Topic 6  Organization and Expression of   Immunoglobulin Genes

Introduction

One of the characteristics of the immune response is its enormous diversity.  Estimates of the number of antibody molecules with different specificities is a given individual range from 10 ⁶ to 10 ⁸.   An understanding of the unique genetic mechanisms by which this remarkable diversity is achieved without using a huge proportion of one’s DNA complement has only recently been acquired.  The lymphocyte cell lineage is the only known vertebrate cell type capable of genomic rearrangement.  Antibody diversity is achieved through recombination of variable germline genes, junction diversity, somatic hypermutation, and random pairing of heavy and light immunoglobulin chains.

This lesson first describes the detailed organization of the immunoglobulin genes, the process of Ig-gene rearrangement, and the mechanisms by which the dynamic immunoglobulin genetic system generates more than 10 ⁸ different antigenic specificities.  Then the mechanism of class switching, the role of differential RNA processing in the expression of immunoglobulin genes, and regulation of Ig-gene transcription are discussed.

Objectives

On completion of this section and the required readings, you should be able to:

n  draw the organization of Kappa light chain genes;

n  draw the organization of Lambda light chain genes;

n  draw the organization of heavy chain genes;

n  describe the V-J joining for light chain genes in terms of the 7-9 and the 12-23 rules;

n  describe the V-D-J joining of heavy chain genes in terms of the 7-9 and the 12-23 rules;

n  explain how allelic exclusion leads to cells that produce immunoglobulins with a single antigen binding site;

n  describe how transcription controls immunoglobulin gene expression;

n  describe at least four mechanisms that contribute to antibody diversity;

n  describe the coexpression of IgM and IdD in terms of RNA processing;

n  describe the mechanisms for class switching;

n  correlate B cell differentiation with immunoglobulin gene rearrangements;

n  describe how the number and organization of immunoglobulin gene segments or exons contribute to the generation of antibody diversity.

Required Reading

Please refer to the textbook key for specific readings for this section.

P Key Words

•    allelic exclusion •    class switching •    combinatorial freedom of chain association •    combinatorial association of gene segments •    Constant (C) gene segments •    Diversity (D) gene segments •    Palindromes •    acceptor junctions •    donor junctions •    enhancers •    exons •    7-9 rule •    12-23 rule •    germline theory

•    somatic theory •    imprecise DNA rearrangement •    Insertion of random N regions •    introns •    Joining (J) gene segments •    junctional diversity •    leader sequence •    Membrane (M) exons •    regions promotors •    recognition sequences •    spacer sequences •    somatic hypermutation •    switch recombination •    switch (S) regions, •    variable(V) gene segments

P Key Concepts

n  Immunoglobulins are constructed through the use of multiple gene segments. 

n  There are three major complexes coding for human Ig molecules - Igh (heavy), Igk (kappa), Igl (lambda).  Each of the Ig complexes consists of three or four regions, V, D, J and C.

n  Diversity in antibody specificity is generated by combinatorial joining of a small number of germline genes, nonuniform joining of variable region segments, occurrence of somatic hypermutation, junctional flexibility, and  the random pairing of light and heavy chains

n  Transcription of immunoglobulin genes is regulated by three types of DNA regulatory sequences: promoters, enhancers, and silencers.

DID YOU KNOW?

A considerable effort has been made over the past 10 years to develop new immunotherapeutic strategies against cancer.  The advances in molecular biology and DNA manipulation techniques along with understanding the genetic basis of antibody diversity allowed development of totally new approaches.  

Potential targets for therapeutic intervention include macromolecules on the surface of cancer cells. One, recently developed strategy, is to use cancer-specific antibodies to target potent cytotoxic substances. The concept of how immunotoxins can be used for cancer therapy is simple:  antibodies specific for antigens found only on cancer cells are connected to toxins, resulting in immunotoxins that selectively bind to and kill cancer cells, while sparing normal cells. There are at least five classes of agents that have been used to mediate the killing of tumor cells, including  protein toxins, cytokines, radioisotopes, small molecular weight chemotherapeutic drugs and enzymes.  In this review we will concentrate on immunotoxins -  proteins engineered into exquisitely cell-type specific toxins.  The immunotoxins can be derived from plant, bacterial or mammalian toxins and include some of the most lethal toxins known, for example:  ricin, human RNase, pseudomonas exotoxin, diphteria toxin and staphylococcal pore-forming alpha toxin.   These are not some unavailable, exotic agents found only in equatorial rain forests, but they are readily available and when expressed  recombinantly can be made in almost endless supply.  The development of immunoglobulin “part” of the immunotoxin was also possible after recombinant DNA technology and transfection have been combined to enable Ig genes to be manipulated so that antibodies can be “tailor made."  Through these procedures, desired Ig genes can be isolated, cloned and recombined.  The “tailor made” genes can then be inserted into suitable vectors and transfected into recipient cells.

In most cases , cDNAs from hybridomas or other cell lines producing a defined antibody are used to clone the variable domains of the antibody that are responsible for antigen binding.  More recently, attempting to recreate the immune repertoire in vitro, combinatorial libraries of antibody variable genes made by phage display have been used for the identification of antibodies with specificities of interest.  The antibody variable genes are subcloned into an expression vector that contains the gene for the toxin and finally large amounts of recombinant protein are produced.

The first-generation immunotoxins, composed of whole antibodies chemically conjugated to plant or bacterial toxin could kill cancer cells in vitro.  They however  showed  very low effectiveness in in vivo studies.  The main problem of these first-generation toxins was their nonspecific toxicity toward the non-cancer cell. Another problem, particularly for the treatment of solid tumors, was that chemically conjugated immunotoxins are large molecules with poor tumor penetration.  Second -generation immunotoxins, which were designed and constructed to be much more specific for cancer  and less toxic towards normal cells, overcome most of these problems.  The size of immunotoxins was  reduced with a concomitant increase in their tumor penetration potential in vivo.  Finally, advanced methods in biotechnology have yielded recombinant immunotoxins of high enough purity and quality for clinical use that could also be produced in sufficient quantities for clinical trials.

The recombinant fragments of antibodies are made in two forms:  F.v. fragments and Fab. F.v. fragments are heterodimers composed of a variable heavy chain (V_H) and a variable light chain (V_L) domain and are the smallest functional fragment of antibodies that maintain the binding and specificity of the whole antibody.  Normally F.v. fragments are unstable.  Stable Fvs can be produced by making recombinant molecules in which the V _H and V_L domains are connected by a peptide linkers so that the antigen-binding site is regenerated in a single molecule. These recombinant molecules are termed scFvs.  An alternative method, which uses antibody engineering strategy, is to stabilize the V_H - V_L heterodimers by an interchain disulfide bond.  These constructs are termed dsFvs. A second recombinant antibody construct is the Fab fragment, which is composed of the light  chain and the heavy chain Fd fragment (V_H and CH1) connected to each other via the interchain disulfide bond between CL and CH1.

As we stated at the beginning recombinant antibodies can be useful not only for tumor diagnosis, but also for the development of chimeric proteins for cancer therapy. Target antigens for immunotoxins in studies up to date have included cell surface markers of the following cancer types:  Leukemia (both T and B cell), liver, breast, stomach, colon, kidney, myeloma, ovary and prostate.  Studies with tissue cultures and experimental animals showed the effectiveness of the immunotoxins.  Regardless of  good initial results there are still outstanding questions that remain.