Immunosuppressive Antibodies

IMMUNOSUPPRESSIVE ANTIBODIES

The development of hybridoma technology by Milstein and Kohler in 1975 revolutionized the antibody field and radically increased the purity and specificity of antibodies used in the clinic and for diagnostic tests in the laboratory. Hybridomas consist of antibody-forming cells fused to immortal plasmacytoma cells. Hybrid cells that are stable and produce the required antibody can be subcloned for mass culture for antibody production. Large-scale fermentation facilities are now used for this purpose in the pharmaceutical industry.

More recently, molecular biology has been used to develop monoclonal antibodies. Combinatorial libraries of cDNAs encod-ing immunoglobulin heavy and light chains expressed on bacterio-phage surfaces are screened against purified antigens. The result is an antibody fragment with specificity and high affinity for the antigen of interest. This technique has been used to develop anti-bodies specific for viruses (eg, HIV), bacterial proteins, tumor antigens, and even cytokines. Several antibodies developed in this manner are FDA-approved for use in humans.

Other genetic engineering techniques involve production of chimeric and humanized versions of murine monoclonal antibod-ies in order to reduce their antigenicity and increase the half-life of the antibody in the patient. Murine antibodies administered as such to human patients elicit production of human antimouse antibodies (HAMAs), which clear the original murine proteins very rapidly. Humanization involves replacing most of the murine antibody with equivalent human regions while keeping only the variable, antigen-specific regions intact. Chimeric mouse-human antibodies have similar properties with less complete replacement of the murine components. The current naming convention for these engineered substances uses the suffix “-umab” or “-zumab” for humanized antibodies, and “-imab” or “-ximab” for chimeric products. These procedures have been successful in reducing or preventing HAMA production for many of the antibodies dis-cussed below.

Antilymphocyte & Antithymocyte Antibodies

Antisera directed against lymphocytes have been prepared spo-radically for over 100 years. With the advent of human organ transplantation as a therapeutic option, heterologous antilympho-cyte globulin (ALG) took on new importance. ALG and antithy-mocyte globulin (ATG) are now in clinical use in many medical centers, especially in transplantation programs. The antiserum is usually obtained by immunization of horses, sheep, or rabbits with human lymphoid cells.

ALG acts primarily on the small, long-lived peripheral lym-phocytes that circulate between the blood and lymph. With con-tinued administration, “thymus-dependent” (T) lymphocytes from lymphoid follicles are also depleted, as they normally par-ticipate in the recirculating pool. As a result of the destruction or inactivation of T cells, an impairment of delayed hypersensitivity and cellular immunity occurs while humoral antibody formation remains relatively intact. ALG and ATG are useful for suppressingcertain major compartments (ie, T cells) of the immune system and play a definite role in the management of solid organ and bone marrow transplantation.

Monoclonal antibodies directed against specific cell surface and soluble proteins such as CD3, CD4, CD25, CD40, IL-2 receptor, and TNF-α (discussed below) much more selectively influence T-cell subset function. The high specificity of these antibodies improves selectivity and reduces toxicity of the therapy and alters the disease course in several different autoimmune disorders.

In the management of transplants, ALG and monoclonal anti-bodies can be used in the induction of immunosuppression, in the treatment of initial rejection, and in the treatment of steroid-resistant rejection. There has been some success in the use of ALG and ATG plus cyclosporine to prepare recipients for bone marrow transplantation. In this procedure, the recipient is treated with ALG or ATG in large doses for 7–10 days prior to transplantation of bone marrow cells from the donor. ALG appears to destroy the T cells in the donor marrow graft, and the probability of severe graft-versus-host syndrome is reduced.

The adverse effects of ALG are mostly those associated with injection of a foreign protein. Local pain and erythema often occur at the injection site (type III hypersensitivity). Since the humoral antibody mechanism remains active, skin-reactive and precipitating antibodies may be formed against the foreign IgG. Similar reactions occur with monoclonal antibodies of murine origin, and reactions thought to be caused by the release of cytok-ines by T cells and monocytes have also been described.

Anaphylactic and serum sickness reactions to ALG and murine monoclonal antibodies have been observed and usually require ces-sation of therapy. Complexes of host antibodies with horse ALG may precipitate and localize in the glomeruli of the kidneys. The incidence of lymphoma as well as other forms of cancer is increased in kidney transplant patients. It appears likely that part of the increased risk of cancer is related to the suppression of a normally potent defense system against oncogenic viruses or transformed cells. The preponderance of lymphoma in these cancer cases is thought to be related to the concurrence of chronic immune sup-pression with chronic low-level lymphocyte proliferation.

Muromonab-CD3

Monoclonal antibodies against T-cell surface proteins are increas-ingly being used in the clinic for autoimmune disorders and in transplantation settings. Clinical studies have shown that the murine monoclonal antibody muromonab-CD3 (OKT3) directed against the CD3 molecule on the surface of human T cells can be useful in the treatment of renal transplant rejection. In vitro, muromonab CD3 blocks killing by cytotoxic human T cells and several other T-cell functions. In a prospective randomized multi-center trial with cadaveric renal transplants, use of muromonab-CD3 (along with lower doses of steroids or other immunosuppressive drugs) proved more effective at reversing acute rejection than did conventional steroid treatment. Muromonab-CD3 is approved for the treatment of acute renal allograft rejection and steroid-resistant acute cardiac and hepatic transplant rejection. Many other mono-clonal antibodies directed against surface markers on lymphocytesare approved for certain indications (see monoclonal antibody sec-tion below), while others are in various stages of development.

Immune Globulin Intravenous (IGIV)

A different approach to immunomodulation is the intravenous use of polyclonal human immunoglobulin. This immunoglobulin preparation (usually IgG) is prepared from pools of thousands of healthy donors, and no single, specific antigen is the target of the “therapeutic antibody.” Rather, one expects that the pool of differ-ent antibodies will have a normalizing effect upon the patient’s immune networks.IGIV in high doses (2 g/kg) has proved effective in a variety of different applications ranging from immunoglobulin deficiencies to autoimmune disorders to HIV disease to bone marrow trans-plantation. In patients with Kawasaki’s disease, it has been shown to be safe and effective, reducing systemic inflammation and pre-venting coronary artery aneurysms. It has also brought about good clinical responses in systemic lupus erythematosus and refractory idiopathic thrombocytopenic purpura. Possible mechanisms of action of IGIV include a reduction of T helper cells, increase of regulatory T cells, decreased spontaneous immunoglobulin pro-duction, Fc receptor blockade, increased antibody catabolism, and idiotypic-anti-idiotypic interactions with “pathologic antibodies.” Although its precise mechanism of action is still unknown, IGIV brings undeniable clinical benefit to many patients with a variety of immune syndromes.

Rh_o(D) Immune Globulin Micro-Dose

One of the earliest major advances in immunopharmacology was the development of a technique for preventing Rh hemolytic disease of the newborn. The technique is based on the observation that a primary antibody response to a foreign antigen can be blocked ifspecific antibody to that antigen is administered passively at the time of exposure to antigen. Rh_o(D) immune globulin is a concen-trated (15%) solution of human IgG containing a higher titer of antibodies against the Rh_o(D) antigen of the red cell.

Sensitization of Rh-negative mothers to the D antigen occurs usually at the time of birth of an Rh_o(D)-positive or D^u-positive infant, when fetal red cells leak into the mother’s bloodstream. Sensitization might also occur occasionally with miscarriages or ectopic pregnancies. In subsequent pregnancies, maternal anti-body against Rh-positive cells is transferred to the fetus during the third trimester, leading to the development of erythroblastosis fetalis (hemolytic disease of the newborn).

If an injection of Rh_o(D) antibody is administered to the mother within 24–72 hours after the birth of an Rh-positive infant, the mother’s own antibody response to the foreign Rh_o(D)-positive cells is suppressed because the infant’s red cells are cleared from circulation before the mother can generate a B-cell response against Rh_o(D). Therefore she has no memory B cells that can activate upon subse-quent pregnancies with an Rh_o(D)-positive fetus.

When the mother has been treated in this fashion, Rh hemo-lytic disease of the newborn has not been observed in subsequent pregnancies. For this prophylactic treatment to be successful, the mother must be Rh_o(D)-negative and D^u-negative and must not already be immunized to the Rh_o(D) factor. Treatment is also often advised for Rh-negative mothers antepartum at 26–28 weeks’ gestation who have had miscarriages, ectopic pregnancies, or abortions, when the blood type of the fetus is unknown. Note:Rh_o(D) immune globulin is administered to the mother and must not be given to the infant.

The usual dose of Rh_o(D) immune globulin is 2 mL intramus-cularly, containing approximately 300 mcg anti-Rh_o(D) IgG. Adverse reactions are infrequent and consist of local discomfort at the injection site or, rarely, a slight temperature elevation.

Hyperimmune Immunoglobulins

Hyperimmune immunoglobulins are IGIV preparations made from pools of selected human or animal donors with high titers of antibodies against particular agents of interest such as viruses or toxins (see also Appendix I). Various hyperimmune IGIVs are available for treatment of respiratory syncytial virus, cytomegalo-virus, varicella zoster, human herpesvirus 3, hepatitis B virus, rabies, tetanus, and digoxin overdose. Intravenous administration of the hyperimmune globulins is a passive transfer of high titer antibodies that either reduces risk or reduces the severity of infec-tion. Rabies hyperimmune globulin is injected around the wound and given intravenously. Tetanus hyperimmune globulin is admin-istered intravenously when indicated for prophylaxis. Rattlesnake and coral snake hyperimmune globulins (antivenoms) are of equine or ovine origin and are effective for North and South American rattlesnakes and some coral snakes (but not Arizona coral snake). Equine and ovine antivenoms are available for rattle-snake envenomations, but only equine antivenom is available for coral snake bite. The ovine antivenom is a Fab preparation and is less immunogenic than whole equine IgG antivenoms, but retains the ability to neutralize the rattlesnake venom.