Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
1998-04-09
2002-01-01
Bansal, Geetha P. (Department: 1642)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C435S069100, C435S070210, C530S387300, C436S536000
Reexamination Certificate
active
06335163
ABSTRACT:
RIGHTS IN THE INVENTION
This invention was made with support from the National Institutes of Health under grant number R01/AI23909 and the United States government has certain rights in the invention.
BACKGROUND OF THE INVENTION
1. Field of the Invention
The invention relates to the creation, interconversion and use of libraries of polyclonal antibodies, cell surface receptors and other proteins with variable regions. These variable regions are linked, cloned into expression vectors which can be maintained, selected and amplified as desired, and the libraries or sub-libraries of variable regions transferred to other expression vectors without loss of overall diversity or complexity. The resulting libraries of variable regions and libraries of whole proteins can be used to treat, prevent or diagnose specific diseases and disorders including neoplasias, malignancies, infections, and genetics defects and deficiencies.
2. Description of the Background
Lymphocytes constitute about 20% of blood leukocytes and are the main components of the mammalian antigen recognition system occurring predominantly in two forms, B cells and T cells. T cells differentiate in the thymus, and possibly other tissues, into cytotoxic (T
c
) cells, helper (T
H
) cells and suppressor (T
s
) cells. These T cells recognize foreign antigen in association with major histocompatibility complex (MHC) antigens via a specific T cell receptor (TcR). This receptor is highly polymorphic and clonally distributed. The typical TcR is a disulfide linked heterodimer consisting of an &agr; and a &bgr; polypeptide which is expressed on the surface of mature T cells. The two chains are similar in size possessing a transmembrane portion encompassed within a constant region and a polymorphic variable region that possesses considerable structural homology with immunoglobulins. Each variable region is composed of a variable (V) segment, a joining (J) segment, and a diversity (D) segment (&bgr; chain only) which assemble into the polymorphic region. Such diversity is necessary for the T cells to respond to a wide variety of antigens.
B cells differentiate in the bone marrow of adult mammals developing from pre-B cells into antibody-producing plasma cells. The importance of B cells to the immune system is highlighted by those rare immunodeficiency diseases in which the patient can only survive by repeated gamma globulin injections. One of the fascinating aspects of B cells is their heterogeneity of antibody expression. It has been estimated from observations that no more than two out of every 10
8
different antibodies could be identical. Not surprisingly, the mechanisms which are responsible for such diversity are not fully understood. Antibodies and TcRs, both having constant and variable regions, fall into what is referred to as the immunoglobulin superfamily.
The process of immunoglobulin expression is initiated with activation of resting B cells. In the T cell independent pathway, mitogen binds to surface receptors of B cells stimulating expression of fairly low affinity IgM monomers. Upon recognition of foreign antigen, these IgM monomers cross-link at the cell surface and are internalized. Antibody expression then switches to the transcription and translation of higher affinity IgG, IgE, IgA, or pentameric IgM. In the T cell dependent pathway, mitogen is again required to stimulate the resting B cell, however, after internalization, antigen is processed within the cell to reemerge on the cell surface in association with MHC class II molecules. As an antigen presenting cell (APC), antigen-MHC complexes are recognized by and stimulate T cell activation and the production of a number of T and B cell soluble mediators. Information received from these surface complexes is transmitted to the B cell's nucleus via second messengers which, among other effects, leads to increased cyclic nucleotide metabolism and protein kinase C activity. In both T cell dependent and independent pathways, B cells develop into functionally mature, antibody producing cells.
Antibodies are bifunctional molecules comprising two heavy (H) chains and two light (L) chains joined with interchain disulfide bonds. Each chain contains constant (C) and variable (V) regions, which can be broken down into domains designated C
H1
, C
H2
, C
H3
and V
H
, and C
L
and V
L
. IgM exists as cell surface monomers or circulating pentamers held together with a 137 amino acid peptide called the J chain. IgA molecules also circulate, but in pairs linked with J chain and contain a small secretory component (SC) which is involved in transport across epithelial membranes. Antibody binds to antigen via the variable region domains contained in the Fab portion and, after binding, interacts with the rest of the immune system through the effector functions of the constant region domain mostly through the Fc portion. Effector functions include activation of the complement cascade, interaction with effector cells such as lymphocytes, and compartmentalization of immunoglobulins. Constant regions are also thought to influence the stabilities of the different immunoglobulins. For example, IgG is relatively stable with a circulating half-life of about 23 days. IgG is also the immunoglobulin responder associated with the secondary immune response. This immunoglobulin fixes complement through the classic complement cascade and has the ability to recruit macrophages and neutrophils. Pentameric IgM is another very strong activator of the classic complement cascade and has a serum half-life of about five days. IgA has a serum half-life of 5-6 days, activates complement through the alternate pathway, and is the principal antibody in mucus secretions.
The antigen binding domains, the variable regions, are encoded in the variable region genes which are somewhat scattered in the genome and must be brought together by a process referred to as somatic recombination. In this process, the V, D and J (not related to the J chain) segments of the host genome are brought together to form a gene region. This region is spliced to the mRNA encoding the antibody's constant region domain to be expressed together as a polypeptide and ultimately as an antibody molecule.
Antibody constant regions are the principal determining features of antibody class for both heavy and light chains, and are encoded on about fifteen different genes. The five classes of heavy chain genes are designated alpha (&agr;), gamma (&ggr;), delta (&dgr;), mu (&mgr;), and epsilon (&egr;), and the two light chain genes, kappa (&kgr;) and lambda (&lgr;). Variable regions, which contain the antigen binding site, are encoded on upwards of one thousand different genetic regions. These regions selectively recombine to create the amino acid combination required to recognize and bind to the target antigen. Binding site variability is not uniformly distributed, but each domain contains a number of highly variable portions called hypervariable regions (HVR), or complementarity determining regions (CDR), and it is these regions which actually interact with antigen.
The generation of binding-site diversity is a combination of several factors; (1) the combination of different V
H
and V
L
domains, (2) the combination of different V, D, and J regions to form the variable domain, (3) the generation of novel diversity at domain junctions referred to as junctional-diversity, and (4) diversity due to somatic mutation. Somatic mutation, to a large extent, is also responsible for maturation of the immune response wherein B cell clones which proliferate during development of the humoral response have an increasingly higher affinity for the antigen. The combination of these processes, in theory, allows an organism to generate a specific immune response to nearly any antigen.
The stimulation of an antibody response is the basis behind most forms of vaccine therapy and prophylaxis. As a prophylaxis, antigen in the form of killed or attenuated microorganism or purified protein is administered to a patient. Administration of this vaccine, it is hoped,
Bansal Geetha P.
Nixon & Peabody LLP
The Trustees of Boston University
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