Drug – bio-affecting and body treating compositions – Immunoglobulin – antiserum – antibody – or antibody fragment,... – Binds hormone or other secreted growth regulatory factor,...
Reexamination Certificate
1995-03-10
2001-02-27
Burke, Julie (Department: 1642)
Drug, bio-affecting and body treating compositions
Immunoglobulin, antiserum, antibody, or antibody fragment,...
Binds hormone or other secreted growth regulatory factor,...
C424S130100, C424S133100, C424S156100, C424S145100, C530S387100, C530S388240, C530S388850, C530S389100, C530S389200, C530S389700
Reexamination Certificate
active
06193969
ABSTRACT:
The present invention relates to the use of immunoglobulin Fab fragments in therapy.
Antibodies are formed as part of the immune response to a microorganism or foreign macromolecule. They are immunoglobulins (Ig) and are used extensively in clinical practice for the diagnosis, monitoring, prevention and treatment of an increasing number of diseases.
The basic unit from which all antibody molecules are formed was elucidated by Porter (1959)
Biochem J.
73, 119-126, using specific proteolytic enzymes. The most important of the immunoglobulins, IgG, comprises two heavy and two light chains with the former being coupled at their hinge region by disulphide linkages. Cleavage with papain above these linkages releases two antibody binding fragments (Fab) and a crystalline fragment (Fc) as shown in FIG.
1
. Cleavage with pepsin, below the hinge results in a somewhat smaller Fc fragment and a single F(ab′)
2
fragment with two binding sites as shown in FIG.
1
. Each Fab fragment contains both a light chain and part of a heavy chain, and includes the sequences responsible for specific binding to a microorganism or foreign macromolecule. The Fc consists of the remainder of the two heavy chains; this is the site to which complement, macrophages and polymorphonuclear white blood cells can bind. The two heavy chains (but not the light chains) are different for each class of antibody ie IgG, IgM, IgA and IgE. IgG is the dominant circulating immunoglobulin in terms of concentration. It consists of a single basic immunoglobulin unit and, characteristically, has a high affinity for its specific antigen. Further details of antibody structure and function are disclosed in Roitt (1991)
Essential Immunology,
7th Edition, Blackwell Scientific Publications, Oxford.
Microbial pathogens can cause deleterious effects by releasing soluble toxins. These include the neurogenic exotoxins released by diphtheria and tetanus bacilli and various endotoxins such as lipopolysaccharide (LPS) from the cell walls of Gram-negative bacteria and peptidoglycans from Gram-positive organisms. In 1890 von Behring showed that exogenous antibodies to soluble antigens were of therapeutic value: the mortality of children with diphtheria was reduced by systemic administration of serum from horses hyperimmunised with diphtheria toxoid. A similar approach was successful in patients with tetanus. Passive immunisation was quickly extended to the victims of snake envenomation by Calmette in 1894, and by others.
The development of the septic shock syndrome involves initiators (such as LPS), mediators (including TNF&agr;, IL-1 and IL-6) and effectors at the cellular level (eg nitric oxide synthase in endothelial cells). An the initiators and mediators are potential antigens; antibodies against these macromolecules can be used for the prevention and treatment of septic shock. Several groups have used polyclonal (PcAb) or monoclonal antibodies (McAb) directed against LPS with varying degrees of success. At the same time, it has been shown that PcAb to TNF&agr; can prevent the lethal effects of this cytokine (Beutler et al (1985)
Science
229, 869-871) in BALB/C mice. Tracey and colleagues ((1987)
Nature
330, 662-664) have shown that McAb against TNF&agr; given one hour before bacterial challenge in baboons afforded partial protection against organ damage and, when given two hours before, more complete protection. In other words the anti-TNF&agr; McAb was used prophylactically.
A few groups have raised PcAb to LPS and to TNF, usually in rabbits, and demonstrated their effectiveness in animal models of septic shock. PcAb to LPS have also been raised in human volunteers and used successfully (Ziegler et al (1982)
New Engl. J. Med.
307, 1225-1230). Although the use of human antibodies avoids the risk of allergic complications, widespread application is precluded by ethical, logistic and other reasons including potential for viral contamination (HIV and hepatitis). Therefore most groups have concentrated their efforts on the production of McAB. McAb offer many advantages, for example homogeneity and the relative simplicity of down-stream processing—often by affinity chromatography with protein A or protein G to separate antibodies from other proteins.
No group involved in developing treatments for septic shock have prepared or used specific Fab fragments. No group has demonstrated the use of anti-TNF Fab fragments in treating humans suffering from septic shock after clinical manifestations of the shock have become apparent.
SUMMARY OF THE INVENTION
We have now demonstrated in humans that administration of Fab fragment reactive towards TNF&agr; benefits patients suffering from “septic shock” symptoms.
We have unexpectedly found that Fab fragments, derived from polyclonal antibodies directed towards TNF&agr;, are more effective at reducing the effects of TNF than are intact IgG directed towards TNF&agr; as disclosed in more detail in the Examples.
Thus, one aspect of the invention provides a method of neutralising TNF&agr; in a patient benefiting from such neutralising, comprising administering to the patient Fab fragment reactive towards TNF&agr;.
Suitably, the patient is suffering from septic shock or from the symptoms of septic shock. Thus, a further aspect of the invention provides a method of preventing or ameliorating septic shock or the symptoms of septic shock in a patient comprising administering to the patient IgG Fab fragments reactive towards TNF&agr;.
The Fab fragments may be generated from substantially pure IgG reactive towards TNF&agr; by methods well known in the art and disclosed in the Examples.
A further aspect of the invention provides IgG Fab fragments reactive towards TNF&agr; for use in medicine.
A still further aspect of the invention provides use of an IgG Fab fragment reactive towards TNF&agr; in the manufacture of a medicament for treating patients benefiting from neutralisation of TNF&agr;.
It is further preferred if the IgG Fab fragments are derived from polyclonal antiserum. Polyclonal antiserum (which includes polyclonal IgG) can be produced by immunizing a sheep, goat, horse or other mammal. It is preferable if the mammal is a sheep, and that it is free of scrapie and zoonotic viruses. Methods of making Fab fragments reactive towards TNF&agr; and derived from polyclonal sheep IgG are described in the Examples.
Fab fragments reactive towards TNF&agr; are useful in the treatment of medical conditions characterised by an increase in the level of circulating TNF&agr;. Such conditions include shock, for example septic shock, and excess TNF&agr; in the context of tumour therapy. Septic shock may occur following bacterial infection, particularly infection with Gram-negative bacteria, and during septicaemia. By shock we also include trauma following an accident or surgery. Further uses include treatment in conjunction with anti-lymphocyte antibody therapy, as taught in WO 89/08460, and in conjunction with cancer chemotherapy, as taught in EP 355 067.
It is known that intravenous infusions of laprine or equine polyclonal antibodies directed against human lymphocytes are very effective in helping to treat acute renal allograft rejection. Such products are still in use. Following clinical trials, Ortho Pharmaceutical Co was granted a licence by the FDA in 1986 for the routine use of a murine monoclonal antibody, OKT3, to help to prevent graft rejection following renal transplant. This product has proved extremely effective in the treatment of acute rejection episodes following kidney, liver and heart transplants and OKT3 remains the only monoclonal-based therapeutic product to have been granted a licence.
OKT3 binds specifically to the CD-3 complex found on all mature T lymphocytes. CD-3 is normally involved in antigen recognition and cell stimulation and both are prevented, due to steric hindrance, by the presence of the murine antibodies.
The hybridoma expressing the monoclonal antibody OKT3 is available from the America Type Culture Collection, 12301 Parklawn Drive, Rockville, Md. 20852 U.S.A. under ac
Burke Julie
Finnegan Henderson Farabow Garrett & Dunner L.L.P.
Helms Larry R
Protherics Inc.
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