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
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.
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.
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.
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.
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.
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.
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.
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.
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.
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. Rho(D)
immune globulin is a concen-trated (15%) solution of human IgG containing a
higher titer of antibodies against the Rho(D)
antigen of the red cell.
of Rh-negative mothers to the D antigen occurs usually at the time of birth of
an Rho(D)-positive or Du-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 Rho(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 Rho(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 Rho(D). Therefore she has
no memory B cells that can activate upon subse-quent pregnancies with an Rho(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 Rho(D)-negative and Du-negative and must not already be immunized to the Rho(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:Rho(D) immune globulin is administered to the mother and must not
be given to the infant.
The usual dose of Rho(D) immune globulin is
2 mL intramus-cularly, containing approximately 300 mcg anti-Rho(D) IgG. Adverse
reactions are infrequent and consist of local discomfort at the injection site
or, rarely, a slight temperature elevation.
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.