Antibody research Essay

Antibodieshttp://www.medscape.com/viewarticle/557205 function as mediators of specific humoral immunity by engaging diverse molecular and cellular effector mechanisms that serve to eliminate the antigen. They are produced by B lymphocytes and plasma cells in the lymphoid organs and the bone marrow, but they perform their effector functions at sites distant from their production.

Like the T cell antigen receptor, antibodies are able to recognize and bind antigens. Diversity and heterogeneity are characteristic features of these molecules. Antibodies, or immunoglobulines (Ig), are a group of glycoproteins present in the serum and tissue fluids of all mammals.

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They are either present in a membrane-bound form on B lymphocytes, where they function as B lymphocyte receptors for specific antigens, or they are present in a secreted form in the blood or lymph, where they carry out their effector functions in the adaptive immune response.

Structure
Mainly with the help of X-ray christallography and immunoglobulin gene sequencing studies, the structures of immunoglobulins became more transparent.

All antibody molecules share the same basic structural characteristics but display remarkable variability in the region that binds the antigen. There are as many as 109 different antibody molecules in every individual, each molecule with a unique amino acid sequence in its antigen-binding sites.
Antibody molecules are Y-shaped proteins composed of two identical heavy chains and two identical light chains that are joined by disulfid linkages. Both light chains and heavy chains contain a series of repeating, homologous units, which fold independently in a globular motif that is called an immunoglobulin domain. All four chains consist of amino-terminal variable (V) regions and carboxyl terminal (C) constant regions.
The variable regions are so named because they contain regions of variability in amino acid sequences that distinguish the antibodies made by one clone of B cells from the antibodies made by another clone. The variable region consists of three short stretches called the hypervariable segments. These stretches are all about 10 amino acids long and form loop structures that contact the antigen. Therefor the variable regions of both the heavy (VH) and the light chains (VL) form the antigen binding site, which is also called Fab region, of the immunoglobulin. Two identical Fab regions are present at the ends of the Y in every immunoglobulin structure, and therefor the molecule has two antigen binding sites.
The effector function of antibodies are associated witht the non-antigen-binding portions, which exhibit relatively few variations among different antibodies. These Fc regions, so-called because it is the fragment of the molecule that most readily crystallized, contain the constant regions, that are separated from the antigen binding sites and do not participate in the antigen recognition. The heavy chain C regions (CH) interact with other effector molecules and cells of the immune system and therefor mediate the effector function of antibodies. The carboxy terminal ends of the heavy chains of membrane bound antibodies, serving as B cell receptor molecules, are anchored in the plasma membrane of B lymphocytes. The C regions of the light chains (CL) do not participate in effector functions and are not attached to cell membranes.

Function
The primary function of antibodies is to bind antigen. B cells initially produce only membrane-bound Ig serving as receptors on B cells that mediate the antigen-triggered activation of B lymphocytes. Following activation, some B cells differentiate into antibody producing plasma cells, where changes in the carboxy terminal end of the antibody lead to the secretion of soluble Ig with the same specificity as the original membrane-bound Ig receptor. The secreted antibodies function as mediators of specific humoral immunity. In some cases this antibody-antigen binding has direct effects – the neutralization of bacterial toxins or prevention of viral attachment to host cells by opsonisation. But most of the effector functions of antibodies are mediated by the binding of heavy chain C regions to different cell surface receptors, also called Fc receptors, on cells including phagocytes, elicting antibody-mediated opsonisation and phagocytosis, or NK cells, inducing the antibody-dependant cell-mediated cytotoxicity (ADCC). Antibodies also bind macromolecules such as the complement system proteins also leading to the eradication of the pathogen.
Receptors for immunoglobulins are therefor expressed on many different cells, including mononuclear cells, neutrophils, natural killer cells, eosinophils and mast cells. They interact with the Fc regions of different classes of immunoglobulins and promote activities such as phagocytosis, tumor cell killing and mast cell degranulation.

Antibody isotypes
During the activation process of the B lymphocyte the cells may undergo a process called isotype switching, in which the type of CH region, and therefor the antibody isotype, produced by the B cell, changes but the V regions, and therefor the antibody-binding site, do not undergo structural changes. As a result different progeny of the original B cell may produce different isotypes and subtypes, which in turn are responsible for different effector functions. Therefor different isotypes of antibodies perform different effector functions.
There are five different classes of immunoglobuline molecules in most higher mammals, namely IgG, IgA, IgM, IgD and IgE. They differ in size, charge, amino acid composition and carbohydrate content. In addition to the differences between these classes, the immunoglobulins within each class are also very heterogeneous.
The class and subclass of an immunoglobulin molecule are determined by its heavy chain type. Thus the four subclasses of human IgG (IgG1-IgG4) have heavy chains called γ1, γ2, γ3 and γ4 that differ slightly, although all are recognizably γ heavy chains (heavy chains are designated by the letter of the Greek alphabet corresponding to the overall isotype of the antibody). There are also known to be subclasses of IgA (namely IgA1 and IgA2) but non have been described for IgM, IgD or IgE. The heavy chain C regions of all antibody molecules of one isotype or subtype have essentially the same amino acid sequence and this sequence is different in antibodies of other isotypes.

Antibody specificity, diversity and affinity
The recognition of antigen by an antibody involves noncovalent, reversible binding (hydrogen bonds, van der Waals forces, or hydrophobic interactions) and the antigen-antibody interactions are always of high specificity. Antibody to the measles virus will only bind the measles virus and confer immunity to this disease, but will not recognize and protect against an unrelated virus, like the polio virus. The specificity of an antiserum is equal to the sum of the actions of every antibody in that serum. The antibody popluation may contain many different antigen-binding sites, each reacting with a different epitope, or even different part of the epitope. However, when some of the epitopes of the antigen “A”, are shared by another antigen, antigen “B”, then a proportion of the antibody pool against antigen “A” will also react with antigen “B” – a phenomenon called cross-reacivity.
In contrast to the T cell receptor, antibodies, either membrane-bound or circulating, recognize the overall shape of an epitope rather than particular chemical residues. Besides the ability to distinguish between small differences in the primary amino acid sequence of protein antigen, they also recognize differences in charge, optical configuration and steric conformation. In contrast to T cells receptors, antibodies are able to bind the antigen in its native form and are therfor able to bind a variety of biological molecules including sugars, lipids, carbohydrates, proteins, and nucleic acids.

Every day the human immune system encounters a great variety of foreign substances (bacteria, viruses, toxins, etc.). In order to perform their crucial role in the line of defense, the antibody repertoire must be extremly diverse. Through a complex process called gene-splicing or somatic recombination the B lymphocytes in humans are capable to produce up to 1 x 109 antibodies that differ in their composition of their binding sites. Somatic recombination or somatic rearrangement is a process of DNA recombination, by which the functional genes encoding the variable regions of the antigen receptor are formed and DNA sequences, initially separated from one another, are brought together by enzymatic deletion and religation. The possibility to combine different sequences in different ways assures the capacity to generate an enormous number of unique binding sites and thereby make antibodies what they are- the perfect line of defense.
The tight binding of antibodies to the respective antigen is achieved by high-affinity and high-avidity interactions. As a late event in T cell-dependant antibody responses a mechanism in antigen-stimulated B lymphocytes, called affinity maturation, generates high-affinity antibodies. Changes in the structure of the V regions of the antibody through point mutations (somatic hypermutations) increase the average binding affinity for an antigen as the humoral response develops.

Monoclonal and polyclonal antibodies
There are two ways to produce antibodies for research or therapy purposes. The production of polyclonal antibodies, obained by the immunization of a mammal, is one way to produce high levels of antibodies. The blood of the immunized mammal is removed and the antibodies can be purified directly from the serum. The polyclonal mixture of resulting antibodies reacts with multiple epitopes on the antigen. In contrast, monoclonal antibodies are antibodies derived from the progeny of a single B cell clone. The development of the technology for producing limitless amounts of monoclonal antibodies in 1975 by Kohler and Milstein revolutionized the uses of antibodies in diagnostics, reseach and therapy. By fusing B lymphocytes with immortal myeloma cells (mostly HAT-sensitive mouse myelomas), and thereby producing the socalled hybridoma cell line, they found a way to culture these cells and produce a homogeneous population of antibodies. These hybridoma cells produce many copies of the exact same antibody specific for the antigen.

Research and therapy
In research, antibodies are used for immunological identification of proteins using techniques including Western Blot, ELISA and flow cytometry. In these assays, antibodies are used to detect proteins. Some common applications of monoclonal antibodies include the identification of phenotypic markers on particular cell types, diagnosis of many infectious and systemic diseases relying on the detection of particular antigens or antibodies in the circulation or the tissue of the patient (immunoassays) or tumor diagnosis and therapy. Tumor-specific monoclonal antibodies are used for the detection of tumors and are also used in tumor therapy to induce elimination of tumor cells. Monoclonal antibodies typically use a combination of mechanisms in directing cytotoxic effects to a tumor cell. They interact with components of the immune system, like the complement system proteins, or alter signal transduction within the tumor cell, or act to eliminate a critical cell-surface antigen. Monoclonal antibodies can also be used to target payloads (radioisotopes, toxins or drugs) to directly kill tumor cells. Finally, monoclonal antibodies, like Cetuximab, an anti-epidermal growh factor antibody, or alemtuzumab, an anti-CD52 antibody, can be used synergistically with traditional chemotherapeutic agents to eliminate tumors.

Autoimmune disorders can often be traced to antibodies that bind the bodies own epitopes. Abnormal antibody production is also recognized as the basis of specific endocrine and neurological diseases and their complications. Myasthenia gravis (MG) is an organ-specific autoimmune disease in which autoantibodies against nicotinic acetylcholine receptors (AChR) at the postsynaptic membrane cause loss of functional AChR and disturbed neuromuscular transmission.
Designed monoclonal antibody therapy is already employed in a number of diseases including rheumatoid arthritis and some forms of cancer. Presently many antibody related therapies are undergoing extensive clinical trials for use in practice. Also in therapies against diseases like multiple sclerosis the suppression of inflammation with short-term monoclonal antibody treatment is discussed as a promissing option.

Links

http://www.antibodyresource.com/antibody.html
http://www.chemicon.com/resource/ANT101/a1.asp
http://www.biology.arizona.edu/IMMUNOLOGY/tutorials/antibody/structure.html
http://www.antibodyresource.com/educational.html
I am doing the group dicussion every weeks. This week i will be the leader of the group on the topic `B cell and Antibiodics Therapies` and will summary the work of my responders at the end and report to the professor on Sunday. I do not know if you can do work on Sunday for me for the Summary part, i am willing to pay extra if you agree to do it for me, just follow the instruction and do it. It does not more than one page though. I will be working on that day also. Now I only ask you to do the origianal message so I can post it on the dicussion board.
Note: Please send me the direct link for what you make the researchs on. Thanks.

Below are the instructions how to achieve the message for the topic:

Original Message
• Prior to Wednesday at 11:55 PM, post your original message to the group based on the discussion topics given by the instructor
• Include answers/thoughts to the questions asked in the discussion topic in your original message based on information you have researched (cite your sources)
• Original messages must be at least 200 words.
• You must use sources other than Wikipedia, and should not copy & paste long passages of material— summarize the information you want to present.
• At the end of the message, direct the discussion by including at least 2 specific questions about your original message or another related topic that you want the responder students to answer
• Send an email to the rest of the your group members once you have posted the message so that they will know to review your message & reply (use Group email function in BB)

http://www.fccc.edu/docs/sci_report/Weiner.pdf

How does monoclonal antibody therapy work in lymphoma?
Professor Peter WM Johnson, Department of Oncology, Royal South Hants and Southampton General Hospital

It is more than 20 years since Kohler and Milstein published the first report describing production of monoclonal antibodies by hybridomas[1], and in that time expectations of their direct application to therapy have risen and fallen repeatedly. It is only recently that an antibody has shown enough therapeutic promise to approach licencing, despite numerous studies in vitro, in animal models and in early clinical trials. Although exciting responses to anti-idiotypic antibody treatment were documented in B-cell lymphomas over a decade ago[2], the escape of antigenic variant clones and the logistic difficulties of raising anti-idiotype monoclonals in murine systems initially restricted this approach.

The other potential obstacles to successful application of antibody therapy (table 1) have been extensively investigated, but despite the improvements in understanding of these, it remains difficult to predict the effect of antibody administration to patients with malignancies. An example of this is the antibody Campath-1H, which appeared highly effective in early clinical studies[3] in keeping with the favourable characteristics of the CD52 antigen, but which has been disappointing in larger clinical trials[4], with the possible exception of studies in T-prolymphocytic leukaemia and to a lesser extent chronic lymphocytic leukaemia[5].

From the earliest days of antibody research, the assumption was made that the principal cytotoxic effect of antibodies in vivo would be through activation of endogenous effector mechanisms. These would include complement activation, or attachment and activation of cells with Fc receptors such as NK cells or macrophages (antibody-dependent cell-mediated cytotoxicity). Recognition of the potential deficiencies in the host effector mechanisms in cancer patients, especially those treated with chemotherapy, has led to the development of alternative effector strategies by conjugation of toxins or radioactive isotopes to the antibodies, and to the use of bispecific antibodies capable of simultaneously binding T-cells. However, recent evidence suggests that antibodies may be less dependent than previously supposed upon host effectors, and that the specific results of binding to cell surface molecules may play an equal or more significant role. This has important consequences for the design of therapy in future, and indicates that the development of appropriate models for testing of antibodies will be a crucial requirement.

The first human B-cell antigen (after surface immunoglobulin) to be identified by a monoclonal antibody was CD20[6]. This is a nonglycosylated 33 to 37 kD integral membrane protein which is thought to act as a calcium channel and to play a role in the regulation of B-cell growth and differentiation[7]. The development of a chimeric antibody with human IgG1 Fc regions and murine variable regions has led to the wide testing of anti-CD20 therapy in human B-cell lymphomas[8], with encouraging results (table 2). In recurrent follicular lymphoma, responses were seen in up to 50% of patients treated with 4 weekly antibody infusions in several trials[9, 10], and in more aggressive lymphomas responses were seen in one third [11]. The combination of anti-CD20 antibodies with chemotherapy [12] or their use in targeting radiotherapy [13, 14] seem particularly effective, with high response rates seen even in heavily pre-treated patients. The response rate to anti-CD20 antibodies correlates to some extent with the level of antigen expression on the B-cells, so that small lymphocytic lymphomas which express lower CD20 levels than follicular lymphomas appear to respond less often. However, mantle cell lymphomas which express the highest levels of CD20 do not appear to have such a high response rate. Given the well-documented chemoresistance of mantle cell lymphoma, this suggests that there may be elements of cross-resistance between the effects of chemotherapy and antibodies, a finding in keeping with the suggestion that intracellular death pathways may play a role in the therapeutic effect.

The evidence for receptor-mediated effects of antibodies comes from several different lines of investigation. The earliest studies using antibodies and peripheral blood B-cells showed that the anti-CD20 antibody 1F5 caused activation of resting cells from G0 to G1 [15], as did anti-immunoglobulin antibodies, whereas the B1 anti-CD20 inhibited progression from G1 to S after mitogen stimulation [16]. Direct comparative studies of 1F5, B1 and a third murine antibody IDEC-2B8 on several different B-cell lymphoma lines demonstrated variation in the degree of growth inhibition, with B1and 2B8 more effective than 1F5 [17]. The humanised antibody IDEC-C2B8 had a similar growth inhibitory effect to 2B8, indicating that the human Fc region was not responsible. Investigation of the mechanisms of CD20 signalling has shown that both tyrosine and serine/threonine kinases may be affected [18], and similar results have been reported with the use of anti-idiotypic antibodies [19]. Other studies have suggested that cross-linking of CD20 molecules may cause increased calcium influx across the plasma membrane and that this may trigger the apoptotic pathway [20]. Certainly apoptosis appears to be a common result of surface molecule cross-linking with antibodies to a variety of molecules including IgM, CD19, CD22 and MHC class II [21, 22].

The results of CD20 cross-linking are to some extent mirrored in studies using anti-CD40 antibodies. CD40 is a member of the TNF receptor family which also includes Fas, a powerful apoptotic signalling molecule. Originally identified as a B-cell proliferation signal, anti-CD40 antibodies have been used to rescue isolated germinal centre B-cells from apoptosis [23], and when presented in an array in the presence of cytokines to sustain proliferation of both normal [24] and malignant [25] B-cells. However, further studies have demonstrated inhibitory effects of anti-CD40 antibodies on transformed B-cell lymphoma lines both in vitro and in mouse lymphoma models [26, 27]. Studies on epithelial cell lines have also yielded the interesting observation that cross-linking of CD40 may induce apoptosis in transformed cells [28], whilst in the normal counterparts it can act as a mitogen [29]. There are clearly important differences in the downstream signalling pathways which dictate the precise response in any particular cell type, and these seem likely to be reflected in the response to antibody treatment.

One further piece of evidence regarding the importance of signalling in determining the response to antibodies comes from work in a syngeneic mouse lymphoma model [30]. In these studies comparing antibodies to CD19, CD22, CD40, CD74, MHC class II and idiotype there was a poor correlation of the binding affinity, complement activation or stimulation of ADCC to the therapeutic effect of the antibodies in vivo. Only anti-idiotype, anti-CD19 and anti-CD40 showed significant activity against lymphomas in the mice, despite equally good or better binding characteristics for other antibodies. Once again this suggests that ligand binding may play a major part in the effect.

In conclusion, the application of monoclonal antibodies for the treatment of lymphoma is reaching an exciting stage after a long gestation. As the mechanisms by which antibodies exert their effects are discovered so it may be that more potent treatments can be developed, particularly capitalising upon the receptor-mediated effects on apoptotic pathways and using an understanding of these to maximise specific cytotoxicity.

Table 1.

Potential difficulties in monoclonal antibody therapy.

1. Inability to deliver the antibody to the malignant cells:

Bulky tumour masses
Non-specific antibody binding to other tissues
Shedding of the antigen from the malignant cells
Modulation of antigen expression by malignant cells
2. Inability to kill all the malignant cells:

Failure of patient’s immune effector mechanisms
Resistance of malignant cells to effector mechanisms
Antigen-negative malignant cells: a priori or emerging subsequently
Non-expression of antigen by malignant stem-cells
3. Toxicity of the treatment:

Allergic reaction
Formation of anti-antibody antibodies
Normal tissue toxicity
Table 2. Results of recent clinical studies with anti-CD20 antibodies:

Study
Patients (N)
Treatment
Response rate
(%)
Response duration
(Months)
Trials in recurrent disease
[9]
WF A -D (50)
375mg/m2 X 4
48
9
[10]
LG (34 )

HG (3)
375mg/m2 X 4
50
10.3
[31]
LG (37)
375mg/m2 X 8
63

[11]
DLCL (30)

Mantle cell (12)

Other WF D-H (10)
375mg/m2 X 8

vs

500mg/m2 X 8
37

33

20

10
Trials of initial therapy
[11]
WF D-H (9)
375mg/m2 X 8

vs

500mg/m2 X 8
33
10
[12]
LG (32)
CHOP X 6 +

375mg/m2 X 6
100
>12
Trials of radioimmunotherapy
[13]
21 recurrent ML

(19 LG, 2 transformed)
CD20-131I-25Gy
86

(76 CR)

[14]
13 LG

8 transformed

7 IG
CD20-131I-0.75Gy
100

75

43

[32]
17 LG
CD20-90Y-0.2-0.4mCi/kg
82

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