2.3.2 Monoclonal Antibodies

2.3.2 Monoclonal Antibodies

The key feature of an mAb is its unique specificity. It recognizes only one particular antigenic determinant (called epitope) on a given molecule – that means it is monospecifi c. All antibodies secreted by an individual hybridoma represent identical immunoglobulin molecules that display identical binding strengths to its antigen (referred to as affi nity) and have identical physicochemical properties (isotype, stability). This homogeneity will give rise to the same immunological effector functions. In principle, mAbs can be produced in unlimited quantities, the hybridoma cell itself survives after cryopreservation at least for decades.

When compared with polyclonal antisera (for instance from rabbit) the affinity of mAbs might sometimes be inferior. Because mAbs consist of homogeneous molecules of the same isotype they may not elicit certain biological responses. The majority of monoclonals are directed against conformational epitopes of an antigen and may lose reactivity when tested on denatured samples, for instances by Western blotting or by immunohistology on paraffin sections. In common with polyclonal reagents, mAbs may show unexpected cross-reactivity with antigens being expressed in unrelated tissues. 

Being glycoproteins, antibodies are potent immunogens when injected into another species. There are three different types of antigenic determinants against which an immune response can be induced: anti-isotype, anti-allotype, and antiidiotype. If a human being is injected several times with a mouse mAb, it is very likely a HAMA response will develop (Khazaeli et al. 1994; DeNardo et al. 2003).

2.4 Production of Monoclonal Antibodies

2.4.1 Immunization

The aim of an immunization is to elicit a strong immune response against a certain antigen.

For mAb production, most commonly mice and rats and less frequently hamsters and rabbits are immunized with antigen by distinct routes of administration. Antigen may consist of cellular components, purified proteins, peptides, carbohydrates, lipids, or nucleic acids and for each of these specific immunization protocols are available (Harlow and Lane 1988; Goding 1996; Coligan et al. 2004). The purity of the antigen used for immunization plays a major role in the outcome of antibody response. If rather impure preparations are used, problems may arise from the possible immunodominance of contaminants. This might occur when complete cells are employed as immunogen; on the other hand intact cells are highly immunogenic. Molecular biology allows the expression of fusion proteins in eukaryotic cells, a method that has largely improved the preparation
of immunizing agents.

Especially for mounting an immune response against soluble antigens, the use of a strong adjuvant is highly recommended. Adjuvants are nonspecific stimulators of the immune system, the most famous representative being Freund’s complete adjuvant (Freund 1956). This consists of mineral oil and inactivated Mycobacterium tuberculosisparticles. When mixed with immunogen, a water-inoil emulsion is prepared that allows the release of antigen over a long period of time. The mycobacteria give rise to an inflammatory response with the production of numerous cytokines. In general, for the first immunization it is appropriate to use a strong adjuvant (complete Freund’s or Bordetella pertussis), whereas the second injection and following challenges can be given with incomplete
Freund’s (mineral oil only) or without any adjuvant. By repeated immunization the response of the animal is shifted against high-affinity antibodies of IgG isotype. Since previously activated lymphoblasts show preferential fusion with myeloma cells, the final booster immunization should be given 3 days prior to fusion to maximize the yield of hybrids

If the amount of chosen protein antigen is short or if the antigen is not available at all, intrasplenic immunization with minute amounts of antigen (Spitz et al. 1984; Grohmann et al. 1991) or DNA immunization (Barry et al. 1994) might offer alternative approaches. During the past years a couple of useful antibodies were produced by immunizing with synthetic peptides coupled to immunogenic
carriers like keyhole limpet hemacyanin (KLH) or bovine serum albumin (BSA). For this, only the amino acid sequence of the protein has to be known, which can be found in several databases. Coupling of peptides carrying an N- or C-terminal cysteine residue to the carrier is achieved by m-maleimidobenzoyl-N-hydroxysuccinimide ester or another heterobifunctional crosslinker (Green et al. 1982). One drawback of the method is that such anti-peptide antibodies often exclusively react with the denatured but not with the native protein. In vitro immunization, invented to prime naive lymphocytes in cell culture, did not fulfill the expectations because in most instances solely an IgM response was induced (Borrebaeck 1983).

2.4.2 Myeloma Cell Lines

Multiple myeloma or plasmacytoma represents a malignancy of plasma cells in which large numbers of antibody-secreting cells residing in bone marrow are produced. They secrete monoclonal immunoglobulin, the specificity of which is usually not known, therefore they are regarded as “antibody without antigen.” All available mouse myeloma lines for fusion are derived from the MOPC-21 tumor that has been induced in BALB/c mice by mineral oil injection into the peritoneal
cavity (mineral oil-induced plasmacytoma) and was then adapted to growth in tissue culture. While the very first hybridomas were made with myeloma fusion partners that endogenously secreted complete antibody, later on loss variants were selected producing solely kappa light chains (e.g. P3-NS1-Ag4-1; Köhler et al. 1976) or no immunoglobulin. Such nonproducer lines are mostly used for cell fusion these days, prominent examples being X63-Ag8.653 (Kearney et al. 1979), Sp2/0-Ag-14 (Köhler and Milstein 1976) and F0 (Fazekas de St. Groth and Scheidegger 1980).

Similarly, for the production of mAbs against mouse antigens some rat myeloma lines have been established from the LOU/C strain. Frequently used are Y3-Ag1.2.3 (secreting kappa light chains; Galfrè et al. 1979) and line IR983F (nonproducer; Bazin et al. 1990). Since rats are not that much easier to handle than mice and since rat hybridomas are sometimes dependent on growth factors, making cell culture more complicated, interspecies hybrids have been constructed. For this, immune rat spleen cells were fused with a murine nonsecretor myeloma cell line. These rat/mouse hybrids turned out to be stable and secreted amounts of mAbs comparable to mouse/mouse hybridomas (Ledbetter and Herzenberg 1979). Mouse interspecies hybridomas have also been created with hamster and rabbit lymphoblasts to obtain respective mAbs (Sanchez-Madrid et al. 1983; Raybould and Takahashi 1988). A more advanced method for the production of mouse antibodies against mouse antigens is the use of knockout mice for immunization. Since they lack expression of the target antigen they are not tolerant and are able to mount a normal immune response.

2.4.3 Cell Fusion

In early experiments cell fusion was facilitated by means of agglutinating viruses like Sendai. The introduction of polyethylene glycol (PEG) as a fusing agent (Pontecorvo 1975) has simplified the procedure drastically and is used throughout the field today. PEG renders the membrane of cells to be fused gluey, so that they stick together. Subsequently plasma membrane fusion occurs, giving rise to a cell with two (or more) nuclei called a heterokaryon. During cell division the nuclear membranes are degraded and the chromosomes are distributed into the daughter cell. These hybrid cells contain only one nucleus but the genetic material of both parents and are named synkaryons. The double set of chromosomes in hybrids causes genetic instability during further mitoses, leading to improper segregation or loss of chromosomes. If chromosomes coding for immunoglobulin heavy or light chain genes (in the mouse chromosomes 6, 12, and 16) are affected, antibody secretion of this hybrid will ultimately stop. To prevent overgrowth of early hybridoma culture by nonproducing variants, immediate cloning of cultures is mandatory. Following many cell divisions the hybrid line is stabilized in its chromosomal inventory. However, prior to mass production of hybridoma cells and in case the antibody titer of culture supernatant declines recloning should be performed (Fig. 2.3).

Factors critically influencing the outcome of a fusion experiment are the choice of fetal calf serum (FCS) and the health status of the immunized mice. Depending on the content of the growth-promoting constituents, some FCS batches evolve better suited for hybridoma growth than others and it is worth testing for an optimal one. Bacterial and viral infections of rodents can cause severe immune suppression and will lead to low yields of hybridomas. Some protocols recommend the use of feeder cells as a source of growth factors as well as lysis of erythrocytes present in spleen cell preparations from immunized animal prior to fusion. In addition, repeated medium changes to remove potentially harmful substances derived from dying cells have been suggested. In my experience a simplified method works best, leaving the fused cells as untouched as possible. If a spleen from an immunized mouse (containing approximately 1–1.2 ×108lymphocytes) is fused with an equal number of myeloma cells and the fusion mixture is distributed into 15–20 96-well microtiter plates (1440–1920 individual cultures), cell density is high enough to support hybridoma growth even without feeder cells and medium exchange (Fig. 2.4)

As an alternative to the PEG procedure described, electrically induced cell fusion has been developed. It is based on the delivery of high-voltage electrical field pulses to physically fuse lymphoblasts and myeloma cells (Ohnishi et al. 1987; Schmitt and Zimmermann 1989). More recently, a method was published allowing the production of mAbs without hybridomas by using transgenic mice harboring a mutant temperature-sensitive simian virus-40 large tumor antigen (Pasqualini and Arap 2004)