Monoclonal Antibodies

MONOCLONAL ANTIBODIES Most antigens such as a bacterial cell possess multiple epitopes and multiple copies of each of those epitopes. Based on this fact, upon injection one would expect a variety of B cells to interact with the various epitopes. The resulting antiserum would be a heterogeneous mixture of antibodies each specific for one epitope among the various epitopes = a polyclonal response. A polyclonal response has advantages for host in that it facilitates the localization, phagocytosis, and complement mediated lysis of antigen. However in experimental situations, antibody heterogeneity often clouds the results and reduces the antiserums efficiency. For most research, diagnostic, and therapeutic purposes a monoclonal antibody is needed-that is, an antibody derived from a single clone and thus specific for a single epitope is preferable. Monoclonal antibody production by somatic cell fusion or hybridoma technology was introduced by Kolher and Milstein in 1975 (got Nobel prize in 1984). Overall: The technique involves fusing a normal antibody producing B cell with a myeloma cell to produce a hybrid cell or hybridoma. The hybridoma would possess the immortal growth properties of the myeloma cell while secreting the antibody produced by the B cell. The resulting hybridoma could be cultured indefinitely thus providing large amounts of homogeneous antibody for research purposes. The usefulness of monoclonal antibodies stems from 3 characteristicstheir specificity of binding, their homogeneity, and their ability to be produced in unlimited quantities. *Additionally, one unique advantage of hybridoma production is that impure antigens can be used to produce specific antibodies. This is based on the fact that you screen with the pure antigen for the antibody of choice and that Ab is produced by isolating a single cell clone!

MONOCLONAL PRODUCTION First mice are injected with an Ag+adjuvant mixture- mice generally receive a primary injection followed by two boosts (14 to 28 days later). Why? After the final boost the mice are euthanized 3 to 5 days later and the spleen is harvested for cell fusion. A single cell suspension from the spleen is made and mixed with the myeloma partner. A fusing agent such as polyethylene glycol is then added. PEG fuses the plasma membranes of adjacent cells forming a single cell with two or more nuclei. This heterokaryon retains these nuclei until the nuclear membranes dissolve prior to mitosis. During mitosis and further rounds of division, the individual chromosomes are segregated into daughter cells. Often chromosomes are lost. Even in the most efficient hybridoma fusions, only about 1% of the starting cells are fused, and only about 1 in 105 form viable hybrids. This leaves a large number of unfused cells still in the culture. The cells from the immunized animal do not continue to grow in tissue culture because they are terminally differentiated cells and thus only capable of limited growth, and so do not confuse further work. However, the myeloma cells are well adapted to tissue culture and must be killed. This is usually done by drug selection. DRUG SELECTION Cells have two pathways for the synthesis of nucleotides, the de novo and salvage pathways. Since cells in tissue culture can survive by using either of these pathways, mutations in the enzymes responsible for nucleotide synthesis have become common and readily manipulated targets for mutagenesis of mammalian cells. The most common target is the enzyme hypoxanthine-guanine phosphoribosyl transferase (HGPRT). HPGRT performs one of the essential steps in the salvage pathway Mutations in the HPGRT gene can be selected by growing cells in the presence of purine analogs such as 8-azoguanine (8-AG). HPGRT will recognize 8-AG as a substrate and convert it to the monophosphate

nucleotide. The 8-AG-containing nucleotide is then processed further and incorporated into DNA and RNA, where it is toxic substitution. Therefore, cells with a functional HPGRT enzyme grown in the presence of 8-AG will die. However, because the HPGRT enzyme is part of a nonessential pathway (the de novo pathway is still available) cells harboring a mutant HPGRT gene can continue to grow. Therefore, selection with 8-AG will kill cells with a wild-type HPGRT but will not affect cells with a mutant HPGRT. The normal rate of mutagenesis is sufficient for treating cells with 8-AG and the surviving cells are HPGRT deficient. SP2/0 myeloma cell line from the ATCC (american type culture collection) The myeloma cell line does not synthesize or secrete any immunoglobulin chains, and is HGPRT-. HAT Selection for Hybridoma Drugs like aminopterin block de novo synthesis of nucleotides. Both purine and pyrimidine synthesis are inhibited so both need to be produced by the salvage pathway and precursors for these pathways (such as hypoxanthine and thymidine) must be included in the selection media. Myeloma fusion partners are deficient in the enzyme required for the salvage pathway of nucleotide synthesis. These cells will die in HAT containing medium because aminopterin blocks normal nucleotide synthesis and the enzyme deficiency blocks utilization of the salvage pathway. If the myeloma and B cell fuse, the resulting hybridoma will live indefinitely in culture because the normal cell supplies the missing enzyme and the myeloma immortalizes the cell line. Unfused normal cells will only survive in tissue culture for approximately 1 week before they die off. After 7-10 days, most of the wells contain dead cells but a few contain clusters of viable cells - each cluster represents a clonal expansion of a hybridoma!

At this point it is necessary to screen your fusion. We use the ELISA (enzyme linked immunosorbent assay) method. Screening at this time point is important because non-Ab producing hybrids will overgrow the slower growing Ab producers! ELISA Coat 96 well plates with antigen at 1 ug/ml in carbonate buffer pH 9.6 (add 100ul per well). Incubate overnight at 4oC. (Polystyrene binds protein noncovalently). Remove the contents of the wells and block plate with 3% NFDM in PBS to block nonspecific antibody binding. Incubate. (Blocking must be performed to prevent nonspecific binding of Igs to any sites on the plate). Wash plate with PBS-tween. Add 100ul of (antibody containing) supernatant from Fusion into Elisa plates. Incubate. Wash plate and add 100ul of anti-Ig labeled with horseradish peroxidase. Incubate then wash plate. Add 100ul of substrate solution to each well. Incubate. Evaluate wells for positives- If an Ag+Ab+enzyme labeled Ab + enzyme substrate is in the well there will be a color reaction! Positive wells contain hybrids making specific Ab to your test Ag! Once clones have been identified, the positive wells need to be expanded and cloned by limiting dilution. You cant be sure that the positive well arose from only one hybrid clone so the cells have to undergo limiting dilution- you dilute out the cells in one well into a series of wells until only one cell is in the well to insure clonality. After each cloning the cells are again screened by ELISA to insure Abs are still being produced. Because hybridoma cells have a very low plating effiency, single cell cloning is normally done in the presence of feeder cells. We use a single

cell suspension of spleen cells-these cells produce growth factors and also provide cell contact for your hybridoma. We usually do limiting dilution 3 consecutive times to one clone to assure clonality. ELISA screening is performed each time. At this point, once you have a clone the cells can be expanded for antibody production and experimental use! Cells can be frozen in a DMSO-FCS mixture and stored in liquid nitrogen for years. * Cells should be frozen down at each step of the cloning procedure in case of contamination. If you do get contamination-you dont have to start all over- you simply retrieve the cell line from the previous cloning and start at that point! CLINICAL USE- Wagner et al. Hybridoma 16(1):33-40 1997 Generated an IgG1 murine monoclonal Ab -anti-idiotypic Ab (Ab2) designated ACA125 which mimics an epitope on the tumor associated antigen designated CA125. This antigen is expressed by most malignant ovarian tumors. Used ACA125 Ab2 for vaccine therapy in 16 patients with advanced epithelial ovarian cancer. Patients with CA125 positive tumors are immunologically tolerant to CA125 -in other words they dont produce an immune response to it. Tolerance - a state in which lymphocytes are present that would normally respond but are somehow prevented from doing so. It affects both the T and B lymphocytes. When the antigen is cleared, and with production of new lymphocytes - you eventually regain response ability- so tolerance is a temporary state. Certain conditions favor tolerance induction: state of the lymphoid system- its easier to induce with immature immune systems or immune systems damaged by irradiation, drugs, ect physiochemical properties of the Ag-size (small, soluble Ags are harder to capture from circulation by the phagocytic system, chemical nature (d-amino acids are toleragens whereas l-

amino acids are immunogens) , epitope density on molecule (too many induces tolerance) route of Ag administration- determines accessibility of Ag to MOs Ag dose- very low and very high doses induce tolerance. Does the tumor display a very low or very high epitope density to induce tolerance? Does it flood the system with soluble Ag? The patients received 3 to 19 injections of 2mg/inj of the Ab2. Patients showed development of specific humoral and cellular immune responses to an otherwise nonimmunogenic tumor antigen. They developed Ab3 (anti-anti-id) and had an increase in IFNg production. (*Remember an increase in IFNg results in an increase of TNF). 9 patients produced the Ab3 with few side effects and displayed a median progression free survival for 11 +/-5.6 (5.4-16.6) months compared to 8+/4.2 (3.8-12.2) months in the Ab3 negative group. Human Hybridomas When mouse monoclonals are introduced into humans they are recognized as foreign and evoke an Ab response. The induced human anti-mouse antibodies quickly reduce the effectiveness of the mouse monoclonal by clearing it from the bloodstream. Also, circulating immune complexes of mouse + human Abs can cause allergic reactions or you can get a buildup of complexes on the basement filter membranes of the kidney- glomerulonephritis. One area of development over the last 5 years has been the development of systems for the production of human hybridomas. Human monoclonal antibodies can be used for clinical applications. The progress has been slow because of the lack of a suitable myeloma partner but several line have been isolated and are now in use. The use of EBV transformed Ab secreting cells has solved some of the problems but these transformants seldom secrete large amounts of Abs. This has been overcome in some cases by fusing the EBV transformed cell with a mouse myeloma cell line to increase Ab secretion- a heterohybridoma.

Also there is the problem of obtaining Ag primed B cells in humans-cant take their spleens! So human hybridomas have to be prepared from B cells in human peripheral blood. Lymphocytes only make up about 20-30% of your WBC population and most (~90%) of the lymphocytes in circulation are T cells. Plus human volunteers can not be just immunized with a range of antigens- have tried in vitro Ag priming of the B cells but conditions are not optimal so only see low affinity IgM production. Immunotoxins Tumor specific monoclonal antibodies with lethal toxins coupled to it are potentially valuable therapeutic reagents. Toxins such as ricin and shigella are so toxic that one molecule can kill a cell. These toxins are composed of two chainsone is the toxin chain and the other is a binding chain that interacts with receptors on the cell surface. Without this binding chain the toxin is harmless since it cannot enter the cell. In theory: the monoclonal Ab with the attached toxin chain will bind specifically to a tumor cell where it will be endocytosed and the toxin will cause death by inhibiting protein synthesis.