Immunoglobulins: structure and function

Antigen adsorbed serum Immunoglobulins Definition: Glycoprotein molecules that are present on B cells (BCR) or produced by plasma cells (antibodies) in response to an immunogen + - albumin Amount of protein globulins α11 α2 2 β γ Immune serum Antigen adsorbed serum Mobility

Immunoglobulin Structure heavy and light chains disulfide bonds inter-chain intra-chain disulfide bond carbohydrate CL VL CH2 CH3 CH1 VH hinge region

complement binding site binding to Fc receptors Immunoglobulin Fragments: Structure/Function Relationships antigen binding complement binding site placental transfer binding to Fc receptors

Immunoglobulin Structure variable and constant regions hinge region hinge region carbohydrate disulfide bond CH1 CL VH CH2 CH3 domains VL & CL VH & CH1 - CH3 (or CH4) oligosaccharides

Ribbon structure of IgG

Immunoglobulin Fragments Structure/Function Relationships Fab antigen binding valence = 1 specificty determined by VH and VL papain Fc effector functions Fc Fab

Immunoglobulin Fragments: Structure/Function Relationships Fab antigen binding Fc effector functions F(ab’)2 - Bivalent! pepsin Fc peptides F(ab’)2

Why do antibodies need an Fc region? detect antigen precipitate antigen block the active sites of toxins or pathogen-associated molecules block interactions between host and pathogen-associated molecules the (Fab)2 fragment can - inflammatory and effector functions associated with cells inflammatory and effector functions of complement the trafficking of antigens into the antigen processing pathways but can not activate (role of Fc region)

Immunoglobulin Structure-Function Relationship cell surface antigen receptor on B cells allows B cells to sense their antigenic environment connects extracellular space with intracellular signalling machinery secreted antibody neutralization opsonization complement fixation

Variability in different regions of the Ig determines Ig classes or specificity isotype allotype idiotype (Classes/subclasses) Sequence variability of H/L-chain constant regions Allelic variants Sequence variability of H and L-chain variable regions (individual, clone- specific)

Human Immunoglobulin Classes encoded by different structural gene segments (isotypes) IgG - gamma (γ) heavy chains IgM - mu (μ) heavy chains IgA - alpha (α) heavy chains IgD - delta (δ) heavy chains IgE - epsilon (ε) heavy chains light chain types kappa (κ) lambda (λ)

PRODUCTION OF IMMUNOGLOBULINS

Antigen A Antigen A and B level of antibodies days secondary response against antigen A primary response against antigen A primary response against antigen B days napok Antigen A Antigen A and B

Polyclonal antibody response Ag Polyclonal antibody Immunserum Set of B-cells Ag Ag Activated B-cells Antibody-producing plasma-cells Antigen-specific antibodies

WHICH KIND OF ANTIBODIES? Gamma-globulin fraction: mixture of policlonal antibodies – normal IgG antibodies ~10 mg/ml albumin Protein amounts globulins immune serum 1 2   antigen adsorbed serum Mobility - + WHICH KIND OF ANTIBODIES? Memory antibodies produced by environmental stimuli REFLECTING TO THE IMMUNE RECORD OF THE INDIVIDUAL Which kind of pathogens we met before

Case study (Multiple myeloma) In 1989, a 55-year-old housewife, who had been in good health her entire life, began to experience excessive fatigue. Her physician did not find abnormalities on physical examination. The blood sample revealed mild anemia; red blood cell count was 3.5 x 106 / ml (normal 4.2-5.0 x 106 / ml), white blood cell count was 3600 / ml (normal 5000 / ml). The sedimentation rate of her red blood cells was 32 mm / h (normal <20 mm / h). (Sedimentation is accelerated when fibrinogen or IgG content of the blood plasma is elevated.) The concentration of IgG was found to be 3790 mg / dl (normal 600 - 1500 mg / dl), that of IgA 14 mg / dl (normal 150 – 250 mg / dl) and that of IgM 53 mg / dl (normal 75 -150 mg / dl).

Case study (Multiple myeloma) Electrophoresis of her serum revealed the presence of a monoclonal protein, which on further analysis was found to be IgG with lambda light chains. normal serum serum from the patient Radiographs of all of her bones did not show any abnormality. No treatment was advised.

Case study (Multiple myeloma) In April 1991 her serum IgG was 4520 mg / dl, and in January 1992 it was 5100 mg / dl. By November 1992, her anemia had worsened and her red blood cell count had fallen to 3.0 x 106 / ml. At the same time her white blood count had fallen to 2600 / ml. In December 1992, she experienced the sudden onset of upper arm pain and headache. Radiographs of the skull and the left upper arm showed ‘punched out’ lesions in the bones.

Case study (Multiple myeloma) She was treated with melphalan (methylphenylalanine mustard), corticosteroids, and irradiation. Her symptoms improved. In April 1993, further chemotherapy was given because of the persisting elevation of her serum IgG. The treatment reduced her serum IgG level from 8200 mg / dl to 6000 mg / dl. In February and in May 1995, she was found to have pneumonia. She was treated successfully with antibiotics. She recovered from this episode in the hospital and remained fully active. She required blood transfusion for her anemia and complained at times of bone pain. Her serum IgG was stable at 6200 mg / dl. Although she was in relative good health as our case history ended, her outlook for survival was very poor. Recently, bone marrow transplants have been used to cure patients with multiple myeloma. /Myeloma proteins have played an important part in the history of immunology. (Bence-Jones protein, subclasses of IgG, amino acid sequence of immuno- globulin molecule)/

Questions The serum IgG from her was assumed to be monoclonal because it migrated as a tight band on electrophoresis in an agarose gel, and because it reacted with antibodies to lambda but not to kappa chains. What other evidence could be brought to bear to prove the monoclonality of this IgG? The IgG could also be shown to belong a single subclass of IgG, that is IgG1, IgG2, IgG3, or IgG4. Further more, it would be possible to show that a single variable-region gene was rearranged to form this IgG. She became anemic (low red blood cell count) and neutropenic (low white blood cell count). What was the cause of this? The proliferation of malignant plasma cells in the bone marrow crowded out blood cell precursors. This creates a limitation on space in the bone marrow.

As her disease progressed, she became susceptible to pyogenic infection; for example, she had pneumonia twice in a short period. What is the basis of her susceptibility to these infections? Although her serum IgG concentration is quite elevated, almost all the IgG is secreted by the myeloma cells and is monoclonal. In fact, she has very little normal polyclonal IgG and has been effectively rendered agammaglobulinemic by her disease. In addition, her white blood cell count is decreased and she has too few neutrophils (<1000 / ml) to ingest bacteria in the bloodstream and lungs effectively. A monoclonal immunoglobulin in the serum is called an M-component (‘M’ for myeloma). Is the presence of an M-component in serum diagnostic of multiple myeloma? No. M-component appear in the blood as people age. About 10% of healthy individuals in the ninth decade of live have M-component. This is called benign monoclonal gammopathy. Without bone lesions and presence of many malignant cells in the bone marrow, the diagnosis of multiple myeloma cannot be made. Some people have IgM M-components in their blood. This is due to another malignancy of plasma cells called Waldenström’s macroglobulinemia, which differs in many ways from multiple myeloma and is a more benign disease.

What happens with the B cells in myeloma multiplex? Healthy individual Myeloma multiplex

B cell tumors

Structures of the ABO blood group antigens Defined by specific enzymes inherited co-dominant genes (Mendelian rules)

Donors and recipients for blood transfusion - + + + - - + + - + - + - - - - The produced antibodies against blood group antigens are IgM isotypes

Pathological consequences of placental transport of IgG (hemolytic disease of the newborn) Passive anti-D IgG

Features of antibody-antigen interaction Valency: numbers of antigens / antibody Affinity: the strength of interaction between a specific antigen and one binding site of the antibody Avidity: sum of affinities of the binding sites of a a given antibody

B Secretory IgA and transcytosis Epithelial cell J ‘Stalk’ of the pIgR is degraded to release IgA containing part of the pIgR - the secretory component J C S s J C S s J C S s IgA and pIgR are transported to the apical surface in vesicles J C S s Epithelial cell pIgR and IgA are internalised Polymeric Ig receptors are expressed on the basolateral surface of epithelial cells to capture IgA produced in the mucosa J C S s B cells located in the submucosa produce dimeric IgA B

Features of polyclonal and monoclonal antibodies Polyclonal antibody Monoclonal antibody (low affinity) (high affinity) Number of recognized antigen determinants several (frequent cross-reactions) one (but frequent cross-reactions) mostly one Specificity polyspecific often polyspecific monospecific Affinity Varying (diverse antibodies) low high Concentration of non-specific immunoglobulines Cost of preparation Standardisability Impossible (or uneasy) easy Amount limited unlimited Applicability method-dependent excellent

Monoclonal antibodies product of one B-lymphocyte clone homogeneous in antigenspecificity, affinity, and isotype - can be found in pathologic condition in humans (the product of a malignant cell clone) advantages against polyclonal antibodies: antibodies of a given specificity and isotype can be produced in high quantity and assured quality. therapeutic usage of monoclonals: anti-TNF-α therapy in rheumatology, tumor therapy

Possible use of monoclonal antibodies Identifying cell types Immunohistochemistry Characterization of lymphomas with CD (cluster of differentiation) markers Isolation of cells Isolation of CD34+ stem cells for autologous/allogeneic transplantation (from peripheral blood!) Blood group determination (with anti-A, anti-B, and anti-D monoclonals) Identification of cell surface and intracellular antigens Cell activation state Targeted chemotherapy CD20+ anti-B-cell monoclonals in non-Hodgkin lymphoma Prevention of organ rejection after transplantation

Monoclonal antibodies as drugs? Mouse monoclonal antibodies may elicit an immune response upon administration in human subjects. (see immunogenicity-determining factors!) How can we solve this problem?

Evolution of monoclonal antibodies Mouse Human Chimeric Humanized The participation in regulatory review allows the researcher reviewer a reality check for the real regulatory impact of the MOA data

Monoclonals as drugs - Tumor therapy Monoclonals can be used for targeted chemotherapy of tumors. It is cell-type specific, but not specific to malignant cells! Immunsuppressive monoclonals Cell-type specific immunsuppression

Monoclonal antibody nomenclature The nomenclature of monoclonal antibodies is a naming scheme for assigning generic, or nonproprietary names to a group of medicines called monoclonal antibodies. This scheme is used for the World Health Organization’s International Nonproprietary Names. Components of nomenclature: Example:Abciximabab- + -ci(r)- + -xi- + -mab, it is a chimeric monoclonal antibody used on the cardiovascular system

Monoclonals in tumor therapy „Naked MAb”, unconjugated antibody Anti-CD20 (rituximab – Mabthera/Rituxan, chimeric): B-cell Non-Hodgkin lymphoma Anti-CD52 (campath – Mabcampath, humanized): chronic lymphoid leukaemia Anti-ErbB2 (trastuzumab – Herceptin, humanized): breast cancer Anti-VEGF (bevacizumab – Avastin, humanized): colorectalis tu. (+ Lucentis!) Anti-EGFR (cetuximab – Erbitux, chimeric): colorectalis tu. (+ Vectibix, rekomb. humán!) 2. Conjugated antibody Anti-CD20 + yttrium-90 isotope (ibritumomab- Zevalin) Anti-CD20 + iodine-131 (tositumomab – Bexxar)

Immunsuppressive antibodies 1. Anti-TNF-α antibodies infliximab (Remicade): since 1998, chimeric adalimumab (Humira): since 2002, recombinant human Etanercept (Enbrel) – dimer fusion protein, TNF-α receptor + Ig Fc-part Not a real monoclonal antibody, no Fab end, the specificity is given by TNF-receptor! Indications of anti-TNF-α therapy: Rheumatoid arthritis Spondylitis ankylopoetica (SPA - M. Bechterew) Psoriasis vulgaris, arthritis psoriatica Crohn-disease, colitis ulcerosa (usually - still – not in the first line!)

Immunsuppressive antibodies 2. Muromonab-CD3 (OKT-3) egér IgG2a Against CD3 pan-T-cell antigen, after transplantation; It is rarely (or not) used nowadays (mouse protein!); ongoing trials in diabetes mellitus, with the humanized version Omalizumab (Xolair): Anti-IgE humanized IgG1k monoclonal Ind.: allergic asthma, Churg-Strauss sy. Daclizumab (Zenapax): anti-IL-2 receptor humanized antibody Ind.: transplantation basiliximab (Simulect): as daclizumab, but chimeric! efalizumab (Raptiva): anti-CD11a, humanized, used in psoriasis

Molecular targeted drugs Name Type Target Indications Alemtuzumab (Mabcampath) Daclizumab (Zenapax) Basiliximab (Simulect) Rituximab (Rituxan/Mabthera) Trastuzumab (Herceptin) Gemtuzumab Ibritumomab (Y90) Edrecolomab Gefitinib Imatinib Monoclonal Ab, humanized Monoclonal IgG1, chimeric Monoclonal IgG1, humanized Monoclonal IgG4, humanized Calicheamicinnel konjugált Monoclonal IgG1, murine Monoclonal IgG2, murine EGFR-TKI KIT-TKI CD52 IL-2 R CD20 HER2/neu CD33 EpCAM EGFR TK TK CLL, CML transplantation Lymphoma Breast cancer, NSC lung cancer leukemia lymphoma CRC NSCLC GIST, CML

Further possibilities with monoclonals Radioimmunotherapy As Zevalin, Bexxar – monoclonal + isotope Antibody-directed enzyme prodrug therapy (ADEPT) An enzyme is linked to the antibody, and the enzyme will make citotoxic drug from the later administered prodrug Immunoliposomes Targeting nucleotides or drugs in liposomes, linked to an antibody Non-immunological targets as abciximab (ReoPro): inhibition of thrombocyte-aggregation

humanized mouse monoclonal antibodies human monoclonal antibodies PASSZÍV IMMUNIZÁLÁS immunization mouse monoclonal antibodies Human immunoglobulin transgenic mouse humanized mouse monoclonal antibodies immunization human monoclonal antibodies PROTECTED SUBJECT serum antibody ENDANGERED SUBJECT immunsystem is not activated prompt effect temporary protection/effect Immunoglobulin degradation

Passive immunization Type Application Intramuscular (less effective due to lower dose) HBV-Ig; Varicella-zoster-Ig; Intravenous (IVIG) Bruton-agammaglobulinaemia; variable and mixed immunodeficiencies with hypogammaglobulinaemia; Anti-venom antibody treatment;