04/12/23 07:02 1

Monoclonal Antibodies

- Antibodies (Abs). Also known as immunoglobulins (Ig).

- Comprised of 2 heavy chains and 2 light chains

- Monoclonal Abs bind specifically to a single site (epitope) on a particular antigen

- Abs are produced by B lymphocytes

- Because of their specificity and ease of generation, they are extensively used as therapeutics (“passive immunotherapy”) and as diagnostic and research tools

-They can be generated in large (unlimited) amounts in vitro

2

B cells develop in the bone marrow hematopoietic stem cells and lymphoid stem cells T-cells and B-cells

progenitor = pro-B cell (B220+)

precursor = pre-B cells: heavy-chain rearranged

immature B cell: IgM + light-chain rearranged

mature B cell: IgM + IgD + an antigen encounter in spleen or lymph nodes; goes to periphery

Terminally differentiated cell = plasma cell, periphery, Ig secretor

Antibodies are made by B-cells

3

Domain structure of an immunoglobulin molecule

} Fc

}FabFragment ,antigen binding

Fragment , crystallizable

disulfidebonds

4

5

Laboratory fragmentation of antibodies

6

ComplementaritydeterminingRegion = “CDR”

Hypervariableregion

Ig molecule showing polarity, disulfides, carbohydrate

7

8

Opsonization

Complement activation

Antibody-dependent cell-mediated cytotoxicity (ADCC)

Transcytosis

Fc functions

9

Opsonization: Direct uptake of bacteria coated with antibody molecules

Complement activation: Activated complement proteins lyse cells by making holes in their mebranes(e.g. bacteria)

Antibody-dependent cell-mediated cytotoxicity (ADCC):Killer T-cells use antibodies on their surface to target cells with an antigen and kill them.

Transcytosis:Antibody-antigen complexes are taken up (endocytosed) on on side of an epithelial cell and directed to theother side, where they are exocytosed

Fc functions

10

Multigene organization of Ig genes

–light chains: V, J (variable) and C (constant)–heavy chain: V, D, J, (variable) C (constant)

Mechanism of Ab gene rearrangement

Recombination signal sequences (RSS)–flank V, D, J gene segments

–V-RSS------RSS-D-RSS---------RSS-J

11IgGkappa gene rearrangement

SPLICING

SPLICING

+ SOMATIC HYPERMUATION

+ SOMATIC HYPERMUATION

12

Antibodies can participate in host defense in three main ways

13

Got this far

14

ADCC = antibody-dependent cell-mediated cytotoxicity

15

MAb therapy targets

Inflammation

Autoimmune disease

Graft rejection

Heart disease (thrombosis)

Cancer

Viral infection

16

Therapeutic strategies

Mabs straight

Mabs fused to other protein binders (e.g., soluble receptors)

Mabs fused to cytotoxic agents (toxins, radionuclides)

Toxins: ricin (stops protein synthesis)calicheamicin (DNA breaks)

Radionuclides:90Y = yttrium111I = indium

17Problems of mouse MAbs

1) Fc portion limited in its ability to interact with Fc receptors of human cells.

2) Lower serum half-life

3) Development of human anti-mouse antibodies (HAMA)

A) Retreatment results in allergy or anaphylactic shock

B) Retreatment is less effective

Solutions via recombinant DNA genetic engineering :

1) Chimeric mouse-human antibodies: Hu V-region fused to mouse C regions

2) Humanized mouse antibodies, Parts of V-region from human interspersed with mouse CDR V-regions

3) Human antibodies (fully), via transgenic mice carrying human immunoglobulin genes(Medarex, Abgenix, Kirin)

Breedveld, Lancet 2000 355:9205

18

Sponsor company Generic name US trade name

mAb type Therapeutic category

US approval

Johnson & Johnson Muromonab-CD3 Orthoclone OKT3

Murine Immunological

‡ 19.06.1986

Centocor Abciximab ReoPro Chimeric Hemostasis 22.12.1994

Biogen IDEC Rituximab Rituxan Chimeric Antineoplastic 26.11.1997

Protein Design Daclizumab Labs |

Zenapax Humanized Immunological 10.12.1997

Novartis Basiliximab Simulect Chimeric Immunological 12.05.1998

MedImmune Palivizumab Synagis Humanized Anti-infective 19.06.1998

Centocor Infliximab Remicade Chimeric Immunological 24.08.1998

Genentech Trastuzumab Herceptin Humanized Antineoplastic 25.09.1998

Wyeth Gemtuzumab ozogamicin

Mylotarg Humanized Antineoplastic 17.05.2000

Millennium/ILEX

Alemtuzumab Campath Humanized Antineoplastic 07.05.2001

Biogen IDEC Ibritumomab tiuxetan

Zevalin Murine Antineoplastic 19.02.2002

Abbott Adalimumab Humira Human Immunological 31.12.2002

Genentech Omalizumab Xolair Humanized Immunological 20.06.2003

Corixa Tositumomab-I131 BEXXAR Murine Antineoplastic 27.06.2003

Genentech Efalizumab Raptiva Humanized Immunological 27.10.2003

Imclone Systems

Cetuximab Erbitux Chimeric Antineoplastic 12.02.2004

Genentech Bevacizumab Avastin Humanized Antineoplastic 26.02.2004

MAbs approved for human therapy

EGF-R colon cancer

HER-2/neu (EGF2) breast cancer

CD33 leukemia (AML)

VEGF colon cancer

IgE asthma

Respiratory infectionSynciitial Virus

IL-2 immunosupressant

19

Monoclonal antibody generation

- Cells needed myeloma cells, mouse spleen cells - antigen administration Kohler and Milstein - hybridoma formation via cell fusion- selection mutants required: defer

- antibody generation cDNA cloning- engineered MAbs expression vectors- refinement chimeric, humanized, human (defer)

20

Monoclonal antibodies via cell hybridization

Selects forrare hybrid cells

21Reducedmyeloma hybrid

Isoelectric focusingimmunoglobulinsmade inhybridoma cells

Georges Kohler

Cesar Milstein

Unreducedmyeloma hybrid

22

Mab Fusion Proteins

Other protein-binding proteins: natural receptors in soluble form

Analogous to MAbs and make use of the Fc portion of the antibody molecule:

Example: Enbrel (etanercept):Anti-rheumatoid arthritis drug Soluble TNF receptor fused to the Fc IgG1 domain (TNF= tumor necrosis factor)

Ties up TNF, blocking its inflammatory functionFc domain dimerizes the receptor, which increases its affinity for TNF.Fc domain increases the half-life of the protein in the bloodstreamAmgen + Wyeth

Still experimental: anti HIV drug PRO 542 Soluble CD4 (HIV receptor) fused to IgG2.

Tetrameric (4 V-regions replaced)Reduced Fc function (since IgG2 < IgG1), Better half-lifeProgenics

23

Single chain antibodies (scFv)

Ag binding site

15 AA linker

Phage display selection of scFvSource of sequence:PCR from genome or mRNA, add randomization