Site-specific drug delivery utilizingmonoclonal antibodies

ContentsI. IntroductionA. ChemistryB. Polyclonals vs. monoclonalsC. Conjugation of antibodiesII. Production of monoclonal antibodiesIII. Drug-monoclonal antibody conjugates for drug targetingA. PrinciplesB. Drug antibody bondingIV. A. Highlights of current research

Introduction In 1976, Kohler and Milstein employed a

method of somatic-cell hybridization in order to successfully generate a continuous “hybridoma” cell line capable of producing monoclonal antibody (MAb) of a defined specificity. Subsequently, several MAbs have exhibited specificity for target sites. It is this property of MAbs that makes them excellent candidates as carriers of therapeutic agents for delivery to specific sites.

Chemistry Antibodies are complex proteins, consisting of

multiple polypeptide chains that contain a variety of reactive chemical groups, such as amino, carboxyl, hydroxyl, and sulfhydryl. Functionally, MAbs possess a molecular polarity based on the joining of an antigen-binding fragment (Fab) to a complement- fixing fragment (Fc). The Fab fragment is responsible for specific antigen binding, whereas the Fc fragment binds to effector cells, fixes complements, and elicits other in vivo biological responses.

Polyclonals vs. monoclonalsPolyclonal antibodies Monoclonal antibodies

Produces large amounts of non specific antibodies

Can produce large amounts of specific antibodies but may be too specific

Recognizes multiple epitopes on any one antigen

Recognizes only one epitope on an antigen

Inexpensive to produce Expensive to produce

Technology required is low High technology required

Skills required are low Training is required for the technology use

Time scale is short Time scale is long for hybridomas

Can be batch to batch variabilityOnce a hybridoma is made it is a constant and renewable source and all batches will be identical

Conjugation of antibodies1. Radioisotope conjugates: 131-I labeled Mab2.Toxin conjugates: Ricin immunoconjugates3.Drug conjugates: Conjugates of methotrexate

with Mab

Production of monoclonal antibodies

Monoclonal antibodies are antibodies which have a single, selected, specificity and which are continuously secreted by "immortalised" hybridoma cells. A hybridoma is a biologically constructed hybrid between an antibody-producing, mortal, lymphoid cell and a malignant, or "immortal", myeloma cell. Hybridomas utilizing hybridoma technology are routinely made in stepwise conventional small-scale culture procedures.

HAT SelectionImmunize mouse

Isolate spleen cells Myeloma cells(HGPRT-)

Non fused spleen cells(s)Non fused myeloma cells (M)

Fused (s-s)Fused(m-m)Fused(s-m)

HAT medium

s-m groW & divideM&M_M; s& s –s can not

divide

Production of monoclonal antibodies

1. The antigen, a foreign substance, such as a lung cancer cell, is injected into a mouse. The spleen is then removed, and the antibody producing cells are collected.

2. Myeloma cells are isolated from a mouse tumor. 3. Spleen and tumor cells are fused together to

form hybridomas. The surviving have the spleen cell’s ability to produce antibodies and the tumor cell’s ability to reproduce. “hybridomas.”

Production of monoclonal antibodies4. Each hybridoma is isolated and allowed to grow into

a large colony of cells that produce a single MAb.5. Each MAb is screened for its ability to attack the

original cancer cells, and the hybridomas producing the desired antibody are kept.

6. The desired hybridoma cells are injected into a mouse where they form a tumor that produces large amounts of concentrated antibody. The first critical step in generating a therapeutic or diagnostic MAb— after initial isolation — is to produce the antibody product.

Drug-monoclonal antibody conjugates fordrug targeting

Use of MAbs in targeting cytotoxic drugs to specific tissues on cancer therapy has been studied considering the followings:

• Number of antigen molecules per cell surface• Number of cells expressing the reactive antigen in

the tumor mass• Size of the tumor mass• Fate of the antigen-antibody complex (stability on

cell surface, internalization, capping, shedding)

Drug-monoclonal antibody conjugates fordrug targeting

• Degree of tumor vascularization• Degree of tumor mass infiltration and necrosis;

presence and reactivity of circulating antigen in the blood

• Duration of MAb binding to cell surface• Isotype of immunoglobulin (IgG subtypes or IgM)• Species of immunoglobulin (murine, human, or

chimeric recombinant)

Drug-monoclonal antibody conjugates fordrug targeting

• Whole immunoglobulin or fragments (Fab, Fab’, F(ab’)2)

• Clearance of Mab from blood, excretion, or reticuloendothelial system

• Dose of MAb used• Route of inoculation of MAb (intravenous,

intraperitoneal, intralymphatic, or intraarterial)

Drug-monoclonal antibody conjugates fordrug targeting

• Development of a human immune response to the administered MAb.

Requirements of antibody-drug conjugates

• The antibody has to be humanized to avoid an immune response

• It has to show selectivity for an antigen which is over expressed in cancer cells

• It needs to be internalized into the cell by receptor-mediated endocytosis

• The link between the antibody & the drug should be stable

Highlights of current researchCorporation Product Usage

Biogen CD4 agent In AIDS

NeoRx Oncotrac Detect spread ofmelanomas

Genentech Oncogene Breast and ovarian cancer

Cetus Prolenkin (interleukin-2) Anticancer

Xoma E5 (Xomen) Septic shock

Questions……?????

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