Serum Sickness

Serum sickness is caused by the administration of large amounts of any foreign protein. Within 7 to 10 days after administration, an immunoglobulin M (IgM) antibody response to the foreign protein develops. When equal molar concentrations of antigens and antibodies are generated, large complexes settle out in the internal elastic lamina of arteries and perivascular regions. Activation of the complement cascade and the release of cytotoxic factors from endothelial cells cause the necrosis of the capillary vessels in the skin and kidneys.

Patients develop clinical manifestations of fever, arthralgia, lymphadenopathy, and skin eruptions. Serious side effects also have been reported as a consequence of antibody therapy. High concentrations of infused antibody (0.5 to 2.0 mg/kg) can cause renal failure, aseptic meningitis, and thrombosis.

Idiopathic Thrombocytopenic Purpura

ITP is a pediatric disease that is associated with low platelet counts. Children often present with bruises all over the body, bleeding gums, and, occasionally, a bleeding nose. Patients with persistently low platelet counts are also at high risk of intracranial hemorrhage.

ITP is an autoimmune disease characterized by antibody-coated platelets. In most cases, coated platelets bind to Fc receptors on macrophages in the spleen and liver. Following phagocytosis, platelets are destroyed by the oxidative killing mechanism. In a small proportion of patients, the liver fails to remove the platelets, and circulating antibody–platelet immune complexes are created. Complexes localize in arterioles and capillaries, occluding vessels and causing tissue ischemia. Vessels in the heart, kidney, brain, pancreas, and spleen are common targets for immune complex deposition.

Kawasaki Disease

Kawasaki disease is a self-limiting, febrile disease that is characterized by vasculitis of peripheral and coronary vessels. Patients present with a persistent rash, conjunctivitis, and lymphadenopathy. Individuals with Kawasaki disease have a high risk of heart failure, pericarditis, mitral or aortic valve insufficiency, and rupture of the coronary artery. In the last 5 years, Kawasaki disease has emerged as the leading cause of heart disease in the United States. Although the etiology of the disease is unclear, an immune response to a viral (parvovirus B19 or cytomegalovirus) or bacterial (Yersinia) infection is believed to activate an autoimmune response to vascular endothelial cells and cardiac myosin.

Erythroblastosis Fetalis

Limitations of Pooled Human Immunoglobulin

Available IVIG products differ widely in immunoglobulin content, the affinity and avidity of antibodies, and IgG subclass distribution. This may reduce the efficacy of the product when used for immunomodulation. Moreover, pooled immunoglobulin is a finite reagent and new lots must be prepared every few years. Variation in efficacy between different batches of the same IVIG lot also occurs. Variations may be caused by differences in the concentrations or the sizes of microaggregates, which reduce the bioavailable antibody concentration.

Safety concerns about the use IVIG do exist. A small percentage of individuals manifest infusion-related adverse effects, including nausea, headache, backache, and muscle pain. Individuals with high levels of anti-IgA antibodies have a high risk of life-threatening anaphylaxis.

Disease transmission is always a concern when blood products are used for therapy. Transmission of HIV, hepatitis B, or Creutzfeldt-Jakob syndrome prions has not been reported. However, several cases of IVIG-transmitted hepatitis C were reported before 1994. Additional antiviral measures included in the manufacturing process have reduced the possibility of hepatitis C transmission.

HAT (Hypoxanthine–Aminopterin–Thymidine) Selection

Selection takes advantage of different nucleic acid synthesis pathways. The classic de novo pathway is a complex pathway that produces folic acid. In turn, folic acid serves as a single carbon methyl donor in the synthesis of nucleotides. The nucleic acid salvage pathway provides a second conduit for DNA synthesis. In this pathway, thymidine and hypoxanthine are recycled into new nucleotides. Recycling requires the presence of an enzyme called hypoxanthine-guanine phosphorylribosyl transferase (HPRT+).

Selection of B cell–myeloma cell hybrids is facilitated by the use of medium that contains hypoxanthine–aminopterin, and thymidine (HAT). Murine B cells have both classic and salvage pathways. As a consequence of several mutational events in the myeloma cells, the HPRT gene is inactivated, and they can only synthesize DNA by the classic pathway. In the HAT selection process, aminopterin (a folic acid inhibitor) blocks the production of DNA by the classic de novo pathway and kills the myeloma cells.

Since B cell–myeloma cell hybrids have both classic and de novo synthesis pathways, they survive the selection process. The surviving cells are known as hybridomas.

Chimeric Antibodies

To produce chimeric antibodies, murine hybridomas are produced by the classic method described above. The genes coding for the murine variable region of heavy and light chains are isolated from the hybridoma and amplified by polymerase chain reaction (PCR). Constant-region genes of human heavy and light chains also are amplified. After insertion of human and murine genes into a plasmid and transfection into bacteria, chimeric antibodies are produced as inclusion bodies. Following lysis of the cells, the chimeric antibodies are purified for in vivo use. Antibody aggregation and limited growth of antibody-producing bacteria restrict the usefulness of bacterial expression systems.

Humanized Antibodies

Humanized monoclonal antibodies are derived from murine hybridomas. However, genes coding for the six murine CDRs (three each on heavy and light chains) are amplified by PCR and inserted into a plasmid vector. Genes for human variable and constant regions also are inserted into the same vector. Transfected bacteria or transformed human B cells produce humanized monoclonal antibodies.

Human Monoclonal Antibodies

It has now become possible to produce human monoclonal antibodies in transgenic mice. In these animals, the murine immune system has been inactivated and replaced by megabase-sized yeast artificial chromosome (YAC) carrying the genes for IgG1–IgG4. Repeated stimulation of the mice produces a robust response of antibodies with high affinity for antigens. B cells isolated from the draining lymph nodes or spleen are fused with myeloma cells. Following HAT selection and hybridoma expansion in 96-well microtiter plates, supernatant fluids are tested for antibody production. These laboratory-created human monoclonal antibodies have high affinity for antigens and have elimination kinetics that are identical to natural antibodies.

Fab Fragments

Using molecular biology techniques, it is possible to construct monoclonal antibody fragments that consist solely of Fab or F(ab′)₂Abciximab, a chimeric Fab fragment, is commonly used to inhibit platelet clumping after angioplasty. Afelimomab, which is undergoing clinical trials for the treatment of sepsis or septic shock, is composed of F(ab′)₂ antibody fragments.

Bi-Specific Antibodies

Bi-specific antibodies can be generated by genetic engineering and the fusion of two antibody-producing hybridomas to form a quadroma. Antibodies produced by the quadroma have a normal Y-shaped IgG configuration. Bi-specific antibodies combine heavy and light chains of one monoclonal antibody with one specificity with the heavy and light chains of another monoclonal antibody with a different specificity. Therefore, a single antibody can react with two different antigens because bi-specific antibodies have two different sets of CDRs.

Bi-Functional Antibodies

Antibodies or antibody fragments can be constructed in a number of different formats by using recombinant DNA technology. Tetravalent antibodies are the most common format used in cancer immunotherapy. Two antibodies react with target molecules on the cancer cell surface, while the other antibodies react with cytotoxic T cells or natural killer (NK) cells. Antibody-mediated cross-linkage of cytotoxic cell receptors activates cytotoxic T and NK cells that lyse tumor cells.

Flex antibodies are a unique subset of tetravalent antibodies that have linker segments between the antigen-combining sites. Because of their increased flexibility, they can react with antigens that are distant from one another on the same cell.

Single-Chain Fab Variable Fragments and Phage Display