Monoclonal Antibodies

Monoclonal Antibodies

1. Introduction:

1.1.Definition:

“The Antibodies which are obtained from single antigen determinant molecule are known as Monoclonal Antibodies”.

Or

“mAbs are antibodies that are made up by identical immune cells that are all clones of unique parent cell”.

1.2.How mono clonal Antibodies differ from polyclonal Antibodies?

 mAbs are obtained from single antigenic determinant molecule while polyclonal antibodies obtained from various antigenic determinant molecules.

 mAbs are made by identical immune cells that are all clones of unique parent cell while polyclonal antibodies are made using several different immune cells.

 mAbs bind only a single epitope of antigen but polyclonal antibodies can bind with different epitopes on the same antigen.

1.3. History of mAbs:

mAbs technique was devised by Kohler and Milstein (for which they shared a Nobel prize in 1984). They developed the methods by which large amounts of a single mab specific for one epitope can be obtained.

1.4.Important terms :

Antibodies: Antibodies (immunoglobulins) are soluble proteins, that are highly specific and extremely sensitive molecules of human immune system that help to defend against entities such as microorganisms /antigens.

Antigen: Antigen is a substance of high molecular weight which lead to activation of any sort of immune system, antigen may also be called as immunogen.

Epitope: Region of the antigen recognized by an antibody. 3

Paratopes: Region of antibody that binds the epitope.

Immune system: The system that protects the body from foreign substances and pathogenic organisms by producing an immune response such as producing antibodies from B lymphocytes or by T lymphocytes or complement system or by phagocytes.

1.5.Production of antibodies by normal human immune system:

Normal immune response is humoral immunity that may also be called as antibody mediated immunity. It is mediated by through B lymphocytes through the production of antibodies.

1.5.1. Mechanism of humoral immunity:

When B-lymphocytes come in contact with foreign antigen, they synthesize RNA and differentiate into plasma cells or immunocytes. These cells secrete antibodies (immunoglobulins). A particular plasma cells produces only one type of antibody. B-lymphocytes are converted into plasma cells. Immune plasma cells are called plasmoblasts. There is development of endoplasmic reticulum and ribosome (5 day). ER are filled with antibodies molecules and plasma secrete antibody. 4

1.6.Structure of antibody (immunoglobulin) and antigen:

  

Fab region: Antigen-binding fragment is a region on antibody that binds to antigens.

Fc region: Fragment crystallizable region is the tail region of an antibody that interacts with cell surface receptors called Fc receptor and some proteins of the complement system. This property allows antibodies to activate immune system.

Hinge region (inter-domain region): It is a flexible amino acid stretch in the central part of the heavy chain of IgG and IgA which links these two chains by disulfide bonds.

1.7.Types of antigens:

Complete antigen: Antigen which cause production of antibodies and react them.

Partial antigen:(hepten) The antigen which can’t produce antibodies but can react them.

Incomplete antigen:(natural) A significant amount of these antigens are required to produce an immune response every e.g in water.In food,in air etc.

1.8.Types of antibodies:

IgG, IgM, IgA, IgE, and IgD they are distinguished by the type of heavy chain they contain.

1.9.Origin of Mab:

 Mab of murine origin

 Mab of human origin

1.9.1. Mab of murine origin: Murine MAbs are obtained from murine hybridoma producedby fusion of B-lymphocytes from immunized mice or rats with murine myeloma cells. A great problem with the therapeutic use of murine monoclonal antibodies in man is the possible induction of antibodies in recipients against murine immunoglobulins (human anti-murine antibody or HAMA).This may result in adverse reactions and limit the duration of effective antibody therapy. In addition antibodies half life is relatively short.

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1.9.2. Mab of human origin: The advantages of human MAbs over murine MAbs are that humans recipients are less likely to develop antibodies against them and that human antibodies are likely to have the full range of biological functions.

1.10. Types and examples of MAbs :

1.10.1. Human MAbs: MAbs that are obtained from human source only by fusion withmyeloma cells of mice called human MAbs these antibodies use the suffix of ‘umab’ e.g. Adalimumab use for crohn disease.

1.10.2. Chimeric MAbs: (human-murine) MAbs that are made by using human Fc Ig components and murine Fb variable region and then fuse with myeloma cells of mice are called chimeric they use the suffix of ‘ximab’ e.g. Abciximab use for myocardialinfarction.

1.10.3. Humanized MAbs: MAbs that are obtained from by using human antibody an small part of mouse /rat monoclonal antibody and then fuse with myeloma cells to form hybridoma called humanized MAbs these MAbs use the suffix of zumab e.g Bevacizumab use for metastaticrenal cancer.

2. PRODUCTION OF MONOCLONAL ANTIBODIES:

Monoclonal antibodies are produced by following technique:

 Hybridoma technology

 In-vitro production

i. Batch wise tissue culture

ii. Semi-permeable membrane base

EBV Immortalization Technique 

2.1.Hybridoma Technology

It is a technology of forming hybrid cell lines (called hybridoma) by fusing a specific antibody producing B-cell with a myeloma cell that is selected for its ability to grow in tissue culture.

The antibodies produced by the hybridoma are all of a single specificity and are therefore known as monoclonal antibodies. 7

STEPS OF HYBRIDOMA TECHNOLOGY:

a. Immunisation of a moue

b. Isolation of B cell from the spleen

c. Cultivation of myeloma cells

d. Fusion of myeloma and B cell

e. Separation of cell lines

f. Screening of suitable cell lines

g. In vitro or in vivo multiplication

h. Harvesting

a) IMMUNIATION OF A MOUSE:

Immuniation of mouse with antigens is performed by injecting microgram or milligram quantities of antigen mixed with an adjuvant (aluminum salt, freund’s complete or incomplete adjuvant) intradermally or subcutaneously at multiple sites repeatedly.

b) ISOLATIONOF B CELLS FROM THE SPLEEN:

After the several week of immunization, blood sample are obtained from the mice for the measurement of the serum antibodies. Serum antibody titer is determined by Enzyme Linked Immunosorbant Assay (ELISA).If the titer is high the cell fusion can be performed. If the 8

titer is too low, mice can be boosted until an adequate response is achieved. When the antibody titer is high enough, the mice is commonly boosted by injecting antigen without adjuvant 3 days before the fusion but 2 weeks after the previous immunization. When the concentration of antibodies is found to be nearly optimal, the animal is sacrificed and the spleen which contains a large number of plasma cells is separated.

c) CULTIVATION OF MYELOMA CELL:

The myeloma cells are selected beforehand to ensure they are not secreting antibody themselves and they lack the hypoxanthine-guanine phosphoribosyltransferase (HGPRT) gene.

d) Fusion Of Myeloma And B Cell:

The fusion is accomplished by using polyethylene glycol or the sendai virus. It is performed by making the cell membranes more permeable.

e) Separation Of Cell Lines:

Removal of the unfused myeloma cells is necessary because they have the potential to outgrow other cells.After fusing the cells are placed in a selection medium of HAT (hypoxanthine-aminopterin-thymidine medium) for roughly 10 to 14 days. Aminopterin blocks synthetic pathways by which nucleotides are made. Therefore cells must use a bypass pathway to synthesize nucleic acids, a pathway that is defective in the myeloma cells.Myeloma cells will die. Whereas B cells have short lifespan, they will also die. Only hybridomas will survive.

f) Screening of suitable cell lines:

The surviving hybridoma cells would have formed against different epitopes. The next step is to select the hybridoma produced against desired antigen. The cultures are diluted to such an extent that only a single cell gets transferred to the wells of microtiter plate. The cells are allowed to multliply. These cells produce antibodies that can be readily detected in supernatant fluids.

g) In vitro or in vivo multiplication:

It is culturing of selected clone cells to grow in invitro or invivo conditions. In invitro technique clone cells are cultured in tissue culture flask with suitable medium.

In invivo technique, selected clones are injected into the peritoneal cavity of mice and allowed to multiply and produce antibody. 9

h) Harvesting:

Monoclonal antibodies are separated from the cells by affinity chromatography.

Diagrammatic representation of mAbs production through Hybridoma Technology

2.2.In Vitro Production of Monoclonal Antibody

Many commercially available devices have been developed for in vitro cultivation. These devices vary in the facilities required for their operation, the amount of operator training required, the complexity of operating procedures, final concentration of antibody achieved, cost, and fluid volume accommodated. 10

Each hybridoma cell line responds differently to a given in vitro production environment.

1. Batch Tissue-Culture Methods

2. Semipermeable-Membrane-Based Systems

2.2.1. Batch Tissue-Culture Methods

The simplest approach for producing mAb in vitro is to grow the hybridoma cultures in batches and purify the mAb from the culture medium.

Fetal bovine serum and fetal calf serum (FCS) are used in most tissue-culture media.

Cell cultures are allowed to incubate in commonly used tissue-culture flasks under standard growth conditions for about 10 days; mAb is then harvested from the medium.

This approach yields mAb at concentrations that are typically below 20 g/ml.

Methods that increase the concentration of dissolved oxygen in the medium may increase cell viability and thus increase mAb concentration. Some of those methods use spinner flasks and roller bottles that keep the culture medium in constant circulation and thus permit nutrients and gases to distribute more evenly.

The gas-permeable bag, a fairly recent development, increases concentrations of dissolved gas by allowing gases to pass through the wall of the culture container. All these methods can increase productivity substantially, but antibody concentrations remain in the range of a few micrograms per milliliter.

Most research applications require mAb concentration of 0.1-10 mg/ml, much higher than mAb concentrations in batch tissue-culture media. The tissue-culture supernatants can be purified by passage over a protein A or protein G affinity column, and mAb can then be eluted from the column at concentrations suitable for most applications.

Merits: Batch tissue-culture methods are technically relatively easy to perform, have relatively low startup costs, have a start-to-finish time (about 3 weeks).

Demerits: The disadvantages of these methods are that large volumes of tissue-culture media must be processed, the mAb concentration achieved will be low (around a few micrograms per milliliter), and some mAb are denatured during concentration or purification.

2.3. Semipermeable-Membrane-Based Systems

Growth of hybridoma cells to higher densities in culture results in larger amounts of mAb that can be harvested from the media. 11

The use of a barrier, either a hollow fiber or a membrane, with a low-molecular-weight cutoff (10,000-30,000 kD), has been implemented in several devices to permit cells to grow at high densities. These devices are called semipermeable-membrane-based systems.

The objective of these systems is to isolate the cells and mAb produced in a small chamber separated by a barrier from a larger compartment that contains the culture media. Culture can be supplemented with numerous factors that help optimize growth of the hybridoma.

Nutrient and cell waste products readily diffuse across the barrier and are at equilibrium with a large volume, but cells and mAb are retained in a smaller volume (1-15 ml in a typical membrane system or small hollow-fiber cartridge).

Expended medium in the larger reservoir can be replaced without losing cells or mAb; similarly, cells and mAb can be harvested independently of the growth medium. This compartmentalization makes it possible to achieve mAb concentrations comparable with those in-vivo.

Two membrane-based systems are available:

The CELLine (Integra Bioscience, Ijamsville, MD): it has the appearance of and is handled similarly to a standard T Flask but is separated into two chambers by a semi-permeable membrane and a gas-permeable membrane is on its underside next to the cell chamber.

The mini-PERM (Unisyn Technologies, Hopkinton, MA): it has a similar design but is cylindrical and comes with a motor unit that functions to roll the fermentor continuously to allow gas and nutrient distribution.

Hollow-fiber bioreactor: In the hollow-fiber bioreactor, medium is continuously pumped through a circuit that consists of a hollow-fiber cartridge, gas-permeable tubing that oxygenates the media, and a medium reservoir. The hollow-fiber cartridge is composed of multiple fibers that run through a chamber that contains hybridoma cells growing at high density. These fibers are semi-permeable and serve a purpose similar to that of membrane-based systems. The hollow-fiber bioreactor is technically the most difficult of in vitro systems, partly because of the susceptibility of cells grown at extremely high density to environmental changes and toxic metabolic-byproduct buildup. The hollow-fiber bioreactor is designed to provide total yields of 500 mg mAb or more. For those reasons, hollow-fiber reactors are used only if large quantities of mAb are needed. 12

                                         Hollow-fiber bioreactor assembly

Merits: The advantage of membrane-based systems is that high concentrations of mAb can be produced in relatively low volumes and fetal calf serum can be present in the media reservoir with only insignificant crossover of bovine immunoglobulins into the cell chamber.

Demerits: A disadvantage is that the mAb may be contaminated with dead cell products. Technical difficulty is slightly more than that of the batch tissue-culture methods but should not present a problem for laboratories that are already doing tissue culture.

The total mAb yield from a membrane system ranges from 10-160 mg according to Unisyn literature.

2.4. Epstein–Barr Virus Immortalization Method

EBV, a member of the herpes virus family is one of the most common viruses found in humans. In most of the Western population each individual maintaining a life- long active immune response against the virus.

In vivo, EBV infects B cells expressing on their surface the CD21 EBV receptor. Under some conditions even other cells, devoid of the receptor, are infected by the virus. In vitro, resting human B lymphocytes infected with EBV show typical viral latency, reflected by the expression of a limited number of viral proteins and two types of non-translated RNA molecules.

The virus-induced-molecules transform the B cells into immortalized diploid cells. EBV efficiently infects the majority of human B lymphocytes, including IgM, IgG, IgA, and IgE cells. The immortalized cells, which grow rapidly as a suspension in standard cell culture media supplemented with a medium.They maintain the characteristics of the initially infected B lymphocytes, including expression and secretion of immunoglobulins. The cells can be cloned with the support of a feeder layer. 13

Depending on each specific cell line, the tissue culture medium derived from cells at saturation contains 0.5–10 μg immunoglobulin/ml. Because of their human origin, these cells cannot be grown as peritoneal ascites in normal mice.

As a rule, peripheral blood lymphocytes are used as a source for establishing antibody-producing cell lines, but even spleen and lymph node-derived cells can serve this purpose.

2.4.1. Materials

 A freshly drawn blood sample (10 ml or more) supplemented with any type of anticoagulant or a buffy coat derived from a blood center, is a suitable source of lymphocytes.

 Phosphate-buffered saline (PBS).

 Sterile Ficoll Isopaque

 A concentrated stock solution of sterile cyclosporin A in PBS

 The EBV reagent harvested from the B95-8 marmoset cell line.

 CpG ODN a nucleotide, is used to activate in vitro the isolated blood lymphocytes.

 RPMI-164O supplemented with penicillin and streptomycin in FCS is used to grow EBV-immortalized lymphoblastoid cell lines.

 Antigen-coated magnetic beads and magnetic device.

 Flat bottom 96-well microplates

 2000 R irradiated blood-derived lymphocytes from any blood donor are used as feeder layer.

 Standard ELISA microplates.

2.4.2. Methods:

A. Isolation of Lymphocytes

i. Carry out the entire procedure under sterile conditions.

ii. Dilute the blood sample with one volume of PBS. If a buffy coat is used, dilute it with 100 ml PBS.

iii. Separate the lymphocytes by layering carefully two volumes of cell suspension onto one volume of Ficoll Isopaque in a test tube,avoiding mixing of the two layers.

iv. Centrifuge the test tubes with no brake. The erythrocytes form a pellet at the bottom of the test tube; the lymphocytes (and platelets too) are located in the interphase above the Ficoll Isopaque layer. 14

v. Use a pipette to discard about 4/5 of the upper layer. Then collect into a test tube the interphase, which contains mainly lymphocytes and platelets. The interphase may be contaminated with some erythrocytes which do not interfere with the process. The recovered cell fraction will also contain some Ficoll Isopaque. Dilute the cell fraction with one volume of PBS and FCS and centrifuge. Discard the supernatant.

vi. Suspend the cells in PBS, FCS and centrifuge. If the supernatant is turbid (reflecting the presence of platelets), washes should be repeated as above (i.e,centrifugation) until the supernatant is clear. Suspend the cells in RPMI-1640, FCS and count. Usually, 106 lymphocytes are recovered from 1 ml blood and about 6 × 108 cells are recovered from the entire buffy coat.

B. Infection with EBV

i. Spin down the lymphocytes, gently resuspend, and add B95-8 virus-containing reagent. Incubate the cells for 1 h at 37 °C. Then spin down the cells and discard the supernatant. Resuspend the cells in RPMI-1640, FCS, cyclosporine A,CpG ODN.

ii. The direct result of the viral infection is that the B cells start to proliferate, with an average doubling time of 24 h. In bulk cultures of EBV-infected non-selected blood-lymphocytes the T cells die out within 1–2 weeks. Cyclosporin A, added to the medium prevents the cytotoxic activity of the T cells against the EBV-immortalized cells.

C. Selection of Lymphocytes and Establishment of Antibody - Secreting Cell Lines

Cells should be selected before EBV infection (option A) or when they have already been immortalized by the virus (option B). Usually selection is performed with the aid of ligand-coated magnetic beads and an appropriate magnetic device.

i. On the day the lymphocytes are isolated and infected with the virus, resuspend them in RPMI-1640 and FCS. It is important to have a single cell suspension and to avoid clumps. The mixture of cells and magnetic beads is now placed in a capped test tube. The entire procedure should be carried out at 4 °C.

ii. Gently suspend the pellet, using a pipette with a relatively wide opening. The magnetic device is then used to isolate the beads with their attached cells. Keep the test tube adjoined to the magnet for 2 min, then open the tube and, while keeping it attached to the magnet, decant the fluid, leaving behind the beads and the beads specifically-bound B cells, still attached to the wall of the test tube. Close the tube, remove the magnet and

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add fresh cold medium as before. Gently resuspend the pellet and repeat the selection procedure five times.

iii. Thoroughly resuspend the pellet and add irradiated feeder cells in RPMI-1640, FCS, CpG ODN. Seed 200μl/well into a flat bottom 96-well micro-plate. Allow the cells to grow for about 4 weeks. There is no need to feed the cells during this period. The magnetic beads do not interfere with the growth of the cells.

iv. Collect 100μl supernatant samples from wells in which there is substantial cell growth and check for the presence of specific antibodies by ELISA or any other rapid assay..

D. Establishment of Cell Lines that Secrete a Specific Antibody Without Selection of Antigen-Committed Cells

i. Sometimes, the number of available PBL is small and, in addition, selection of antigen-committed cells is not feasible: the antigen may not be available as a pure reagent or it is not possible to coat the beads with the antigen used for selection. In such cases, a different strategy is called for (option C).

ii. A small number of virus-immortalized antigen-non-selected cells are allowed to grow as oligoclones in flat-bottom micro wells together with irradiated allogeneic lymphocytes as feeder layer. When cell saturation is reached, the supernatants should be tested (usually by ELISA) for the presence of a specific antibody.

E. Cloning of EBV-Immortalized Cells

i. EBV-immortalized cells cannot develop from a single cell unless supported by a feeder cell layer. Moreover, technically it is very difficult to get the cells to grow as monoclonal colonies in soft agar. The cells are therefore cloned by limiting dilution in 96-well microplate, in the presence of 2000 R irradiated allogeneic blood–derived lymphocytes. These cells can be prepared in advance and kept frozen until used as feeder.

ii. Wrap the microplates with aluminum foil and keep them in the incubator for 4 weeks without refeeding.

iii. Eventually the outgrowth of the LCL is clearly observed under an inverted microscope.

iv. Transfer the cells collected from the wells to round bottom tissue culture test tubes containing RPMI-1640 and FCS.

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3. APPLICATION OF MONOCLONAL ANTIBODIES:

The application of monoclonal has been evaluated for more than 10 year. The use of

monoclonal antibodies falls mainly under diagnostic, therapeutic and catalytic categories.

The earliest studies were focused on cancer therapy, but now a day’s use of monoclonal antibodies to also another human disease. Some monoclonal antibodies are now commercially products under development and many other are still undergoing evaluation.

Clinical application of monoclonal antibodies

• Diagnostic applications

• Therapeutic applications 17

For both of these application localization of monoclonal antibodies is very important. Many factors involve in localization of monoclonal antibody. For effective localization, the monoclonal antibodies should have a right affinity for the target antigen. The reactivity should be minimized with other non targeted tissue.

Human MAB have a lower rate of blood clearance. The blood clearance is depending upon the molecular size.If the molecular weight is lower the clearance is faster. The fragment MAB have a lesser half lifethan the intact body. Due to smaller size the fragments can diffuse into tissue. But for areaction with immune effectors function the MAB should be intact.The dose of antibody also has an effect on clearance rate. If the dose is high (more that10mg) saturation of non specific sites in live andother organ occur, there by reducing the clearance rate.Routes of administration, other than the IV routealso alter the biodistribution of MAB of the antibody.

a. Diagnostic application of MAB

MAbs are utilized in diagnostic kits for the diagnosis of various infectious diseases, monitoring drug levels, Detecting pregnancies matching histocompatibility antigen detecting diabetes,cancer and in immunoscintigraphy.Radiolabelled MAB are used in diagnostic purpose. The technique is called immunoscintigraphy. In this technique a planergamma camera is used to detect the distribution of gamma entitling radioisotopes conjugated with mAb in a two dimensional manner. The imaging has been applied successfully for cardiovascular disease infection disease and cancer disease.

FDA licensed a new diagnostic imaging agent that can determine the extent of disease in

patients diagnosed with small cell lung cancer.

Because these agents can detect tumor in different part of the body at one time, it can help physician to advice certain patients with advanced forms of the disease about treatment option without requiring further diagnostic tests. The new agent, Nofetumom Ab, is a fragment of a monoclonal antibody that when tagged with the radioisotope technique, can detect a protein found on the surface of most small lung cancer cells.

1.Cardiovascular disease:

MAB is used in myocardial infarction disease Myoscint is the first MAB based imaging agent in market in much European country. The product consists of a kit containing 0.5 mg of antimyocin 18

fab fragment conjugate with chelator DTPA (diethyl triamine penta acetic acid) this is labeled by mixing with (indium chloride) after incubation of 10 minute. In labeled MAB is ready for IV injection. Imaging is done after 24-28 hours. About 600 patients have been tested without adverse effect. The product has high degree of sensitivity and can detect location and extent of necrotic heart function.

2-Infectious Disease

mAbs are being tried to image the sites of infection. Antibodies directed against bacterial antigen have proven successfully. Inflammatory leukocytes which accumulate at the site of infection have shown high sensitivity and specificity for detection of localized infection.

3- Cancer

mAbs are being evaluated for detection of different type of cancer like breast carcinoma, ovarian carcinoma, and lung carcinoma. These mAbs can be targeted against many type of tumor. Monoclonal antibody fab fragment is labeled with radioactive isotope in its chemical composition .Isotopic labeling is a technique used to track the passage of an isotope(an atom with a detectable variation through a reaction, metabolic pathway or cell. The reactant is labeled, the nuclides used in isotopic labeling may be stable nuclides or radionuclides, the presence of labeling isotopes can be detected through their mass, vibrational mode or radioactive decay. when these isotopes decay they emit radiations, for these purpose useful type of radioactive decay is positron emission .when a positron collides with an electron, it releases two high energy photons travelling in diametrically opposite directions. If the positron is produced within a solid object ,it is likely to do this before travelling more than a millimeter. If both of these photons can be detected,the location of the decay event can be determined very precisely.

Detection of pregnancy :

Home pregnancy tests can find the presence of a pregnancy hormone HCG in a sample of urine. HCG level increases quickly. The kit contains 3 zones in it, test zone, reaction zone and control zone. After conception level of HCG increases in body that appears in urine too, urine is collected and placed on test zone, antibodies are present there that grasp HCG ,a large no of antibodies are still free. These antibodies are attached with dye activating enzyme, antibody-HCG complex is sandwich by another antibody, this is known as sandwich assay. This complex 19

activates dye activating enzyme that produces a sharp red color that proves that the result is positive .The last one is control zone that confirms the test.

Therapeutic applications

Improving the outcome of bone marrow transplantation by using CD52 mAbs to prevent

Graft-Versus-Host disease and Graft rejection. Alemtuzum Ab is the monoclonal antibody used for this purpose. Graft-versus-Host Disease (GVHD) is a major cause of mortality and morbidity after allergenic bone marrow transplantation, but can be avoided by removing T-lymphocytes from the donor bone marrow. However, T-cell depletion increases the risk of graft rejection. This study examined the use of CD52 mAb to eliminate T- cells from both donor marrow and recipient to prevent both GVHD and rejection.

Abciximab (ReoPro®) is a chimeric human-murine monoclonal antibody Fab fragment that inhibits platelet aggregation by binding platelet glycoprotein IIb–IIIa receptors. Abciximab is used for myocardial infarction, ischemia, and angina. Platelet aggregation is almost completely inhibited 2 hrs aft er the initiation of abciximab therapy. The major complication of abciximab infusion is dose-related bleeding. 20

Adalimumab (Humira®) is a human mAb that binds TNF-_ and in the presence of complement lyses cells that express surface TNF-_. Adalimumab is used to treat autoimmune disorders, such as Crohn’s disease, psoriatic arthritis, and moderate-to-severe rheumatoid arthritis.

Alemtuzumab (Campath-1®) is used for immunosuppression in organ transplant by targeting CD52 on T cells and monocytes. Campath-1® is also used to treat chronic lymphocytic leukemiaand T-cell prolymphocytic leukemia where it is dosed 3 g daily as 2-hrs IV infusions, then increased to 30 mg three times per week when tolerated.

Muromonab-CD3 (Orthoclone-OKT3®) is an immunosuppressive mAb targeted to the CD3 glycoprotein on human T cells. Lymphocytes containing CD3, CD4, CD8, and CD11 levels fall quickly after administration, and after 2 to 7 days, lymphocytes containing CD4, CD8, and CD11 return to circulation. Muromonab-CD3 is used to reverse acute renal allograft rejection.

Bevacizumab (Avastin®) is a humanized mAb that binds VEGF (vascular endothelial growth factor), thus inhibiting angiogenesis in metastatic tissue. Bevacizumab is used to treat metastatic colorectal cancer and non-squamous, non-small cell lung cancer; HER-2 negative breast cancer; progressive glioblastoma; and metastatic renal cell cancer. Unlabeled uses include agerelated-macular degeneration, recurrent ovarian and cervical cancer, and soft tissue sarcomas.

Catalytic applications

The antibodies are extremely efficient at binding ground states of the target molecule while enzymes obtained their catalytic efficiency from tight binding of the transition state for the reaction. Thus antibodies can be made efficient catalysts if they are made for reaction transition state. Lemer and his co-workers explored the probability of enzyme like action of antibodies by producing hapten-carrier complex where the hapten structurally resembled transition state and anti-hapten mAbs generated, gave catalytic activity.

• The hydrolysis of substrate increased thousand fold after incubation.

• ABZYMES are MAb used for this purpose. 21

REFERENCES

1. Taha Nazir (2014). Applied Molecular Cell Biology, Lahore: Study Aid Foundation For Excellence.

2. National Institutes of Health (NIH) (1999).Monoclonal Antibody Production, Washington, DC: NATIONAL ACADEMY PRESS.

3. Steinitz, Michael (2014). Human Monoclonal Antibodies, Totowa, NJ: Humana Press.

4. Steinitz M, Klein G, Koskimies S, Mäkelä O (1977) EB virus-induced B lymphocyte cell lines producing specifi c antibody. Nature 269: 420–422.

5. Kuppers R (2003) B cells under infl uence: transformation of B cells by Epstein-Barr virus. Nat Rev Immunol 3:801–812.

6. Ehlin-Henriksson B, Gordon J, Klein G (2003) B-lymphocyte subpopulations are equally susceptible to Epstein-Barr virus infection, irrespective of immunoglobulin isotype expression. Immunology 108:427–430.