Production of Monoclonal Antibodies and its application : Rituximab

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Production of Monoclonal Antibodies

and its application : RituximabPresented by:Sakshi Saxena

ASU2013010200124IBT Vth

Antibodies are large Y-shaped proteins.

They are recruited by the immune system to identify and neutralize foreign objects like bacteria and viruses.

Each antibody has a unique target known as the antigen present on the invading organism.

This antigen is like a key that helps the antibody in identifying the organism.

The specific region on an antigen that an antibody recognizes and binds to is called the epitope, or antigenic determinant.

Antibodies

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Understanding Monoclonal Antibodies

Y Y YYY

Different B cells produce different antibodies

For producing single kind of antibody, Specific to one antigen

Y

Y

Y YY Y

Monoclonal antibodies

Monoclones

One kind of B cells producing specific antibodies

Production process

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Step 1. Immunization of mouse

Mice are immunized every 2-3 weeks with an antigen that is prepared for injection

https://www.youtube.com/watch?v=c_krTc9M1WU

Step 2: Screening of Mice for Antibody Production

Blood samples are obtained from mice for measurement of serum antibodies whose titer is determined with various techniques, such as enzyme-linked immunosorbent assay (ELISA) and flow cytometry.

Step 3 : Isolation of antibody producing spleen cells

Isolation of antibody producing spleen cells

•When the antibody titer is high enough, mice are commonly boosted by injecting antigen without adjuvant intra peritoneally or intravenously (via the tail veins) 3 days before fusion but 2 weeks after the previous immunization.

•If the titer is too low, mice can be boosted until an adequate response is achieved, as determined by repeated blood sampling.

•Then the mice are euthanized and their spleens removed for in vitro hybridoma cell production.

Step 4 : Production of hybridomas

Culture medium Culture medium

A week before cell fusion, myeloma cells are grown in 8-azaguanine (analog of guanine, acts as competitive inhibitor).

Myeloma cells lack HPGRT (hypoxanthine phospho ribosyl transferase) enzyme, which is responsible for synthesis of nucleotides.

The cells are then screened in HAT (hypoxanthine-aminopterin-thymidine) medium which blocks the pathway for nucleotide synthesis.


Step 5 : Screening of hybridomas

Myeloma cells B cells Hybridomas

HAT (hypoxanthine-aminopterin-thymidine )medium

Nucleotide synthesis pathway blocked




HPGRT gene presentHPGRT gene present

Nucleotides fail to get synthesized

Nucleotides get synthesized Nucleotides get synthesized

Step 5 : Screening of hybridomas

Myeloma cells B cells Hybridomas

HAT (hypoxanthine-aminopterin-thymidine )medium

Nucleotides fail to get synthesized

Nucleotides get synthesized Nucleotides get synthesized

Cells die due to lack of nucleotide synthesis

Cells die due to short life span

Cells survive


Step 6 : Culturing hybridoma cells – Monoclones production

Hybridomas are separated and individually cultured : 1 cell per well

These cells are called as clonal culture. Because each cell in the well is derived from singe cell and are therefore identical.

After few weeks, when growing cultures can be seen, further screening can be done for desired antibody.

Step 7 : Screening for desired antibodies

Antigens are immobilized in the wells and the antibodies are transferred (one per well) so that they bind to the complementary antigen.

*Different antibodies react to different epitopes on the same antigen. https://www.youtube.com/watch?v=c_krTc9M1WU

Step 8 : Selection and culture of screened antibodies

Finally, the desired antibodies are grown in mass culture and are frozen for storage.


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Chimeric monoclonal antibody

Rituximab (Trade names :

Rituxan, MabThera and Zytux)

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IntroductionRituximab (is a chimeric monoclonal antibody against the protein CD20, which is primarily found on the surface of immune system B cells.

It destroys B cells and is therefore used to treat diseases which are characterized by excessive numbers of B cells, overactive B cells, or dysfunctional B cells.

This includes manylymphomas, leukemias, transplant rejection, and autoimmune disorders.

Mechanism of actionThe antibody binds to CD20 which is widely expressed on B cells, from early pre-B cells to later in differentiation, but it is absent on terminally differentiated plasma cells. CD20 does not shed, modulate or internalise.

Rituximab tends to stick to one side of B cells (where CD20 is present) forming a cap and drawing proteins over to that side.

The presence of the cap changed the effectiveness of natural killer (NK) cells in destroying these B cells.

When an NK cell latched onto the cap, it had an 80% success rate at killing the cell. In contrast, when the B cell lacked this asymmetric protein cluster, it was killed only 40% of the time.


•Fever, chills, body aches, flu symptoms, feeling weak or tired;•Ongoing cold symptoms such as stuffy nose, sneezing, sore throat;•Headache, earache, painful mouth ulcers, skin sores, warmth or swelling with skin redness;•Pain or burning when you urinate, urinating less than usual;•Severe skin rash with blistering, itching, peeling, or pus;•Weak pulse, fainting, overactive reflexes;•Muscle weakness, tightness, or contraction; or•Lower back pain, blood in your urine, numbness or tingly feeling around your mouth

•Severe infusion reaction.•Cardiac arrest•Cytokine release syndrome•Tumor lysis syndrome, causing acute renal failure•Infections•Hepatitis B reactivation•Other viral infections•Progressive multifocal leukoencephalopathy (PML)•Immune toxicity, with depletion of B cells in 70% to 80% of lymphoma patients•Pulmonary toxicity•Bowel obstruction and perforation

Severe side effects

1. “Production of Monoclonal Antibodies” ;Wayne M. Yokoyama, Michelle Christensen, Gary Dos Santos,Diane Miller, Jason Ho, Tao, Wu, Michael Dziegelewski, Francisca A. Neethling ; Current Protocols in immunology, unit 2.5

2. “Production of monoclonal antibodies: Strategy and tactics “; St. Groth, Doris Scheidegger ; Journal of Immunological Methods, Volume 35, Issues 1–2, 15 July 1980, Pages 1-21

3. “Rituximab (monoclonal anti-CD20 antibody): mechanisms of action and resistance” , Mitchell R Smith ; Oncogene (2003) 22, 7359–7368. doi:10.1038/sj.onc.1206939

4. “Rituximab: mechanism of action”; George J. Weiner , Semin Hematol. 2010 Apr; 47(2): 115–123.

5. “Monoclonal Antibody Production” , NATIONAL ACADEMY PRESS Washington, DC 1999

6. Selecting Myeloma Cells for HGPRT Mutants with 8-Azaguanine Retrieved on 20th November 2015 from http://antibodiesmanual.org/index.php?prt=101

7. Rituxan Side Effects Center (2015), John P. Cunha, DO, FACOEP Retrieved on 21st Nov. 15 from http://www.rxlist.com/rituxan-side-effects-drug-center.htm

8. “Rituximab, an Anti-CD20 Monoclonal Antibody: History and Mechanism of Action” , M. D. Pescovitz ; American Journal of Transplantation. 2006;6(5):859-866.

9. https://www.youtube.com/watch?v=c_krTc9M1WU

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Production of Monoclonal Antibodies and its application : Rituximab