Hybridoma Monoclonal Antibody Techniques

In a humoral immune response, B-lymphocyte-derived plasma cells produce antibodies with variations in chemical structure. Biologically, these variations extend the utility of the secreted antibody. These variations are caused by affinity maturation, the tendency for the affinity of antibody for antigen to increase with each challenge, and mutation at the time of somatic recombination. These phenomena produce antibodies with slightly different specificities. Because the clones of antibody-producing cells provide more than one structural type of antibody, they are called polyclonal antibodies. Another type of antibody consists of highly homogeneous populations of hybrid proteins produced by one clone of specially prepared B lymphocytes. These antibodies, lacking structural variations, are highly "focused" on their antigenic counterparts' determinants or epitopes, and are called monoclonal.

A problem with creating MAbs is that one cannot simply prepare an antibody-producing B lymphocyte and propagate it. Such cells live only briefly in the laboratory environment. Instead, antibody-producing cells are fused with an immortal (tumor) cell line to create hybridomas—long-lived, antibody-secreting cells. The trick is to select the monoclonal cells that produce the desired antibody. The hybridoma technique has opened the door to new therapeutic antibodies, imaging agents, radiological diagnostic test kits, targeted ra-dionuclide delivery agents, and home test kits.

In the hybridoma method (Fig. 4.14), a mouse or other small animal is sensitized with an antigen. When a high enough titer of antibody against the selected antigen has been attained, the animal is sacrificed and its spleen cells are collected. The spleen cells contain a large number of B lymphocytes, and it is certain that some will be able to produce antigen-specific antibodies. Because the spleen cells are normal B lymphocytes, they have a very short lifetime in cell cultures. Therefore, a method must be used to extend their lifetime.

To produce MAbs, B cells are fused with immortal myeloma cells in the presence of fusogens such as PEG. This procedure produces genetically half-normal and half-myeloma cells. Because the myeloma cells are immortal, the longevity problem is solved. The selection process depends on two different myeloma cell lines: one lacking the enzyme hypoxanthine-guanine phosphoribosyl transferase (HGPRT), a key enzyme in the nucleotide salvage pathways, and the other lacking the Tk gene, a key gene in the pyrimidine biosynthetic pathways. The spleen B cells are HGPRT and Tk (+), whereas the myeloma cells are HGPRT and Tk (—). This myeloma cell line cannot survive in a medium containing aminopterin, a thymidylate synthetase inhibitor, because it cannot synthesize pyrimidines. The HGPRT (—) cell line cannot use the purine salvage pathways to make nucleotides, forcing it to use thymidylate synthetase. With thymidylate synthetase inhibited, the cell dies. After fusion, cells are maintained on a medium containing hypoxanthine, aminopterin, and thymidine (HAT). Only cells that are "correctly" fused between one spleen cell (HGPRT [+ ]) and one myeloma cell (immortal), that is, a hybridoma, can survive in HAT medium. Fused myeloma cells (myeloma-myeloma) lack the correct genes and cannot survive. Fused spleen cells (spleen-spleen) cannot grow in culture. Thus, only the fused hybridoma (myeloma-spleen) survives. Hypoxanthine and thymidine furnish precursors for the growth of HGPRT (+) cells. Aminopterin suppresses cells that failed to fuse. Hybridomas can be isolated in a 96-well plate and transferred into larger cultures for proliferation. The culture medium will eventually contain a high concentration of MAb against the original antigen. This antibody can be purified to homogeneity.

MAbs, being proteins, tend to be highly immunogenic in humans. This is especially true of the MAbs produced in mouse culture. Humans begin to develop antibodies to

Figure 4.14 • General method for the preparation of monoclonal antibodies, using hybridomas and HAT medium. (Ab, antibody; Ag, antigen.)

Figure 4.14 • General method for the preparation of monoclonal antibodies, using hybridomas and HAT medium. (Ab, antibody; Ag, antigen.)

mouse MAbs after a single dose. This is natural. The human host is mounting an antibody response to a foreign antigen. The human antimouse antibody (known as HAMA) response has tended to limit the use of monoclonals in human therapy.

In developing a method for making MAbs useful in humans, it is necessary to remove the mouse immunogenic characteristics from the MAb. The antigen-recognition region (Fab) of the MAb must retain its ability to bind to the antigen, however. If this feature is altered, the antibody will likely be useless. Within the light and heavy chains of the Fab portions of antibody molecules are regions that are called complementarity-determining regions or CDRs. Each chain possesses three of these. One of the CDRs, CDR3, is located at the juncture of the variable and common domains. CDR3 is also referred to as the hypervariable region because most of the variability of the antibody molecule is concentrated there. These must be intact for specific antigen-antibody binding. Immune responses against murine MAb are directed against not only the variable regions, but also the constant regions. Hence, to decrease the immunogenicity of an MAb one must create antibodies that have been "humanized." In MAb production, usually the VH and VL domains of a human antibody are replaced by the corresponding regions from the mouse antibody, leaving the specificity intact, but using human constant regions that should not be immunogenic. Antibodies like these are called chimeric, and they are less immunogenic and have a longer half-life in human patients. Examples of chimeric MAbs are abciximab, rituximab, infliximab, and basiliximab.

Methods are available for the development of MAbs with 95% to 100% human sequence. By using transgenic mice, all of the essential human antibody genes can be expressed.

Monoclonal Antibody Drugs


Rituximab (Rituxan, Chimeric) is an MAb directed against the CD20 antigen expressed on the surfaces of normal and malignant B lymphocytes. The MAb is produced in mammalian (CHO) suspension culture and is a chimeric (murine/human) MAb of the IgG1 k type. The protein is composed of murine light and heavy chain variable regions and human constant regions. Rituximab is indicated for the treatment of patients with relapsed or refractory, low-grade or follicular, CD20(+) B-cell non-Hodgkin lymphoma. Rituximab binds specifically to antigen CD20 (human B-lymphocyte-restricted differentiation antigen, a hydropho-bic transmembrane protein expressed on pre-B and mature B lymphocytes). CD20 is a protein of 35 to 37 kDa, and it may play a role in B cell activation and regulation and may be a calcium ion channel. The antigen is also expressed on more than 90% of non-Hodgkin lymphoma B cells but is not found on hematopoietic stem cells, pro-B cells, normal plasma cells, or other normal tissues. CD20 regulates the early steps in the activation process for cell cycle initiation and differentiation.


Gemtuzumab ozogamicin (Mylotarg, fusion molecule) is an MAb derived from the CD33 antigen, a sialic acid-dependent adhesion protein expressed on the surface of leukemia blasts and immature normal cells of myelomono-cytic origin but not on normal hematopoietic stem cells. CD33 binds sialic acid and appears to regulate signaling in myeloid cells. The antibody is recombinant, humanized IgG4 k, linked with the cytotoxic antitumor antibiotic ozogamicin (from the calicheamicin family). More than 98.3% of the amino acids of gemtuzumab are of human origin. The constant region of the MAb contains human sequences, whereas the CDRs derive from a murine antibody that binds CD33. The antibody is linked to N-acetyl-y-calicheamicin via a bifunctional linker.

Gemtuzumab ozogamicin is indicated for the treatment of patients with CD33-positive acute myeloid leukemia in first relapse among adults 60 years of age or older who are not considered candidates for cytotoxic chemotherapy.

Gemtuzumab ozogamicin binds to the CD33 antigen expressed by hematopoietic cells. This antigen is expressed on the surface of leukemic blasts in more than 80% of patients with acute myeloid leukemia. CD33 is also expressed on normal and leukemic myeloid colony-forming cells, including leukemic clonogenic precursors, but it is not expressed on pluripotent hematopoietic stem cells or nonhematopoi-etic cells. Binding of the anti-CD33 antibody results in a complex that is internalized. On internalization the calicheamicin derivative is released inside the lysosomes of the myeloid cells. The released calicheamicin derivative binds to the minor groove of DNA and causes double-strand breaks and cell death.


Alemtuzumab (Campath) is humanized MAb (Campath-1H) that is directed against the 21- to 28-kDa cell surface glyco-protein CD52. CD52 is expressed on the surface of normal and malignant B and T lymphocytes, NK cells, monocytes, macrophages, and tissues of the male reproductive system. The Campath-1H antibody is an IgG1 k form with humanized variable and constant regions and CDRs from a rat MAb, Campath-1G.

Alemtuzumab is indicated for the treatment of B-cell chronic lymphocytic leukemia in patients who have been treated with alkylating agents and who have failed on this therapy. Alemtuzumab binds to CD52, a nonmodulating antigen that is present on the surface of essentially all B and T lymphocytes; most monocytes, macrophages, and NK cells, and a subpopulation of granulocytes. The proposed mechanism of action is antibody-dependent lysis of leukemic cells following cell surface binding.


Basiliximab (Simulect, Chimeric) is an MAb produced by a mouse monoclonal cell line that has been engineered to produce the basiliximab IgG1 k antibody glycoprotein. The product is chimeric (murine/human). Basiliximab is indicated for prophylaxis of acute organ rejection in patients receiving renal transplantation when used as part of a regimen of immunosuppressants and corticosteroids. Basiliximab is also indicated in pediatric renal transplantation.

Basiliximab specifically binds to the IL-2 receptor a-chain (the CD25 antigen, part of the three-component IL-2 receptor site). These sites are expressed on the surfaces of activated T lymphocytes. Once bound, it blocks the IL-2a receptor with extremely high affinity. This specific, high-affinity binding to IL-2a competitively inhibits IL-2-mediated activation of lymphocytes, a critical event in the cellular immune response in allograft rejection.


Molecularly, daclizumab (Zenapax, Chimeric) is an IgG1 MAb that binds specifically to the a subunit of the IL-2 receptor (the complete, high-affinity activated IL-2 receptor consists of interacting a, S, and y subunits). IL-2 receptors are expressed on the surfaces of activated lymphocytes, where they mediate lymphocyte clonal expansion and differentiation. Daclizumab is a chimeric protein (90% human and 10% mouse) IgG1. The MAb targets only recently activated T cells that have interacted with antigen and have developed from their naive form into their activated form. It is at this time that the IL-2 receptors are expressed. The human amino acid sequences of daclizumab derive from constant domains of human IgG, and the variable domains are derived from the fused Eu myeloma antibody. The murine sequences derive from CDRs of a mouse anti-IL2a antibody.

The indications for daclizumab are prophylaxis of acute organ rejection in patients receiving renal transplants, as part of an immunosuppressant regimen including cy-closporine and corticosteroids. The mechanism of action is the same as that of basiliximab.


Muromonab-CD3 (murine, Orthoclone-OKT3) is an unmodified mouse immunoglobulin, an IgG2a, monoclonal. It binds a glycoprotein on the surface of mature T lymphocytes. Mature T cells have, as part of the signal transduction machinery of the T-cell receptor complex, a set of three gly-coproteins that are collectively called CD3. Together with the protein zeta, the CD3 molecules become phosphorylated when the T-cell receptor is bound to a peptide fragment and the major histocompatibility complex. The phosphorylated CD3 and zeta molecules transmit information into the cell, ultimately producing transcription factors that enter the nucleus and direct the T-cell activity. By binding to CD3, muromonab-CD3 prevents signal transduction into T cells.

Muromonab-CD3 blocks the function of T cells that are involved in acute renal rejection. Hence, it is indicated for the treatment of acute allograft rejection in heart and liver transplant recipients resistant to standard steroid therapies.


Abciximab (ReoPro, chimeric) is an MAb engineered from the glycoprotein IIb/IIIa receptor of human platelets. The preparation is fragmented, containing only the Fab portion of the antibody molecule. This MAb is a chimeric human-mouse immunoglobulin. The Fab fragments may contain mouse variable heavy- and light-chain regions and human constant heavy- and light-chain regions.

Abciximab is indicated as an adjunct to percutaneous transluminal coronary angioplasty or atherectomy for the prevention of acute cardiac ischemic complications in patients at high risk for abrupt closure of a treated coronary vessel. Abciximab appears to decrease the incidence of myocardial infarction.

Abciximab binds to the intact GPIIb/GPIIIa receptor, which is a member of the integrin family of adhesion receptors and the major platelet-specific receptors involved in aggregation. The antibody prevents platelet aggregation by preventing the binding of fibrinogen, the von Willebrand factor, and other adhesion molecules on activated platelets. The inhibition of binding to the surface receptors may be a result of steric hindrance or conformational effects preventing large molecules from approaching the receptor.


Trastuzumab (Herceptin, humanized) is an MAb engineered from the human epidermal growth factor receptor type 2 (HER2) protein. This MAb is a human-murine immunoglob-ulin. It contains human structural domains (framework) and the CDR of a murine antibody (4D5) that binds specifically to HER2. IgG1 k is the type structure, and the antibody is monoclonal. The protein inhibits the proliferation of human tumor cells that overexpress HER2.

Trastuzumab is indicated for use as a single agent for the treatment of patients with metastatic breast cancer whose tumors overexpress the HER2 protein and who have not received chemotherapy for their metastatic disease.

The HER2 proto-oncogene encodes a transmembrane receptor protein of 185 kDa that is structurally related to the epidermal growth factor receptor HER2. Overexpression of this protein is observed in 25% to 30% of primary breast cancers. Trastuzumab binds with high affinity to the extracellular domain of HER2. It inhibits the proliferation of human tumor cells that overexpress HER2. Trastuzumab also mediates the process of antibody-mediated cellular cy-totoxicity (ADCC). This process, leading to cell death, is preferentially exerted on HER2-overexpressing cancer cells over those that do not overexpress HER2.


The MAb infliximab (Remicade, chimeric) is produced from cells that have been sensitized with human TNFa. The MAb is a chimeric human-mouse immunoglobulin. The constant regions are of human peptide sequence and the variable regions are murine. The MAb is of type IgG1 k.

Infliximab is indicated for the treatment of moderately to severely active Crohn disease to decrease signs and symptoms in patients who had an inadequate response to conventional treatments. Infliximab binds specifically to TNFa. It neutralizes the biological activity of TNFa by binding with high affinity to soluble and transmembrane forms of the TNF. Infliximab destroys TNFa-producing cells. An additional mechanism by which infliximab could work is as follows: by inhibiting TNFa, pathways leading to IL-1 and IL-6 are inhibited. These interleukins are inflammatory cy-tokines. Inhibiting their production blocks some of the inflammation common to Crohn disease.

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