Mice Have Been Engineered with Human Immunoglobulin Loci

It is possible to functionally knock out, or disable, the heavy-and light-chain immunoglobulin loci in mouse embryonic stem (ES) cells. N. Lonberg and his colleagues followed this procedure and then introduced large DNA sequences (as much as 80 kb) containing human heavy- and light-chain gene segments. The DNA sequences contained constant-region gene segments, J segments, many V-region segments, and, in the case of the heavy chain, DH segments. The ES cells containing these miniature human Ig gene loci (miniloci) are used to derive lines of transgenic mice that respond to anti-genic challenge by producing antigen-specific human antibodies (Figure 5-23). Because the human heavy- and light-chain miniloci undergo rearrangement and all the other diversity-generating processes, such as N-addition, P-addition, and even somatic hypermutation after antigenic challenge, there is an opportunity for the generation of a great deal of diversity in these mice. The presence of human heavy-chain minilocus genes for more than one isotype and their accompanying switch sites allows class switching as well. A strength of this method is that these completely human antibodies are made in cells of the mouse B-cell lineage, from which antibody-secreting hybridomas are readily derived by cell fusion. This approach thus offers a solution to the problem of producing human monoclonal antibodies of any specificity desired.

SUMMARY

■ Immunoglobulin k and X light chains and heavy chains are encoded by three separate multigene families, each containing numerous gene segments and located on different chromosomes.

■ Functional light-chain and heavy-chain genes are generated by random rearrangement of the variable-region gene segments in germ-line DNA.

■ V(D)J joining is catalyzed by the recombinase activiating genes, RAG-1 and RAG-2, and the participation of other enzymes and proteins. The joining of segments is directed by recombination signal sequences (RSS), conserved DNA sequences that flank each V, D, and J gene segment.

■ Each recombination signal sequence contains a conserved heptamer sequence, a conserved nonamer sequence, and either a 12-bp (one-turn) or 23-bp (two-turn) spacer. During rearrangement, gene segments flanked by a one-turn spacer join only to segments flanked by a two-turn spacer, assuring proper VL-JL and VH-DH-JH joining.

Mouse embryonic stem cells (ES cell) Knockout mouse and K

Mouse embryonic stem cells (ES cell) Knockout mouse and K

|l/K-knockout ES cells

|l/K-knockout ES cells

Transfert into ES cells

Transfert into ES cells

Vh genes D genes J genes C^ C y1 Germ-line human heavy-chain minilocus

Vh genes

Jk genes Ck

Mouse ES cells incorporating human H and L miniloci

Inject into host embryo

Germ-line human k light-chain minilocus

Mouse ES cells incorporating human H and L miniloci

Inject into host embryo

Blastocyst

Chimeric mouse Breed

Blastocyst

Human miniloci

00000-

Chimeric mouse Breed

FIGURE 5-23

Grafting human heavy- and light-chain miniloci into mice. The capacity of mice to rearrange Ig heavy- and light-chain gene segments was disabled by knocking out the C^ and Ck loci. The antibody-producing capacity of these mice was reconstituted by introducing long stretches of DNA incorporating a large part of the human germ-line k and heavy-chain loci (miniloci).

Miniloci Nontransgenic transgenic mouse offspring

Immunize

YYYY

Human antibodies

Chimeric mice were then bred to establish a line of transgenic mice bearing both heavy- and light-chain human miniloci. Immunization of these mice results in the production of human antibody specific for the target antigen. [N. Lonberg et al., 1994, Nature 368:856.]

■ Immunoglobulin gene rearrangements occur in sequential order, heavy-chain rearrangements first, followed by light-chain rearrangements. Allelic exclusion is a consequence of the functional rearrangement of the immunoglobulin DNA of only one parental chromosome and is necessary to assure that a mature B cell expresses immunoglobulin with a single antigenic specificity.

■ The major sources of antibody diversity, which can generate >1010 possible antibody combining sites, are: random joining of multiple V, J, and D germ-line gene segments; random association of heavy and light chains; junctional flexibility; P-addition; N-addition; and somatic mutation.

■ After antigenic stimulation of mature B cells, class switching results in expression of different classes of antibody (IgG, IgA, and IgE) with the same antigenic specificity.

■ Differential RNA processing of the immunoglobulin heavy-chain primary transcript generates membrane-bound antibody in mature B cells, secreted antibody in

Go to www.whfreeman.com/immunology i . Self-Test Review and quiz of key terms plasma cells, and the simultaneous expression of IgM and IgD by mature B cells.

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

■ Growing knowledge of the molecular biology of im-munoglobulin genes has made it possible to engineer antibodies for research and therapy. The approaches include chimeric antibodies, bacteriophage-based combinatorial libraries of Ig-genes, and the transplantation of extensive segments of human Ig loci into mice.

References

Chen, J., Y. Shinkai, F. Young, and F. W. Alt. 1994. Probing immune functions in RAG-deficient mice. Curr. Opin. Immunol. 6:313.

Cook, G. P., and I. M. Tomlinson. 1995. The human immunoglobulin VH repertoire. Immunol. Today 16:237.

Dreyer, W. J., and J. C. Bennett. 1965. The molecular basis of antibody formation. Proc. Natl. Acad. Sci. U.S.A. 54:864.

Fugmann, S. D., I. L. Lee, P. E. Shockett, I. J. Villey, and D. G. Schatz. 2000. The RAG proteins and V(D)J recombination: Complexes, ends and transposition. Annu. Rev. Immunol. 18:495.

Gavilondo, J. V., and J. W. Larrick. 2000. Antibody engineering at the millennium. Biotechniques 29:128.

Hayden, M. S., L. K. Gilliand, and J. A. Ledbetter. 1997. Antibody engineering. Curr. Opin. Immunol. 9:201.

Hesslein, D. G., and D. G. Schatz. 2001. Factors and forces controlling V(D)J recombination. Adv. Immunol. 78:169.

Hozumi, N., and S. Tonegawa. 1976. Evidence for somatic rearrangement of immunoglobulin genes coding for variable and constant regions. Proc. Natl. Acad. Sci. U.S.A. 73:3628.

Maloney, D. G., et al. 1997. IDEC-C2B8 (Rituximab) anti-CD20 monoclonal antibody therapy in patients with relapsed low-grade non-Hodgkin's lymphoma. Blood 90:2188.

Manis, J. P., M. Tian, and F. W. Alt. 2002. Mechanism and control of class-switch recombination. Trends Immunol. 23:31.

Matsuda, F., K. Ishii, P. Bourvagnet, Ki Kuma, H. Hayashida, T. Miyata, and T. Honjo. 1998. The complete nucleotide sequence of the human immunoglobulin heavy chain variable region locus. J. Exp. Med. 188:2151.

Max, E. E. 1998. Immunoglobulins: molecular genetics. In Fundamental Immunology, 4th ed., W. E. Paul, ed. LippincottRaven, Philadelphia.

Mills, F. C., N. Harindranath, M. Mitchell, and E. E. Max. 1997. Enhancer complexes located downstream of both human im-munoglobulin C alpha genes. J. Exp. Med. 186:845.

Oettinger, M. A., et al. 1990. RAG-1 and RAG-2, adjacent genes that synergistically activate V(D)J recombination. Science 248:1517.

Tonegawa, S. 1983. Somatic generation of antibody diversity. Nature 302:575.

Van Gent, D. C., et al. 1995. Initiation ofV(D)J recombination in a cell-free system. Cell 81:925.

Winter, G., and C. Milstein. 1990. Man-made antibodies. Nature 349:293.

USEFUL WEB SITES

http://www.mrc-cpe.cam.ac.uk/imt-doc/public/ INTRO.html#maps

V BASE: This database and informational site is maintained at the MRC Centre for Protein Engineering in England. It is an excellent and comprehensive directory of information on the human germ-line variable region.

http://www.mgen.uni-heidelberg.de/SD/SDscFvSite.html

The Recombinant Antibody Page: This site has a number of links that provide interesting opportunities to explore the potential of genetic engineering of antibodies.

http://www.ebi.ac.uk/imgt/hla/intro.html.

The IMGT site contains a collection of databases of genes relevant to the immune system. The IMGT/LIGM database houses sequences belonging to the immunoglobulin super-family and of T cell antigen receptor sequences.

Study Questions

Clinical Focus Question The Clinical Focus section includes a table of monoclonal antibodies approved for clinical use. Two, Rituxan and Zevalin, are used for the treatment of non-Hodgkins lymphoma. Both target CD20, a B-cell surface antigen. Zevalin is chemically modified by attachment of radioactive isotopes (yttrium-90, a p emitter or indium-111, a high energy 7 emitter) that lethally irradiate cells to which the monoclonal antibody binds. Early experiments found that Zevalin without a radioactive isotope attached was an ineffective therapeutic agent, whereas unlabeled Rituxan, a humanized mAB, was effective. Furthermore, Rituxan with a radioactive isotope attached was too toxic; Zevalin bearing the same isotope in equivalent amounts was far less toxic. Explain these results. (Hint: The longer a radioactive isotope stays in the body, the greater the dose of radiation absorbed by the body.)

1. Indicate whether each of the following statements is true or false. If you think a statement is false, explain why.

a. VK gene segments sometimes join to Cx gene segments.

b. With the exception of a switch to IgD, immunoglobulin class switching is mediated by DNA rearrangements.

c. Separate exons encode the transmembrane portion of each membrane immunoglobulin.

d. Although each B cell carries two alleles encoding the im-munoglobulin heavy and light chains, only one allele is expressed.

e. Primary transcripts are processed into functional mRNA by removal of introns, capping, and addition of a poly-A tail.

f. The primary transcript is an RNA complement of the coding strand of the DNA and includes both introns and exons.

2. Explain why a VH segment cannot join directly with a JH segment in heavy-chain gene rearrangement.

3. Considering only combinatorial joining of gene segments and association of light and heavy chains, how many different antibody molecules potentially could be generated from germ-line DNA containing 500 VL and 4 JL gene segments and 300 VH, 15 DH, and 4 JH gene segments?

4. For each incomplete statement below (a-g), select the phrase(s) that correctly completes the statement. More than one choice may be correct.

a. Recombination of immunoglobulin gene segments serves to

(1) promote Ig diversification

(2) assemble a complete Ig coding sequence

(3) allow changes in coding information during B-cell maturation

(4) increase the affinity ofimmunoglobulin for antibody

(5) all of the above b. Somatic mutation of immunoglobulin genes accounts for

(1) allelic exclusion

(2) class switching from IgM to IgG

(3) affinity maturation

(4) all of the above

(5) none of the above c. The frequency of somatic mutation in Ig genes is greatest during

(1) differentiation of pre-B cells into mature B cells

(2) differentiation of pre-T cells into mature T cells

(3) generation of memory B cells

(4) antibody secretion by plasma cells

(5) none of the above d. Kappa and lambda light-chain genes

(1) are located on the same chromosome

(2) associate with only one type of heavy chain

(3) can be expressed by the same B cell

(4) all of the above

(5) none of the above e. Generation of combinatorial diversity among im-munoglobulins involves

(1) mRNA splicing

(2) DNA rearrangement

(3) recombination signal sequences

(4) one-turn/two-turn joining rule

(5) switch sites f. A B cell becomes immunocompetent

(1) following productive rearrangement of variableregion heavy-chain gene segments in germ-line DNA

(2) following productive rearrangement of variableregion heavy-chain and light-chain gene segments in germ-line DNA

(3) following class switching

(4) during affinity maturation

(5) following binding of TH cytokines to their receptors on the B cell g. The mechanism that permits immunoglobulins to be synthesized in either a membrane-bound or secreted form is

(1) allelic exclusion

(2) codominant expression

(3) class switching

(4) the one-turn/two-turn joining rule

(5) differential RNA processing

5. What mechanisms generate the three hypervariable regions (complementarity-determining regions) of immunoglobu-lin heavy and light chains? Why is the third hypervariable region (CDR3) more variable than the other two (CDR1 and CDR2)?

6. You have been given a cloned myeloma cell line that secretes IgG with the molecular formula 72X2. Both the heavy and light chains in this cell line are encoded by genes derived from allele 1. Indicate the form(s) in which each of the genes listed below would occur in this cell line using the following symbols: G = germ line form; R = productively rearranged form; NP = nonproductively rearranged form. State the reason for your choice in each case.

a. Heavy-chain allele 1 d. K-chain allele 2

b. Heavy-chain allele 2 e. X-chain allele 1

c. K-chain allele 1 f. X-chain allele 2

7. You have a B-cell lymphoma that has made nonproductive rearrangements for both heavy-chain alleles. What is the arrangement of its light-chain DNA? Why?

8. Indicate whether each of the class switches indicated below can occur (Yes) or cannot occur (No).

9. Describe one advantage and one disadvantage of N-nucleotide addition during the rearrangement of im-munoglobulin heavy-chain gene segments.

10. X-ray crystallographic analyses of many antibody molecules bound to their respective antigens have revealed that the CDR3 of both the heavy and light chains make contact with the epitope. Moreover, sequence analyses reveal that the variability of CDR3 is greater than that of either CDR1 or CDR2. What mechanisms account for the greater diversity in CDR3?

11. How many chances does a developing B cell have to generate a functional immunoglobulin light-chain gene?

12. Match the terms below (a-h) to the description(s) that follow (1-11). Each description may be used once, more than once, or not at all; more than one description may apply to some terms.

Terms a. _RAG-1 and RAG-2 e. _P-nucleotides b ._Double-strand break f. _N-nucleotides repair (DSBR) enzymes g. _Promoters c. _Coding joints h. _Enhancers d ._RSSs

Descriptions

(1) Junctions between immunoglobulin gene segments formed during rearrangement

(2) Source of diversity in antibody heavy chains

(3) DNA regulatory sequences

(4) Conserved DNA sequences, located adjacent to V, D, and J segments, that help direct gene rearrangement

(5) Enzymes expressed in developing B cells

(6) Enzymes expressed in mature B cells

(7) Nucleotide sequences located close to each leader segment in immunoglobulin genes to which RNA poly-merase binds

(8) Product of endonuclease cleavage of hairpin intermediates in Ig-gene rearrangement

(9) Enzymes that are defective in SCID mice

(10) Nucleotide sequences that greatly increase the rate of transcription of rearranged immunoglobulin genes compared with germ-line DNA

(11) Nucleotides added by TdT enzyme

13. Many B-cell lymphomas express surface immunoglobulin on their plasma membranes. It is possible to isolate this lymphoma antibody and make a high affinity, highly specific mouse monoclonal anti-idiotype antibody against it. What steps should be taken to make this mouse monoclonal antibody most suitable for use in the patient. Is it highly likely that, once made, such an engineered antibody will be generally useful for lymphoma patients?

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Responses

  • tim
    What is the arrangement of its k light chain dna?
    8 years ago
  • Aimee-leigh
    When is the frequency of somatic mutation of immunoglobulin gene segments are greatest?
    8 years ago
  • Derek
    What are the 2 alleles of the immunoglobulin heavy chain?
    8 years ago
  • blake
    Do serparate exons encode the transmembrane portion of each membrane ig?
    8 years ago
  • maik
    How many different antibody molecules could be generated from germ line dna containing?
    8 years ago
  • rasmus holappa
    Why Vh gene segment cannot join directly to a Jh gene segment.?
    4 years ago
  • barry
    Why a Vh segment can not join directly with a Jh segment in heavy chain gene rearrangement?
    3 years ago
  • Aman Ambessa
    Why Vh segment cannot join directly with Jh segment in heavy chain gene arrangement?
    3 years ago
  • makda
    Do mice have immunoglobing?
    5 months ago

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