIB / IIIa was diluted 1: 500 in PB and added to Nunc Maxisorp plates (0.25 ml / well). Plates were incubated overnight at 4 ° C. Standards and samples were then added to the plates (0.25 ml / well) and incubated for 45 minutes at room temperature. Finally, p-nitrophenyl was added (4 mg / m in DEA) and changes in absorption versus time were recorded with a SpectraMax Microplate reader. The plate was washed 3 times with PB-Tween between each step of the assay. Standards were made to final concentrations of 0, 1, 2, 2.5, 5, 10, and 20 ng / ml in 1% mouse plasma. Inter-animal assay variability was <15% for the quantity control sample within the standard range curve.

At a dose of 8 mg / kg, 7E3 caused rapid and severe thrombocytopenia in rats. As can be seen in FIG. 1, pretreatment of rats with IVIG significantly changed the platelet count time course after administration of 7E3 (P = .031). Statistically significant differences (P <.01) from controls were observed at 1 and 3 hours for the 2-g / kg IVIG group and 3 hours for the 1-g / kg IVIG group. Because of the large degree of initial absolute platelet count variability, this model used the percent platelet count to assess the effect of 7E3. However, each group has a mean initial platelet count that can be compared to the control, 0.4-, 1-, and 2-g / kg IVIG groups, 326 ± 62, 323 ± 137, 272 ± 111, and 301 ± 69 × 10. Each had an absolute initial number of ⁹ platelets / L. Since absolute platelet count can be important in assessing the risk of bleeding, we also measured the platelet count nadir value as a metric to determine the IVIG effect in this model. After 7E3 treatment alone, the animals had an absolute platelet bottom point of 48 ± 28 × 10 ⁹ platelets / L, corresponding to an average initial count of 14% ± 8%. Using IVIG pretreatment, 121% to 279% increase in the lowest percent platelet count (compared to control) was observed (P = .044), 0.4, 1-, and 2-g / kg IVIG administration, respectively With values of 31% ± 26%, 44% ± 24%, and 53% ± 27%. Each IVIG treatment group was significantly different from the control (P <.05). However, IVIG was not completely effective at disrupting thrombocytopenia, even at the highest doses. The percentage of rats reaching the threshold for thrombocytopenia (<30% of the initial number) decreases with dose for animals pretreated with IVIG, and is 0.4-, 1-, and 2-g / kg. 75%, 50%, and 25% of rats in the IVIG group had nadir platelet counts less than the initial 30%.

  These results indicate that pretreatment of rats with IVIG attenuated 7E3-induced thrombocytopenia. IVIG pretreatment reduces the average degree of thrombocytopenia achieved after 7E3 treatment (as measured by the mean percent platelet count at the lowest point), and animals exhibiting severe thrombocytopenia Fraction decreased.

[Example 3]
This example describes the pharmacokinetics of the effect of IVIG on 7E3. To determine this, the 7E3 plasma concentration after pretreatment of rats with IVIG was measured. As can be seen from FIG. 2 and Table 1, IVIG was observed to enhance the clearance of 7E3. ANOVA revealed a highly significant difference between the clearance values calculated for the four treatment groups (P <.001). Differences in 7E3 clearance were shown to be statistically significant for all parts of the treatment group, except for a comparison of data from animals receiving 0.4 g / kg IVIG versus those receiving 1 g / kg IVIG (Tukey Multiple comparison test). Significant differences from controls were observed for 7E3 concentrations at 12 hours and longer for the 2-g / kgIVIG group and at least 48 hours for the 0.4- and 1-g / kgIVIG groups .

  As demonstrated by this example, IVIG altered the pharmacokinetics of 7E3. Our data demonstrate a trend towards decreased terminal half-life of 7E3 with IVIG administration (P = .06), comparing half-life in control animals to that seen in animals receiving 1 g / kg IVIG Statistical significance was achieved in (P <.05). More importantly, IVIG was found to induce a dramatic increase in clearance of antiplatelet antibodies (P <.001). Since the effect of IVIG on elimination rate (and half-life) can be expected to decrease with time after IVIG administration, clearance to meet the time-and-concentration-average measure of 7E3 excretion is 7E3 excretion It is an excellent measurement for the evaluation of the IVIG effect.

[Example 4]
This example demonstrates that IVIG does not bind to anti-FcRn antibodies. Goat anti-human-IgG (1: 500 diluted in PB, 0.25 mL / well) was added to a Nunc® Maxisorp® 96-well multiplate (Nunc® model No. 4-42404, Roskilde, Denmark) and then the plates were allowed to incubate overnight at 4 ° C. IVIG (25 mg / mL) and 7E3 (0, 0.01, 0.05 and 0.10 mg / mL) were combined in test tubes and incubated for 2 hours at 37 ° C. The positive control sample consists of IVIG incubated with the same concentration of mouse anti-human IgG (Pierce®) as shown for 7E3. Samples and controls were diluted to 1% BSA in PBS to 1000, then added to microplates (0.25 mL / well) and incubated for 2 hours at room temperature. Alkaline phosphatase-labeled anti-mouse IgG (diluted 1: 500 in PB, 0.25 mL / well) was then added to the plate and incubated for 45 minutes, again at room temperature. Finally, p-nitrophenyl phosphate (4 mg / mL in diethanolamine buffer, pH 9.8) was added at 0.2 mL / well and the plate was plated at 405 nm with a plate reader (Spectora Max® 340PC, Molecular Devices® Trademark), Sunnyvale, CA). Plates were recorded over 10 minutes and assay response (dA / dt) was assessed using the slope of the absorption versus time curve. After each sample is assayed in triplicate, the response is shown as mean ± SD. Between each step of the assay, the wells of the microplate were washed 3 times with PB-Tween.

  In vitro binding of 7E3 to IVIG could not be detected. FIG. 3 shows the results obtained from experiments designed to detect 7E3-IVIG binding. IVIG and 7E3 were incubated in vitro at 37 ° C. for 2 hours. Following this incubation, the samples were diluted and added to microplates coated with anti-human IgG. That is, if 7E3 binds to IVIG, secondary anti-mouse IgG will detect the presence of 7E3. There was no statistically significant difference between the assay responses for the 7E3-containing sample versus the negative control (IVIG alone), with P = .164. However, there was a significant difference in the assay response (at each concentration) for the positive control antibody, with P <.001. The concentration ratio of 7E3 / IVIG in this experiment was designed to be the same as expected in in vivo experiments.

To determine whether this effect of IVIG is specific for the antiplatelet antibody, 7E3, we identified the pharmacokinetics of the secondary monoclonal antibody, AMI, in the presence and absence of IVIG. Rats (n = 3 / group) were administered 2 g / kg IVIG (or saline for control) via the jugular vein cannula followed by AMI (8 mg / kg). Blood samples were collected over a week and plasma was analyzed for AMI concentration by ELISA. Pharmacokinetic analysis was performed as described above for 7E3. Figure 4 shows that IVIG also increased the clearance of AMI, and the AMI clearance increased from 0.44 ± 0.05 to 1.17 ± 0.05 mL hr- ¹ kg ^-1 from the control to the IVIG treatment group (P <.001). Show. Furthermore, the relative degree of clearance increased by IVIG treatment was similar between groups, with a 2.37-fold increase in clearance observed for 7E3 and a 2.66-fold increase in clearance observed for AMI followed by 2-g / kg IVIG treatment. Accompanied.

[Example 5]
This example describes a qualitative and quantitative test to determine whether IVIG can inhibit 7E3 binding to human platelets. For quantitative studies, 10 μg / mL 7E3 was incubated for 1.5 hours with human platelets (1 × 10 ⁷ platelets / mL) in the presence or absence of IVIG (2.5 mg / mL). Control mouse IgG was a negative control. Samples were centrifuged at 4000 rpm for 6 minutes, washed with PBS (2 times), and then incubated with 100 μL of a 1:10 dilution (in PBS) of FITC-labeled anti-mouse IgG solution for 45 minutes. Samples were washed again and resuspended in PBS and then subjected to analysis by flow cytometry (Flow Cytometry Core Facility, Huntsman Cancer Institute, Salt Lake City, UT). In quantitative inhibition studies, the potential of IVE inhibition of 7E3 platelet binding was studied in considerable detail. Human platelets (8.2 × 10 ⁸ / mL) were incubated with 7E3 (4.8-72.5 μg / mL) in the presence or absence of IVIG (25 mg / mL) for 2 hours. The sample was then centrifuged at approximately 3000 g for 6 minutes to obtain a platelet pellet. A portion of each supernatant was obtained and assayed for unbound 7E3 concentration. Binding to 7E3 platelets in the presence and absence of 7E3 was analyzed by fitting the data to the following binding curves. :

In the above equation, F _f is the free fraction of the 7E3, K _A is an apparent about 7E3 platelet binding, [7E3] _f is the unbound molar 7E3 concentrations, and R _t are, receptor concentration of total It is. Using Micromath Scientist®, a nonlinear minimum binary analysis of the data was created, the parameter values and the reported SDs are from the software output.

The results of qualitative flow cytometry analysis are shown in FIG. In the fluorescence histogram, no shift was observed in the presence of IVIG. The results from the quantitative test are shown in FIG. The binding curves are almost identical in the presence and absence of IVIG. There was no significant difference in binding parameters K _A and R _t− . Without the presence of IVIG is, K _A is ^{4.9 ± 0.7 × 10 8 M -1} , R t was ^{7.5 ± 0.4 × 10 -8 M (} 55000 ± 3000GP / platelets). With IVIG, K _A is ^{5.5 ± 1.2 × 10 8 M -1} , R t was ^{7.6 ± 0.7 × 10 -8 M (} 56000 ± 5000GP / platelets).

[Example 6]
In this example, the effect of IVIG on clearance of antiplatelet antibodies was tested in FcRn knockout mice. β-2-microglobulin knockout mice (those lacking FcRn expression) and C57B1 / 6 control mice, 21-28 g were obtained from Jackson Laboratory (Bar Horbor, ME). 3-5 mice per group were administered IVIG (1 g / kg) or saline via the carotid artery cannula followed by 8 mg / kg 7E3. At each time point, a 20 μl blood sample was taken from the saphenous vein of the mouse over the course of 4 days for the knockout mice and 30-60 days for the control mice. Plasma 7E3 concentration was determined by ELISA as described in Example 2.

Standard non-compartmental pharmacokinetic analysis was performed and 7E3 clearance and terminal half-life for various treatment groups (11) were determined using WINNONLIN software (Pharsight Corp., Palo Alto, Calif.). Unpaired t-tests were performed using GraphPad Instat (GraphPad software, Inc., San Diego, Calif.).
The effect of IVIG on 7E3 pharmacokinetics in B-2-microglobulin knockout and control C57BL / 6 mice is shown in FIG. IVIG increased 7E3 clearance in control mice (P <0.0001), and IVIG treatment was observed to fail to increase 7E3 clearance in mice lacking FcRn expression (see Table 2), in anti-platelet antibody clearance It is established that the effect of IVIG is mediated by the FcRn receptor.

[Example 7]
An example of an agent suitable for specifically inhibiting the binding of an antiplatelet antibody to the FcRn receptor is a monoclonal anti-FcRn antibody. Hybridomas secreting monoclonal anti-FcRn antibodies were obtained from American Type Culture Collection (ACTT #: CRL-2437, designation: 4C9). Hybridoma cells were grown in standard medium supplemented with medium, 1% calf serum. The culture supernatant was collected, centrifuged, and then subjected to protein-G chromatography to purify IgG. As shown in FIG. 8, administration of a specific anti-FcRn antibody preparation of ~ 60 mg / kg led to a ~ 400% increase in the clearance rate of anti-platelet antibodies in the thrombocytopenic animal model from Example 1. It was. In contrast, in this same model, 2 g / kg IVIG led only to a ˜100% increase in antiplatelet antibody clearance. This demonstrates that the agent used to affect the clearance of 7E3 in this example, i.e. a specific inhibitor of FcRn, is more potent and more effective than IVIG. It is considered to be a non-specific inhibitor.

[Example 8]
This example describes the effect of 4C9 on other antibodies AMI. 175-275 g female Sprague-Dawley rats were fitted with a jugular vein cannula under ketamine / xylazine anesthetic (75/15 mg / kg). Two days after surgery, animals were treated with 0, 3, 15, and 60 mg / kg of 4C9 injected via the jugular cannula (3-4 rats per group). Four hours after 4C9 administration, AMI (8 mg / kg) was administered via cannula and blood samples (150 μl) were collected at 1, 3, 6, 12, 24, 48, 72 and 96 hours. The cannula patency was retained for washing with approximately 200 μl of heparinized saline. The blood was centrifuged at 13,000 g for 3-4 minutes, then the plasma was isolated and stored at 4 ° C. until analysis. Plasma AMI concentration was determined by ELISA.

  As shown in FIG. 9, the clearance of AMI is up to 99% after 4C9 administration, from 0.99 ± 0.14 ml / h / kg in control animals to 1.97 ± 0.49 ml / kg in animals pre-treated with 60 mg / kg 4C9. Increased to h / kg (p <0.05). Thus, these data demonstrate that anti-FcRn antibodies can be used to increase the clearance of IgG antibodies in vivo.

[Example 9]
This example shows the production of monoclonal antibodies against human FcRn. Using a human FcRn light chain (i.e., human beta-2-microglobulin, Sigma Chemical, St. Louis, Mo.) emulsified in Freund's incomplete adjuvant (Sigma Chemical), 6 Balb / c mice (Harlan , Indianapolis, IN) was repeatedly immunized. Animals were bled from the saphenous vein 7-10 days after immunization and antibodies directed against human FcRn light chains were detected using an antigen capture enzyme-linked immunosorbent assay (ELISA). The animals with the highest ELISA response were selected for use as splenocyte donors and fusions were performed with murine SP20 myeloma cells (ATCC, Manassas, VA). Briefly, mice were sacrificed with ketamine (150 mg / kg) and xylazine (30 mg / kg) and the spleen was quickly removed using aseptic technique. Splenocytes are removed from spleen tissue using a sterile 22-gauge needle, suspended in RPMI 1640 and then standard methods with polyethylene glycol (eg Harlow E and Lane D. 1988. Antibody: Research Fused with SP20 cells by centrifugation using a laboratory manual, New York: Cold Spring Harbor Laboratory. Fusion cells were selected by application of HAT selection medium (Sigma Chemical) and cloned by the method of limited dilution. Tissue culture supernatants were assayed for anti-FcRn activity by assessing an ELISA response to human beta-2-microglobulin.

  91 viable hybridoma clones were identified and tissue culture supernatants were obtained from cultures of each clone and probed for the presence of anti-human FcRn light chain antibodies. Briefly, human FcRn light chain was coated overnight on a 96-well microplate at 4 ° C. The plate is then washed and then secreted with phosphate buffered saline (PBS, negative control), culture supernatant obtained from the hybridoma, or antibody directed against the light chain of 4C9 hybridoma cells-rat FcRn (Raghavan et al., Immunity 1 (4): 303-315, 1994). After 2 hours incubation at room temperature, the plates were washed and a goat anti-mouse Fab specific antibody conjugated with alkaline phosphate was added and incubated for 1 hour. Finally, the plate was washed and then p-nitrophenyl phosphate was added. Absorption changes were monitored over time (over 10 minutes) at 405 nm with a microplate reader. From 91 viable potential anti-human FcRn clones, 8 positive clones were identified. These clones were 1H5, 4B10, 6D10, 7C7, 7C10, 10E7, 11E4 and 11F12. The response of human FcRn to the light chain is summarized in FIG. 10 (plotted is the net assay response: eg, the raw reaction minus the PBS control assay reaction). One-way ANOVA revealed significant differences in assay responses (p <0.0001), and the assay response for 8 positive clones was found to be significantly different from that of controls (p <0.01 for each clone). , Dunnet multiple comparison test). Furthermore, this assay revealed that the 4C9 antibody directed against the rat FcRn light chain does not show significant binding to the human FcRn light chain.

[Example 10]
This example describes the effect of anti-FcRn light chain antibodies on the binding of human IgG to 293 cells expressing human FcRn. To prove this, 293 cells expressing human FcRn were obtained from Dr. Neil Simister at Brandeis University. Human IgG was labeled with FITC by standard methods. Tissue culture supernatant was obtained from a culture of four hybridomas (11E4, 11F12, 1H5, 10E7) (Example 9) that were found to secrete antibodies directed against the light chain of human FcRn. .

293 cells were treated with trypsin: EDTA and suspended in the medium. The cell suspension was centrifuged at 300 g for 5 minutes, resuspended in buffered saline, and then the cells were counted with a hemocytometer. Approximately 3.6 × 10 ⁶ cells / ml of 293 cells were added to each centrifuge tube in pH 6 or 8 buffered saline. Cells were incubated with buffered saline alone or with FITC-IgG at a concentration of 1 μg / ml in the presence or absence of cell culture supernatants obtained from hybridoma cells. The reaction mixture was incubated at room temperature for 1.5 hours, then the cells were washed and resuspended in buffered saline. Cell bound fluorescence was analyzed on a fluorometer using a set of excitation and emission wavelengths of 494 and 520 nm, respectively.

  Consistent with the known pH-dependent binding of human IgG to human FcRn, cell-bound fluorescence is at 253000 and 10800 for 293 cells incubated at pH 6.0 and 8.0 respectively with 1 μg / ml FITC-human-IgG. It was recognized that there was. In contrast, for cells incubated in the absence of FITC-IgG, cell bound fluorescence was observed to be 5220 and 5300 at pH 6.0 and 8.0, respectively. For cells incubated with FITC-IgG and cell supernatant at pH 6.0—obtained for cells secreting anti-FcRn antibodies—cell-bound fluorescence was reduced to 80-84% ( (See Table 3 below).

  These results indicate that binding of human IgG to 293 cells expressing human FcRn is pH-dependent, indicating stronger binding at pH 6.0 compared to that observed at pH 8.0. Indicates that Culture supernatants from hybridomas secreting antibodies directed against the human FcRn light chain can inhibit the binding of human IgG to FcRn.

[Example 11]
This example further demonstrates that the antibodies of the invention are non-competitive inhibitors of IgG binding to FcRn. Binding of mouse IgG to 293 cells expressing hFcRn was determined as follows in the presence or absence of anti-hFcRn antibody. 293 cells with PBS, with cell culture supernatant from two hybridomas identified as secreting anti-human FcRn light chain antibody, and secreting monoclonal anti-methotrexate mIgG1 (AMI as negative control) The cells were incubated with the cell culture supernatant obtained from the cells. This incubation was done in pairs with or without simultaneous incubation with human IgG (1 mg / ml). After this incubation, the cells were incubated with anti-mouse IgG antibody labeled with FITC (ie, the presence of murine anti-FcRn antibody bound to human FcRn on the surface of 293 cells). To detect). Cells were washed and then cell-bound fluorescence was assessed with a fluorometer. All incubations were performed at pH 7.4.

  The results (FIG. 11) show significant binding of mouse IgG to 293 cells expressing hFcRn following cell incubation with culture supernatants from hybridoma cells (11E4 & 1H5 from Example 9). . These binding data indicate that co-incubation with human IgG did not lead to significant changes in the assay response, which is consistent with “non-competitive” binding (ie, anti-reactivity for hFcRn). The apparent affinity of the FcRn antibody was not changed by the presence of the natural ligand-human IgG).

  Also shown are the results from the incubation of 293 cells (ie, as a negative control) with supernatant from cells secreting murine monoclonal IgG1 antibody directed against methotrexate. Incubation of 293 cells with anti-methotrexate antibody did not lead to a significant assay response. This (also) is consistent with the assumption that certain anti-hFcRn antibodies are responsible for the significant binding observed following cell incubation with 11E4 & 1H5 supernatant.

[Example 12]
This example describes the production of monoclonal antibodies with specificity for human FcRn. A peptide sequence GEEFMNFDLKQGT (Invitrogen Corp., Carlsbad, Calif.), Selected from the primary sequence of human FcRn heavy chain, is combined with mussel hemocyanin (KLH) (Pierce Biotechnology Inc., Rockford, IL) and Freund's incomplete adjuvant ( Emulsified in Sigma Chemical) and then used to repeatedly immunize 6 Balb / c mice (Harlan, Indianapolis, IN). Animals were bled from the saphenous vein 7-10 days after immunization and antisera were evaluated for activity to inhibit binding of human IgG to cells expressing human FcRn using a cell binding assay. Briefly, serum samples from animals were incubated with 293 cells expressing human FcRn and with 50 μg / ml FITC-labeled human IgG at pH 6, 37 ° C. for 2 hours. The mixture was then centrifuged at 250 g for 5 minutes, and the cells were then washed with pH 6 PBS. After centrifugation, the cells were resuspended in pH 7.4 PBS. Fluorescence was assessed by fluorometry. Excitation and emission wavelengths were set at 494 nm and 520 nm, respectively. Animals associated with anti-sera showing the greatest inhibition of the fluorescent signal were selected for use as splenocyte donors. Spleen cells were harvested and fused with murine SP20 myeloma cells (ATCC, Manassas, VA). Briefly, mice were sacrificed with ketamine (150 mg / kg) and xylazine (30 mg / kg) and then the spleen was quickly removed using aseptic technique. Splenocytes are drawn from spleen tissue using a sterile 22-gauge needle, suspended in RPMI 1640, and centrifuged using polyethylene glycol by standard methods (Harlow E and Lane D. 1988. Antibody: Laboratory). Manual, New York: as described in Cold Spring Harbor Laboratory). Fusion cells were selected by application of HAT selection medium (Sigma Chemical) and then cloned by the method of limited dilution. Tissue culture supernatants were assayed for inhibition of binding of human IgG to 293 cells expressing human FcRn using techniques modified from those described above. Thirty-two viable hybridoma clones were identified and tissue culture supernatants were taken from each clone culture and probed for the presence of anti-human FcRn antibodies. Briefly, tissue culture supernatants from all viable clones were incubated with human FcRn-infected 293 cells and 30 μg / ml FITC-labeled human IgG at pH 6, 37 ° C. for 2 hours. After incubation, the cells were washed and resuspended in pH 7.4 PBS. Sample fluorescence was analyzed by fluorometry. From 32 viable potential anti-human FcRn clones, 2 positive clones were identified. These clones were 1D6 and 11C1 (Figure 13). One-way ANOVA revealed a significant difference in the assay response (p <0.0001) and the assay response for the two positive clones was found to be significantly different from that of the control (for each clone). p <0.01, Dunnett multiple comparison test). Furthermore, this assay revealed that the negative control AMI antibody, a murine IgG1 antibody with high affinity for methotrexate, does not inhibit the binding of human IgG to human FcRn-infected 293 cells.

[Example 13]
To test whether 1D6 or 11C1 binds non-competitively to human FcRn (hFcRn) (ie with respect to human immunoglobulin), culture supernatants from W8 (control), 1D6 and 11C1 were treated with hFcRn-infected The cells were incubated for 2 hours at pH 7.4 and 37 ° C., optionally in the presence of 1 mg / ml pooled human immunoglobulin (IVIG, Gamunex®, Bayer). After washing and centrifugation, 293 cells were incubated with 100 μg / ml FITC-labeled goat anti-mouse IgG at pH 7.4 and 37 ° C. for 1.5 hours. Cells were washed and centrifuged and then cell bound fluorescence was analyzed by fluorometry. The results are shown in FIG. 12 (n = 3).
The data show that high concentrations of human immunoglobulin do not inhibit the binding of hFcRn-infected cells to 1D6 or 11C1. Thus, these data support the assumption that both 1D6 and 11C1 bind to hFcRn in a non-competitive manner (with respect to pooled human IgG).

[Example 14]
This example describes the evaluation of 1D6 and 11C1 binding to human beta-2-microglobulin to further support that 1D6 and 11C1 are directed against the heavy chain of FcRn. To assess this, 96-well microplate wells were coated with human beta-2-microglobulin (b2m, 3 μg / ml in phosphate buffer, 250 μl). Plates were incubated overnight at 4 ° C. The plate was then washed with phosphate buffered saline and DDW. Cell supernatants from 11E4 (anti-human beta-2-microglobulin), 4C9 (anti-rat beta-2-microglobulin), 1D6 and 11C1 were added to the plate and incubated for 2 hours at room temperature. Plates were washed with PBS and DDW. Goat-anti-mouse IgG-alkaline phosphatase conjugate (1: 500 in phosphate buffer, 250 μl) was added to the plate and the plate was incubated for 1 hour at room temperature. Plates were washed with phosphate buffered saline and DDW. Paranitro-phenyl-phosphate (4 mg / ml, 200 μl) in DEA buffer was added to the plate and the absorbance at 405 nm was measured with a plate reader for 10 minutes.
The results are shown in Table 4 below.

[Example 15]
This example describes the construction and expression of mouse / human chimeric antibodies. Total RNA was prepared by SV total RNA isolation system (Promega) from pelleted hybridoma cells expressing 1D6 and 11C1 monoclonal antibodies. cDNA was synthesized using oligo dT primer and reverse transcriptase (Invitrogen). The variable regions of 1D6 heavy and light chains were first analyzed using Taq DNA polymerase with 35 cycles of PCR (1 cycle is 93 ° C for 1 minute, 45 ° C for 30 seconds, and 72 ° C for 1 minute). Amplified from strand cDNA. The variable region of the 11C1 heavy chain was first strand cDNA using Taq DNA polymerase, using 35 cycles of PCR (1 cycle is 93 ° C for 1 minute, 56 ° C for 30 seconds, and 72 ° C for 1 minute). Amplified from The variable region of the 11C1 light chain was extracted from the first strand cDNA using 35 cycles of PCR (1 cycle at 93 ° C for 1 minute, 60 ° C for 30 seconds, and 72 ° C for 1 minute) using Taq DNA polymerase. Amplified. All PCR products were purified and cloned into PCR2.1-TOPO vector.

The PCR2.1-TOPO vector containing the 1D6 and 11C1 variable regions was sequenced and the results were confirmed by alignment analysis. Baculovirus cassette vector, using pAC-K-CH _3, the heavy and light chain variable region genes were cloned. To clone the variable region pAC-K-CH ₃ vector was amplified by PCR using primers containing a heavy chain variable region XhoI and NheI cleavage site. The light chain variable region was amplified by PCR using primers containing SacI and HindIII cleavage sites. PAC-K-CH ₃ containing both heavy and light chain variable regions was sequenced to confirm insertion. Alignment analysis showed that the heavy and light chains are all coupled to the pAC-K-CH ₃ vector.

The pAC-K-CH ₃ and single stranded baculovirus DNA comprises the variable regions from both the heavy and light chains were co-transformed into Sf9 insect cells. Recombinant baculovirus was prepared by homologous recombination using the BaculoGold transfection kit (BD Biosciences) according to the manufacturer's instructions. Recombinant baculovirus was recovered from the supernatant of SF9 cell culture medium 7-8 days after transformation. The subsequent two rounds of amplification were performed to obtain a high titer recombinant virus.
Sf9 cells are infected with a recombinant virus expressing IgG antibodies, then grown in serum free medium (Orbigen) and in T75 flasks at 27 ° C., approximately 50-60% dead cells Incubated until observed.

  The descriptions of the specific embodiments are for illustrative purposes and are not intended to be limiting. From the teachings of the present invention, various modifications and changes can be made by those skilled in the art without departing from the spirit of the invention.

Effect of IVIG on the time course of 7E3-induced thrombocytopenia. Rats received 8 mg / kg 7E3 after receiving IVIG (or saline). Panel A shows individual untreated platelet counts for animals given saline (1), 0.4 g / kg IVIG (2), 1 g / kg IVIG (3) or 2 g / kg IVIG (4). Indicates time data. Panel B shows the average percent of initial platelet count data. Symbols indicate IVIG treatment groups (n = 4 rats / group): saline (●), 0.4 g / kg (■), 1 g / kg (▲), and 2 g / kg (♦). IVIG and 7E3 were administered intravenously and platelet counts were obtained using a Serdyne 1700 multi-parameter blood analyzer. Error bars indicate the standard deviation for the mean. IVIG attenuated the course of thrombocytopenia in a dose-dependent manner. The difference in treatment was statistically significant (p = 0.031). Pharmacokinetics of plasma 7E3 following IVIG treatment. Rats (3-4 per group) were intravenously administered IVIG (0-2 g / kg) followed by 7E3 (8 mg / kg). Panel A shows plasma 7E3 pharmacokinetic data for each animal given saline (1), 0.4 g / kg IVIG (2), 1 g / kg IVIG (3), or 2 g / kg IVIG (4). Indicates. Panel B shows mean plasma pharmacokinetic data for animals receiving 7E3 and IVIG. Treatment groups are shown below: brine (●), 0.4 g / kg (■), 1 g / kg (▲) and 2 g / kg (♦). The concentration of 7E3 was determined by ELISA. Error bars indicate the standard deviation for the mean concentration at each time point. IVIG treatment significantly increased the clearance of 7E3, calculated from the concentration versus time characteristics shown in this figure (p <0.001). IVIG does not bind directly to 7E3. 7E3 (or control IgG) and IVIG were combined in vitro, at a constant IVIG concentration (25 mg / m), and varying the 7E3 concentration (0-0.1 mg / ml). The positive control was mouse anti-human IgG. Samples were then added to microplates covered with anti-human IgG. Murine IgG binding was visualized using a secondary anti-mouse IgG-alkaline phosphatase conjugate. p-Nitrophenyl phosphate was added and the plate was read at 405 nm (kinetic assay over 10 minutes). The assay response to 7E3 was not different from the control (p = 0.164), but the positive control was significantly different from the control (p <0.001). Plasma AMI pharmacokinetics following IVIG treatment. Rats (3 per group) were given saline (●) or 2 g / kg (♦) IVIG intravenously followed by AMI (8 mg / kg). AMI concentration was determined by ELISA. Error bars indicate the standard deviation for the mean concentration at each time point. IVIG treatment significantly increased AMI clearance calculated from the concentration versus time characteristics shown in this figure (p <0.001). The effect of IVIG on antibody pharmacokinetics is not specific for 7E3. Effect of IVIG on 7E3-platelet binding as determined by flow cytometry. 7E3 was incubated with human platelets in the presence or absence of IVIG. The histogram plots platelet count versus relative fluorescence intensity. The lower panel shows the fluorescence histogram obtained for control mouse IgG incubated with platelets (median fluorescence intensity (MFI) was 1.3). The middle panel shows 7E3 incubated with platelets (MFI = 246) and the upper panel shows 7E3 incubated with platelets in the presence of IVIG (MFI = 284). No decrease in MFI was observed for 7E3 binding to platelets in the presence of IVIG. Effect of IVIG on 7E3 platelet binding curve. Since the 7E3 concentration increased in the presence (◯) or absence (▽) of IVIG, the total platelet concentration remained constant. Free (ie unbound) 7E3 concentration was determined by ELISA. The data was fit as described in the text. The lines show the best fit of the data set (continuous line = IVIG, break line = no IVIG) and are essentially superimposed. The parameters obtained from the fit (K _A and R _t ) were not significantly different. In the absence of IVIG, K _A is ^{4.9 ± 0.7 × 10 8 M -1} , R t was ^{7.5 ± 0.4 × 10 -8 M (} 55000 ± 3000GP / platelets). With IVIG, K _A is ^{5.5 ± 1.2 × 10 8 M -1} , R t was ^{7.6 ± 0.7 × 10 -8 M (} 56000 ± 5000GP / platelets). IVIG does not prevent 7E3 from binding to platelets. 7E3 pharmacokinetics after IVIG treatment in control and FcRn-deficient mice. Mice (3-5 per group) were intravenously administered IVIG (1 g / kg) followed by 7E3 (8 mg / kg). Treatment groups are designated as follows: 7E3 + saline (●) in control mice; 7E3 ± IVIG (■) in control mice; 7E3 + saline (◯) in knockout mice; and 7E3 + IVIG (□) in knockout mice. The 7E3 concentration was determined by ELISA. Error bars indicate the standard deviation for the mean concentration at each time point. IVIG treatment significantly increased 7E3 clearance in control mice (p <0.001) but not in FcRn-deficient mice. Changes in anti-platelet antibody pharmacokinetics following administration of anti-FcRn monoclonal antibody. Rats were intravenously administered with monoclonal anti-platelet antibody (7E3, 8 mg / kg) with or without pretreatment with monoclonal anti-FcRn antibody (4C9, 60 mg / kg). Black circles indicate 7E3 plasma concentrations observed in animals receiving only 7E3 (n = 4), red triangles pretreated with monoclonal anti-FcRn antibody (4.5 hours prior to administration of 7E3 7E shows plasma concentration observed in rats (administered). As shown, pretreatment with monoclonal anti-FcRn antibody led to a dramatic increase in antiplatelet antibody excretion (ie, 7E3 clearance increased to ~ 400%). The 7E3 concentration was determined by ELISA. Error bars indicate the standard deviation for the mean concentration at each time point. Pharmacokinetics of plasma AMI following various doses of 4C9. Rats (3-4 animals per group) were intravenously administered 4C9 (0-60 mg / kg) 4 hours prior to administration of AMI (8 mg / kg, intravenous). Blood samples were taken and then plasma samples were analyzed by ELISA for AMI concentrations. Treatment groups are designated as follows: : Brine (●), 3 mg / kg (■), 15 mg / kg (▲), 60 mg / kg (◆). Error bars indicate the standard deviation for the average AMI concentration at each time point. 15 and 60 mg / kg significantly increased AMI clearance compared to subjects (p <0.01). Reactivity of hybridoma supernatant to human FcRn. A hybridoma secreting an antibody against the light chain of hFcRn was prepared. Plates were coated with human FcRn light chain and incubated with supernatant from designated hybridomas. Positive clones were identified using goat anti-mouse Fab fragments conjugated to alkaline phosphatase. Eight hybridomas producing antibodies specific for the human FcRn light chain were identified. Effect of the presence of IgG on the reactivity of anti-hFcRn to FcRn. 293 cells expressing hGcRn were incubated with anti-FcRn antibody, optionally with human IgG. Binding was detected with a secondary antibody conjugated to FITC. Cell fluorescence was assessed with a fluorometer. Effect of the presence of IgG on the reactivity of anti-hFcRn (heavy chain) to FcRn. 293 cells expressing hFcRn were incubated with anti-hFcRn (heavy chain) antibody, optionally with human IgG. Binding was detected with a secondary antibody conjugated to FITC. Cell fluorescence was assessed with a fluorometer. The binding of human IgG to 293 cells expressing human FcRn is reflected by sample fluorescence (as shown in the y-axis). Tissue culture supernatants obtained from hybridoma clones 1D6 and 11C1 showed highly significant inhibition of human IgG binding to 293 cells at pH 6.0.

Claims (20)

Human FcRn is a human FcRn heavy chain or fragment thereof, which functions as a non-competitive inhibitor of IgG binding to human FcRn and does not bind to b2-microglobulin. An antibody or fragment thereof that binds. 2. The antibody of claim 1, wherein the antibody is a murine antibody. 2. The antibody of claim 1, wherein the antibody is selected from the group consisting of polyclonal and monoclonal. 2. The antibody of claim 1, wherein the antibody is chimeric or humanized. The fragment according to claim 1, wherein the fragment is selected from the group consisting of Fab, F (ab) ′ ₂ , Fv and ScFv. 7. The antibody of claim 6, wherein binding to FcRn is pH independent throughout the pH range of 6.0 to 8.0. 4. The antibody of claim 3, wherein the antibody is a monoclonal antibody. The antibody according to claim 7, wherein the antibody is a monoclonal antibody produced by a hybridoma selected from the group consisting of 1DG and 11C1. A method for reducing the concentration of pathogenic antibodies in an individual comprising the step of administering to the individual a therapeutically effective dose of the antibody or fragment thereof according to claim 1. The method of claim 9, wherein the antibody is a polyclonal or monoclonal antibody. 11. The method of claim 10, wherein the fragment of the antibody is selected from the group consisting of Fab, F (ab) ′ ₂ , Fv and ScFv. 10. The method of claim 9, wherein the antibody or fragment thereof is administered in a pharmaceutically acceptable carrier. The method of claim 9, wherein the individual is a human. 10. The method of claim 9, wherein the antibody is administered with an adjuvant. A method for reducing IgG binding to FcRn in an individual comprising:
Providing an antibody or fragment thereof that binds to human FcRn that is made against the heavy chain of human FcRn or a fragment thereof, is a non-competitive inhibitor of IgG binding to human FcRn, and does not bind to b2-microglobulin ;and,
Administering the antibody or fragment thereof to an individual in an amount sufficient to inhibit binding of IgG to FcRn in the individual. 16. The method of claim 15, wherein the individual has an autoimmune or alloimmune disease. 17. The method of claim 16, wherein the autoimmune disease is immune thrombocytopenia. The method of claim 17, wherein the individual is a human. 16. The method of claim 15, wherein the antibody is administered at a dose of 1 mg / kg to 2 g / kg. 20. The method of claim 19, wherein the antibody is administered at a dosage of 1 mg / kg to 200 mg / kg.

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