Key Notes
Standardized procedures involving fusion of an immortal cell (a myeloma tumor cell) with a specific predetermined antibody-producing B cell are used to create hybridoma cells producing monospecific and monoclonal antibodies (mAb). These mAb are standard research reagents and many have significant clinical utility.
Most mAbs developed have been mouse, and although useful as research and diagnostic tools, they are not ideal therapeutics because of their
immunogenicity in humans. This has been dealt with by humanizing these murine Abs or by making fully human mAbs.
By randomly fusing heavy (H) and light (L) chain variable (V) region genes from B cells, Fv libraries containing a vast number of binding specificities can be generated and used as a source for creation of specific mAbs.
Related topics
In 1975, Kohler and Milstein developed a procedure (for which they received the Nobel Prize) to create cell lines producing predetermined, monospecific and monoclonal antibodies (mAb). This procedure has been standardized and applied on a massive scale to the preparation of antibodies useful to many research and clinical efforts. The basic technology involves fusion of an immor- tal cell (a myeloma tumor cell) with a specific predetermined antibody-produc- ing B cell from immunized animals or humans (Fig. 1). The resulting hybridoma cell is immortal and synthesizes homogeneous, specific mAb, which can be made in large quantities. Thus, MAbs have become standard research reagents and have extensive clinical applications.
The vast majority of mAbs have been developed in mice, and although useful as research and diagnostic tools, they have not been ideal therapeutic reagents at least partly because of their immunogenicity in humans. That is, a murine Ab introduced into a patient will be recognized as foreign by the patient’s immune system and a Human Anti-Mouse Ab (HAMA) response will develop that compromises the therapeutic utility of the Ab. This has been dealt with in two basic ways.
Humanized antibodies
Murine mAbs can be genetically modified to be more human (Fig. 2). In particu- lar the constant region of the murine IgG heavy (H) and of the murine light (L) chain can be replaced at the DNA level with the constant region genes of Humanization and
chimerization of mAbs
Monoclonal antibodies
Lymphocytes (C1) Immunodiagnosis (N4)
Immunotherapy of tumors with antibodies (N6)
Monoclonal antibodies
Humanization and chimerization
of mAbs
Fv libraries
human IgG1 H and L chains to create a chimeric Ab where only the variable (V) regions are murine. This significantly decreases but does not eliminate the immunogenicity of the Ab. Another approach involves sequencing the V regions of the mouse Ab VH and VL regions and then inserting the DNA sequences of the hypervariable regions of these chains into human IgG VH- and VL-chain genes. The resulting Ab is 95% human with only the binding regions being murine.
Fully human mAbs
Human Abs have been made by fusing human B cells with myeloma cells, although this has been very difficult and usually requires immortalizing the B cells using Epstein–Barr virus before fusing. This approach is not ideal as a virus is used, the specificity of the mAbs produced is limited and the yield of the Abs produced is poor. More recently, a human antibody mouse has been created by replacing the genes for mouse immunoglobulins with genes for human immunoglobulins. Thus, when the mouse is immunized it makes fully human Abs against the Ag and the B cells making these Abs can be fused with myeloma cells to generate hybridomas making the human mAb.
Fv libraries Another way of preparing mAbs involves Fv libraries. This approach initially involves obtaining mRNA for the VH and VL regions from a large number of
D5 – Monoclonal antibodies 81
Serum Spleen cell suspension
Cell 1 Cell 2 Cell 3 ...Cell n Cell 3
Cell n Cell 1 Ab1
Ab2
Ab3 Abn
Cell 2
Ab1 Ab2 Ab3 ...Abn Antigen Immunization
Cell 1 Cell 2 Cell 3 Cell n
...
Myeloma cells Cell fusion
(immortalization of spleen cells) Hybrid
cells
Mixing of spleen
and myeloma
cells
Cloning of hybrid
cells
Clone 1 (cell 1 → Ab1) Clone 2 (cell 2 → Ab2) Clone 3 (cell 3 → Ab3) Clone n (cell n → Abn)
Glycol
Polyethylene
Fig. 1. Preparation of monoclonal Abs.
B cells. From this mRNA, cDNA for each H-chain V region is prepared and joined to the cDNA for each L-chain V region (Fig. 3) to create all combinations, and thus genes encoding a vast number of different antigen-combining sites (Fv regions). These are cloned into cells for production of the antibody-binding site they encode. For example, they can be cloned into bacteriophage (viruses that infect bacteria) and selected for their specificity. Thus, Fvs can be expressed in a replicating bioform and used as a source from which specific mAbs can be created.
82 Section D – Antibodies
Mouse antibody
SS SS
SS
Chimeric antibody
SS SS
SS
Humanized antibody
SS SS
SS
Replace mouse C regions with human C regions
Insert DNA for mouse Lv and Hv regions into human L and H chain genes Mouse H chain C region Mouse H chain V regions with mouse Hv regions Mouse L chain
V regions with mouse Hv regions Mouse L chain C region
Human C regions Mouse Lv and Hv regions Mouse L and
H chain V regions Human L chain C region Human H chain C region
Fig. 2. Humanizing and chimerizing mouse monoclonal antibodies. Chimeric mAbs are created by replacing the murine genes for the constant region of the light (L) and heavy (H) chain with the corresponding human constant region genes. Humanized mAbs are created by inserting the gene sequences for each of the hypervariable (Hv) regions of the mouse antibody into the corresponding place in the genes for the L and H chains for a human antibody.
mRNA for L chain V region (VL) mRNA for H chain V region (VH)
cDNA for VL
cDNA
for VH VH VL
VL VH
S S
Fv (Antigen combining
site) cDNA for Fv
Ligation with spacer (S)
Insert into cell for production B cell
Fig. 3. Fv preparation. mRNA for the V regions of L- and H-chains is prepared from B cell mRNA using the polymerase chain reaction. From this mRNA, cDNA for each H chain V region is prepared and joined to the cDNA for each L chain V region, with a spacer between. This yields a gene encoding the antigen binding region of the antibody, which is inserted into a cell for production of a protein, Fv, that is the combining site of an antibody.
Section D – Antibodies