Hybridoma Technology for Monoclonal Antibodies
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Questions and Answers

What is the purpose of immunizing laboratory animals with a chosen antigen in hybridoma technology?

To stimulate B-cell differentiation into plasma B-cells and memory B-cells.

What is the role of polyethylene glycol (PEG) in the fusion process of hybridoma technology?

To facilitate the merging of the plasma membranes of B-cells and myeloma cells to create hybrid cells.

Why are hybridoma cells selected in HAT medium?

To inhibit unfused cells' nucleotide synthesis while allowing hybrid cells with functional HGPRT genes to thrive and divide.

What is the purpose of screening hybridoma cells in hybridoma technology?

<p>To identify cells that produce antibodies against specific epitopes.</p> Signup and view all the answers

What are the two methods used to clone and propagate selected hybridomas?

<p>In vivo methods involving mice and in vitro methods in controlled lab conditions.</p> Signup and view all the answers

What is the final product of hybridoma technology?

<p>Monoclonal antibodies (mAbs) that are highly specific and pure.</p> Signup and view all the answers

What is the result of fusing spleen cells that produce anti-TNF-α antibodies with SP2/0 cells?

<p>Hybridoma cells</p> Signup and view all the answers

What is the purpose of the radioimmunoassay in the production of monoclonal antibodies?

<p>To verify the production of antibodies against the antigen</p> Signup and view all the answers

What is the role of the murine A2 hybridoma's variable genes in the construction of plasmids?

<p>They provide DNA sequences for the construction of plasmids encoding the heavy chain or light chain</p> Signup and view all the answers

What is a key observation related to the mechanisms of infliximab in responders?

<p>The rapid induction of T cell apoptosis and the specific induction of M2-type wound-healing macrophages.</p> Signup and view all the answers

What is the therapeutic application of infliximab, a monoclonal antibody against TNF-α?

<p>Treatment of various inflammatory conditions, including Crohn's disease and ulcerative colitis</p> Signup and view all the answers

What is the goal of developing biosimilars like Remsima?

<p>To provide a cost-effective alternative to the original biologic drug.</p> Signup and view all the answers

What is thought to be the underlying cause of chronic relapsing intestinal inflammation in inflammatory bowel disease?

<p>An aberrant immune response to gut microbes, influenced by individual genetic susceptibility</p> Signup and view all the answers

How do variations in biosimilar manufacturing affect the drug's efficacy and safety?

<p>Variations in three-dimensional folding, glycosylation, charge, and impurity presence can impact antigen binding and immunogenicity.</p> Signup and view all the answers

What is the purpose of using Protein A-Sepharose in the production of monoclonal antibodies?

<p>To purify the monoclonal antibody from the hybridoma's supernatant</p> Signup and view all the answers

What is the purpose of PEGylation in modifying infliximab?

<p>To reduce enzymatic degradation and increase the half-life of the antibody.</p> Signup and view all the answers

What changes can be introduced to enhance the stability of infliximab?

<p>Differences in terminal amino acid sequences and post-translational modifications.</p> Signup and view all the answers

What was demonstrated by the development of Remsima as a biosimilar of infliximab?

<p>That it is possible to create a drug with comparable safety, immunogenicity, and pharmacokinetics to the original.</p> Signup and view all the answers

What was the severe adverse reaction observed in human volunteers during the phase I trial of TGN1412?

<p>Systemic inflammatory response syndrome and life-threatening multiorgan failure</p> Signup and view all the answers

What is the primary goal of the study comparing TGN1412 binding in humans and macaques?

<p>To investigate the influence of the Fc domain and C region structure on the in vitro immunomodulatory activity of TGN1412</p> Signup and view all the answers

What were the recombinant subclass variants generated from TGN1412 in the study?

<p>IgG1, IgG2, and IgG4</p> Signup and view all the answers

What was the binding pattern observed for TGN1412 in both human and macaque species?

<p>Strong binding to the high-affinity FcγRIa receptor, but minimal or no binding to other Fc receptors</p> Signup and view all the answers

Why did the study compare TGN1412 binding to IgG1 and IgG4 in humans?

<p>To investigate the differences in binding affinity due to variations in dimer content and glycosylation</p> Signup and view all the answers

What was the purpose of examining CD28 binding properties in the study?

<p>To exclude the possibility that binding was significantly altered by the manipulations</p> Signup and view all the answers

How does the binding of Aqueous phase and biotinylated F(ab’) 2 fragments compare to TGN1412 in terms of binding to biotinylated rCD28/Fc?

<p>Aqueous phase and biotinylated F(ab’) 2 fragments bind slightly less biotinylated rCD28/Fc than TGN1412.</p> Signup and view all the answers

What is the ranking of CD28 binding ability among TGN1412, F(ab’) 2, and Fab fragments?

<p>TGN1412 &gt; F(ab’) 2 &gt; Fab</p> Signup and view all the answers

Do the subclasses IgG1 and IgG4 display equivalent CD28 binding properties to TGN1412?

<p>Yes, IgG1 and IgG4 display equivalent CD28 binding properties to TGN1412.</p> Signup and view all the answers

How does the binding of IgG2 to lymphocytes compare to TGN1412 and IgG4 using Flow cytometry?

<p>IgG2 shows brighter staining of lymphocytes than TGN1412 and IgG4 using Flow cytometry.</p> Signup and view all the answers

How does the Ab Fc affect binding to cell surface CD28, and are there any exceptions?

<p>Ab Fc does not significantly affect binding to cell surface CD28, with the exception of NIB(28SA)-G2.</p> Signup and view all the answers

What is the ranking of binding to recombinant FcgRIa among IgG1, TGN1412, IgG4, and IgG4 mutants?

<p>IgG1 &gt; TGN1412 &gt; IgG4 + IgG4 mutants &gt; IgG2</p> Signup and view all the answers

Study Notes

Hybridoma Technology

  • Hybridoma technology is used to produce monoclonal antibodies (mAbs) through the immunization of laboratory animals, such as mice, with a chosen antigen.
  • This process involves the fusion of activated B-cells with immortal myeloma cell lines to form hybridoma cell lines that produce mAbs against a specific antigen.

Production of Anti-TNF-α Antibodies

  • Spleen cells that produce anti-TNF-α antibodies are fused with SP2/0 cells (mouse myeloma cells) to create hybridoma cells.
  • The production of antibodies against the antigen is verified through radioimmunoassay, and the monoclonal antibody is purified from the hybridoma's supernatant using Protein A-Sepharose.

Plasmid Construction and SP2/0 Transfection

  • Plasmids encoding the heavy chain or light chain are constructed, incorporating DNA sequences from the murine A2 hybridoma's variable genes along with DNA encoding human constant regions.
  • These plasmids are transfected into SP2/0 cells to produce chimeric antibodies against human TNF-α.

Infliximab and Its Mechanism

  • Infliximab is a therapeutic monoclonal antibody used to treat inflammatory bowel disease (IBD), including Crohn's disease and ulcerative colitis.
  • Infliximab works by rapidly inducing T cell apoptosis and specifically inducing M2-type wound-healing macrophages in responders.

Modification to the Antibody (Biosimilar)

  • Biosimilars, such as Remsima, are developed to provide a cost-effective alternative to the original biologic drug.
  • Modifications like PEGylation can be applied to enhance the efficacy of infliximab by reducing enzymatic degradation and increasing its half-life.

TGN1412 Investigation

  • The phase I trial of TGN1412 resulted in severe adverse reactions in human volunteers, including systemic inflammatory response syndrome and life-threatening multiorgan failure.
  • The study investigates the binding of TGN1412 to the FcγRIa receptor and its immunomodulatory activity in humans and macaques.

Study Results

  • TGN1412 showed strong binding to the high-affinity FcγRIa receptor but minimal or no binding to other Fc receptors in both species.
  • The binding affinity of TGN1412 varied compared to other subclasses, with IgG1 showing stronger binding and IgG4 showing weaker binding.
  • The F(ab')2 and Fab fragments of TGN1412 were created, and their binding properties were compared to TGN1412.
  • The study found that the Ab Fc did not significantly affect binding to cell surface CD28, with some exceptions.

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Related Documents

MBS1202_Study.docx

Description

Learn about the process of producing monoclonal antibodies using hybridoma technology, from immunization to B-cell isolation. Understand the role of plasma B-cells and memory B-cells in this process.

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