Archive for the ‘Immunology’ Category

Therapeutic antibody generation

Posted: April 15, 2013 in Immunology

Hybridomas

First developed and most widely used technology for generation of monclonal antibodies = the use of mouse hybridomas. It is generated from stable fusion of immortalised myeloma cells with B cells from immunnized mice.

Method:

  1. mice first exposed to antigen(Ag) to which AB of interest is specific for –> by series of injections of Ag
  2. B cells then isolated from spleen
  3. myeloma cells are aslo selected to ensure that they themselves do not secrete AB and lack hypoxanthine guanin phosphoribosyltransferase (HGPRT) gene , making them sensitive to HAT medium
  4. myeloma cells + B cells are fused using polyethylene glycol
  5. fused cells incubated in HAT((hypoxanthineaminopterinthymidine) medium ,
  6. aminopterin blocks nucleotide synthesis. Therefore cells forced to use the purine salvage pathway to synthesise purine nucleotide which require HGPRT  , as B cells have this gene , so only B cells that have fused will be able to survive.
  7. unfused myeloma cells is removed for its potential to outgrow other cells
  8.  B-cell myeloma hybrids produce AB  that are immortal
  9. the incubated medium is diluted into multi well plate to extent that each well contains one cell
  10. so AB produced in single cell , were generated from single B cells = directed towards same epitope and thus monoclonal AB
  11. B cells that produces desired AB then cloned  in supplemental media containing IL-6
  12. once hybridoma colony established –> continue to grow in culture medium like RPM1-1640( fetal bovine serum+ antibiotics) and produce AB

The ubiquitous use and broad success of monoclonal antibody in research , is sharp contrast to 3% success rate in drug development.

This high infrequent success rate reflects:

  • the high immunogenicity of these foreign proteins in humans as well as,
  • weak interactions that mouse antibodies typically have with human complement and FcγRs,which results in inefficient effector functions(fi,g 1)
  • mouse AB does not bind human salvage receptor FcRn, resulting in terminal half-life (typically less than 20 hours)

These limitations of mouse AB have largely been overcome by their chimerization or humaization.

ab

Chimerization and humanization 

Chimerization involves joining the variable( antigen binding region) domains of mouse monoclonal antibody to the constant domains of human antibody. This process requires the appreciation of structure and function of immunoglobulin domains.

  1. Starting point= hybridoma cell line generating AB against desired epitope
  2. reverse trascriptase PCR used to generate copies of DNA–> complementary to mRNA coding for variable region of murine mAB
  3. Recombinant DNA techniques allow this cDNA for variable region to be combined with DNA coding for human constant region
  4. recombinant DNA introduced into myeloma cell line using vectors
  5. chimeric mAB is expressed
  6. because original murine variable region= bind to same epitope
  7. because human constant region= less immunogenic and interact with human receptors to elicit therapeutic effects e.g (ADCC, ADCP,CDC)

Rituximab is an example of a chimeric mAb that is used clinically to treat non-Hodgkins lymphoma . Unfortunately, even the murine variable region can still be recognised as foreign—leading in many cases to the same limitations as murine mAbs.

The simplest of many humanization strategies involves transfer of complementarity determining regions (CDR) from mouse AB to human IgG. For generation of high affinity binding , generally requires additional transfer of one or more framework-region residues from parent mouse AB.

Route for completely human Ab

Transgenic mice – human AB generated by target antigen immunization of mice transgenic for human immunoglobulin (Ig) genes and disrupted Ig heavy  chain and Igκ light-chain loci.

Subsequent progress in research enabled the inclusion the expression of more V gene segments by transgenic mice, thereby  expanding the potential repertoire of recovered AB.

  • B cells that express human AB are isolated from immunized mice.
  • then the B cells are cloned as for hybridomas, similar to generation of mouse monoclonal AB
  • binding affinity of human AB from transgenic mice = often high, reflecting  in vivo affinity maturation= integral to secondary  immune response.
  • initial output of transgenic mice is human IgG, allowing early screening  of biological function.
  • hybridoma cell lines generated from the transgenic mice= produce AB easily for screening through pre-clinical development.
  • recombinant cell lines such as CHO (chinese hamster ovary) or NSO mouse myeloma cell = produce higher AB titres ( than hyridoma) –> chosen for later  stage of clinical development if not all.

Phage-display libraries–  phage encoding single chain V domain AB fragment (scfv) first described 1990.

  • Diverse human Ig heavy chain V (Vh) gene segments and light chain V gene segment were prepared.  How? using amplification PCR  from peripheral blood lymphocytes of non immunized donors.
  •  scFv generated randomly combining the variable heavy and light chain gene segments using PCR,
  • Combinatorial library(fewer than 107 scFv genes) was then cloned for display on surface of phage,
  • then used to identify scFv that bind target antigens.
  • More commonly,antibodies need to be optimized to meet the design goals of the project, and this is readily accomplished by selection for high-affinity variants from phage-display
    libraries.

One human antibody isolated by phage-display technology has been approved widely for therapeutic use: adalimumab ( TNF- specific monoclonal AB) used for treatment of rheumatoid and psoriatic arthritis.

A particular strength of phage-display libraries, in contrast to hybridoma technology, is that direct
selection for exquisitely specific binding properties, such as species crossreactivity,  is sometimes possible( allowing the biological function of the antibodies to be evaluated in animal models of disease.)

antibodyengineering

Antibody(AB) is useful tool for research and medicine ( diagnosis & therapy). In fact it emerged in the mid 1990’s as a new drug class .   AB was approved for therapeutic use in clinical setting such as

  • oncology;
  • chronic inflammatory diseases;
  • transplantation;
  • infectious diseases and
  • cardiovascular medicine

One of the biggest advantage of AB use is the high success rate from first use in humans.

  • 29%  chimeric AB
  •  25% humanized AB
  • this is compares favorably with 11% success rate of small molecule drugs

In general AB are well tolerated by humans although infusion reactions( fever, chills e.g patients treated with rituximab CD20 specific monoclonal AB) particularly for first dose are common but manageable. And key strength of AB as therapeutics is their clinical potential can be readily be increased by improving their existing properties.

AB limitations of therapeutics

  1.  restriction of targets to those  on surface or exterior of host cells or invading pathogens
  2. AB drugs are expensive which limits its use to serious medical conditions. Many factors contribute to high cost a) large expense of drug development in general, b) high cost of manufacturing and c) large total doses that are often required.
  3. although AB therapeutics often safe and well tolerated, rare but serious adverse events have been reported.

Goal for AB therapeutics

One need is to improve the efficacy of AB therapeutics e.g anticancer therapy, for which AB are seldom( if ever) curative. Therefore in oncology, one of the goals of increasing efficacy of AB is to improve, antitumour activity and improve patient survival.

  • How? by increasing frequency of partial responses or more preferably , complete responses and by extending the duration of responses.

To improve safety, might be useful to focus on limiting first infusion reactions or more serious target related adverse events

  • e.g  acute and severe influenza- like syndrome associated with adminstration of CD3 specific monoclonal AB= due to Fc-mediated and can be overcome…
  • how? by attenuating the interaction between Fc region of AB and receptors for AB(e.g IgG receptors: FcγRs) expressed by patient

Increasing the potency of AB or extending its half-life in plasma might allow dose or frequency of administration to be reduced, which might have associated benefits  e.g improved quality of life and/or convenience for patient and or reduced cost of drug.