mAB Drugs
Email Kathy


Surfing Biotech's Second Wave: Monoclonal Antibody drugs
September 15, 2009

 

Homework Note: The notes below are AGAIN in OUTLINE form and require you to do a bit of homework (~2 hours of time before class). For each drug listed below, go to the link provided and read about the drug to determine:
(1) The Target: what human protein is recognized by the mAb?
(2) Indication: why the mAb is prescribed and what it does it do in the body?

Monoclonal antibodies: be sure you are familiar with these terms and procedures:

A question before we begin: What do all these words have in common?
* Alemtuzumab
* Gemtuzumab ozogamicin
* Rituximab
* Trastuzumab
* Ibritumomab tioxetan

 

Answers (circle all that apply):

  1. They are all relaxation chants used in Hatha Yoga
  2. They all sound like "Number 9, Number 9" if you pronounce them backwards
  3. They are all code words for admission to a secret society of M.D.s
  4. They all end in -mab, shorthand for monoclonal antibody
  5. They are all names of monoclonal antibody based drugs targeted to cancer cells!
If you guessed 4 and 5 = you're right!

Monoclonal antibodies: Antibodies are proteins formed by animals when they encounter a foreign protein or substance. Interaction of the foreign protein (antigen) with the antibody results in the destruction of the antigen, and further protection of the organisms from related antigens.

  • Due to their ability to specifically recognize and target antigens, antibodies have also become useful therapeutic tools.
  • For instance, if a cancer cell expresses an aberrant protein, and an antibody can be made against that protein and injected into a patient, then the antibody itself becomes a therapeutic drug and can be used as a 'magic bullet' to find and destroy cancer calls.
  • HOWEVER, antibody secreting cells (B-cells) in culture die out after a few generations, due to the finite growth potential of any somatic cell.
  • In addition, the immune system does not normally just make an antibody to just ONE part of an antigen, but makes 'polyclonal antibodies' synthesized to recognize a many different features of the antigen. While this serves an animal well in fighting off a foreign protein, as a therapeutic, it can lead to problems in specificity and recognition of related proteins.

IN 1975 Cesar Milstein, Georges Kohler and Niels Jeme developed monoclonal antibody technology by fusing immortal tumor cells (myeloma cells) with antibody-producing B lymphocyte cells to produce "hybridomas," that continuously synthesize identical (or "monoclonal") antibodies. Milstein, Kohler and Jeme were awarded the 1984 Nobel Prize in Medicine.

 

Monoclonal antibodies are preparations of a single antibody-secreting B cell (from the spleen of a mouse immunized with the target protein) fused with myeloma cells (cancerous B-cells with unlimited growth that have lost the ability to make their own antibodies).

 

The procedure:

A. Obtain mouse. Immunize. Obtain spleen = Antibody specificity (B cell)

B. Obtain HGPRT- Myeloma Cells (cancerous B-cells) = Unlimited Proliferation

Myeloma cells lack the ability to synthesize purines (HGPRT- = lack hypoxanthine-guanine phosphoribosyl transferase - catalyzes the first step in the purine pathway). Myeloma cells cannot grow in a selective, purine-free media (HAT media contains purine precursors hypoxanthine, aminopterin and thymidine)

C. Fuse cells with PEG to make a Hybridoma

 

1. Culture in HAT media

  • Unfused myeloma cells (HGPRT-) die out because they cannot make purines.
  • Unfused spleen cells (containing B-cells) die out because they have a limited life span
  • The only cells that will survive are the fused B-cell: myeloma cells = hybridomas

2. Test fused cells for antibodies by ELIZA
3. Subculture and clone positives.
4. Test supernatants for antibodies
5. "Expand' or scale up the best clone (all derived from a single cell = monoclonal)

6. Hybridoma cultures producing monoclonal antibodies can be maintained indefinitely in vitro or in vivo

7. MAbs can be developed into drugs, and injected into a person to seek out, bind, and target for destruction the antigen against which the antibody was raised.

 

The combination of antibody specificity (B cell) + unlimited proliferation (myeloma):

 

Because the human body eventually develops an immune response against the monoclonal antibody (made from a mouse B-cell), most monoclonal antibody-based drugs today are so-called humanized monoclonal antibodies: the mouse antibody is carefully digested to release just the antigen-binding variable region, which is then swapped into a human antibody missing its variable region. This prevents an immune response to the antibody itself.

 

A fully humanized monoclonal antibody has the antigen binding murine complementarity-determining regions interspersed within the variable regions of the light (light gray) and heavy (dark gray) chains of the Fab portion of the engineered antibody. A chimeric antibody has the entire antigen-binding murine component of the variable region of the Fab section is maintained integrally (handout).

 

See figures from Access Excellence and for humanized monoclonal antibodies.

T cells (lymphocytes) are WBCs that mature in the thymus and expresses antigen-specific receptors like CD4 or CD8, and the CD3 T-cell receptor needed for T-cell activation and proliferation. (CD4+ cells are = helper T-cells a; CD8+ are cytotoxic (killer) T cells. Memory T-cells can be either C4+ or C8+ = "cellular immunity")
B cells (lymphocytes) are WBCs that are derived from the bone marrow and spleen. B cells develop into plasma cells, which produce antibodies. "Humoral immunity"

Major Competitors in the Transplantation Antibody Therapy Market:

 
Target: what human protein recognized by the mAb
Indication: why the mAb is prescribed and what it does in the body
Other notes
1. Orthoclone OKT3
(Muromonab) 
 T-cell CD3
receptor 		Blocks CD3+T-cell activation
Prevents organ transplant rejection		 [1986!] First mAb
OrthoBiotech
2. Simulect
(Basiliximab)
 T-cell IL-2 receptor Blocks IL-2 mediated T-cell activation
Prevents organ transplant rejection		 [Nov 1997] Novartis;
3. Zenapax
(Daclizumab)
 T-cell IL-2 receptor Blocks IL-2 mediated T-cell activation
Prevents organ transplant rejection		 [1997] Roche; 90% human, 10%murine

 

All three: Immunosuppressive therapy to block T-cell activation/proliferation. MHC antigens on transplanted organs continually activate T-cells within the recipient's immune system to rid the body of the foreign MHC antigens (organ rejection, GVHD). CD3 is part of the T-cell receptor (TCR) needed for T-cell activation, IL-2 is a potent stimulator of T-cell activation / proliferation), Typically only a few doses are given, just BEFORE transplant and then for a few weeks after treatment; other immunosuppressives are taken for life. Simulect and Zenapax have also been tested for people with MS.

 

More about T-cell proliferation in organ rejection: A Review of Interleukin-2 receptor Antagonists in Solid Organ Transplantation From MedSccape/WebMD

Major Competitors in the Oncology Market:

Several THEMES:

Inhibiting Angiogenesis: Interfering with cancer's blood supply
HER Signaling Pathway: Interrupting growth signaling in HER overexpressing tumors
Depleting B Cells: Targeting CD20 and other B-cell-specific proteins


 
Target: what human protein recognized by the mAb
Indication: why the mAb is prescribed and what it does in the body
Other notes
1: Rituxan
(Rituximab)
 CD20+ antigen
B-cell		 Non-Hodgkin's Leukemia Binds & selectively depletes CD20+ B cells NHL [Nov 1997] Genentech/
Biogen IDEC 1st cancer mAb; MOA image
2: Zevalin
(Ibritumomab)
CD20+ antigen
B-cell		 Non-Hodgkin's Leukemia1st radioimmunotherapy*
(beta radiation = cytotoxic)		 [Feb 2002] Biogen IDEC
mAb + 111Indium & 90Ytrium = Yikes!
3: Bexxar
(Tositumomab)
CD20+ antigen
B-cell
(not a front-line therapy) 		Non-Hodgkin's Leukemia Indicated for chemo- or Rituxan-refractory, CD20+ B cells [June 2003] Corixa;
mAb + 131Iodine - (beta/gamma) 2 I.V. doses over 2 weeks
4: Herceptin
(Trastuzumab)
 Her2 receptor
(EGFreceptor)		 Breast Cancer: Targets Her2 receptor overexpression (transmembrane gf receptor) [Sept 1998] Genentech
MOA - go here
5: Avastin
(Bevacizumab)
 VEGF overexpression
(tumor-derived GF)
 Metastatic colo-rectal cancer: VEGF produced by cancer cells (Judah Folkman) [Feb 26 2004] Genentech
MAb + 5-FU, MOA
6: Erbitux
(Cetuximab)		 Her1 receptor
(EGFreceptor)		 Metastatic colo-rectal cancer (with irinotecan,or irinotecan-refractory, or as single agent) [Feb 12 2004] ImClone
1st mAb for MCRC
Martha!; not pancreatic
7: Campath
(Alemtuzumab)
 CD52 antigen
B and T cells. 		B-cell chronic lymphocytic leukemia (B-CLL) (in patients who have failed fludarabine therapy) [May 2001] Genzyme/Berlex
Destroys cells in both blood and bone marrow.
8: Mylotarg
(Gemtuzumab) 		CD33+ antigen
Leukemic blast cells
(immature WBC)		 Acute Myelogenous Leukemia: Selectively targets leukemic blast cells and delivers calicheamicin.

[May 27, 2000]
Weyth MOA. Also called "GO" Way cool!
mAb + calicheamicin.

 

Double Whammys: watch out cancer cells...
*Zevalin, "the first FDA-approved radioimmunotherapy, combines the targeting power of monoclonal antibodies with the cell-damaging ability of localized radiation. Radioimmunotherapies like Zevalin are made by linking a monoclonal antibody (to the CD20 antigen) to radioactive isotopes (Indium-111 and Yttrium-90). When infused into a patient, these radioactive antibodies circulate in the body until they locate CD20+ B cells, and then deliver their cytotoxic radiation directly to cells. Zevalin binds to malignant and normal B cells. Normal B cells are replenished within nine months following therapy." Wicked side effects but impressive remission rates! Bexxar works about the same way...and both are between $26K -$30 K per regimen!


Mylotarg (gemtuzumab ozogamicin, GO, Wyeth) is a humanized anti-CD33 monoclonal antibody linked to a highly potent cytotoxic antibiotic, calicheamicin, which induces cell death by cleaving double-stranded DNA at specific sequences. "Mylotarg indicated for bone marrow cancer (CD33 positive acute myeloid leukemia) in patients who are 'relapsing after the first treatment, 60 years of age or older, or not considered able to take standard leukemia chemotherapy. This double whammy drug contains a monoclonal antibody that binds to the cell-surface CD33 antigen abundant on AML hematopoietic cells, linked to an extremely potent chemotherapy agent calicheamicin. Once Mylotarg is internalized, calicheamicin is released inside the lysosomes of the myeloid cell and binds to DNA which results in DNA double strand breaks and cell death. As a result, the leukemic cells are destroyed but the cells that are responsible for replenishing normal blood cells are spared'. [Quote] Way cool! Nature Oncogene 2007

 

This is NOT one of our Biotech drugs, but every year Med Students tell me I need to talk about this one!

  • Gleevic (Imatinib) - a competitive inhibitor of the TK bcr-abl indicated for CML
  • In chronic myelogenous leukemia (CML), the Philadelphia chromosome leads to a fusion protein of abl with bcr (breakpoint cluster region), termed bcr-abl - now a continuously active TK.
  • Gleevic binds to the active site that would normally bind ATP, preventing ATP from phosphorylating the substrate, decreasing bcr-abl activity & stopping cell division!
  • $32,000 per year for a 400 mg/day dose

Major Competitors in the T-Cell Autoimmune Disease Market:

Backgrounder on Autoimmune Disease:

  • Over 80 serious, chronic illnesses that affect nearly every organ system in humans.
  • In T-cell autoimmune disease, the body’s T-lymphocytes destroy the body's own organs.
  • More than 100 million patients worldwide suffer from T-cell mediated autoimmune disorders, such as psoriasis, rheumatoid arthritis (RA), ulcerative colitis, Crohn's disease, and multiple sclerosis (MS).
  • T-cell autoimmune diseases are triggered by the overproduction of cytokines (signalling molecules that include interleukins) secreted by macrophages or lymphocytes that lead to the invasion and inflammation of the target organ.
  • For example, overproduction of the inflammatory cytokine TNF in the joints of people with RA result in invasion of the joint space by destructive macrophages, neutrophils, and T cells, causing inflammation, joint pain and swelling.
  • Psoriasis alone affects about 80 million people worldwide and 4.5 million U.S. adults,
  • Women more affected possibly due to hormones, genetic component.

******************
Recently, a number of drugs have been developed to treat T-cell autoimmune diseases. Each work differently, but the net result of all of these particular drugs is to decrease either T-lymphocytes or cytokines like TNF. Each is being investigated as a way to treat psoriasis (P), rheumatoid arthritis (RA) , ulcerative colitis, Crohn's disease, Psoriatic Arthritis (PsA), Ankylosing spondylitis (AnkS) and/or multiple sclerosis (MS). [Don't have to remember which!]

Note: Enbrel and Amevive are NOT mAbs but fusion proteins, where the protein of interest is attached to the Fc portion of an IgG1 antibody heavy chains, and the resulting 'fusion protein' subsequently purified. As a drug, the Fc portion initiates the immune system function of the antibody, eliminating the fusion protein-antigen complex via binding to phagocytes or NK cells.

 
Target: what human protein recognized by the mAb
Indication: why the mAb is prescribed and what it does in the body
Other notes
1: Enbrel *
(Etanercept)
 excess circulating TNF Scavenges excess TNF in RA, PsA, P, AnkS [1998] Amgen;
Fusion ptn: IgG1 + TNF receptor
2: Amevive *
(Alefacept)
CD4+ CD8+ T-lymphocytes Suppresses T-lymphocytes involved in Psoriasis (immunosuppressive) [Jan 2003] Biogen;
Fusion ptn: IgG1 + LFA-3
3: Remicade
(Infliximab)
excess circulating TNF Scavenges excess TNF in RA, Crohn's [1998] Centocor
mAB to TNF
4: Humira:
(Adalimumab)
 excess circulating TNF Scavenges excess TNF in RA [Dec 2002] Abbot
1st Fully Human mAb
5: Raptiva
(Efalizumab)
CD11+ T-lymphocytes Suppresses T-lymphocytes activation, proliferation, migration, interaction with cells, and cytokine release; PsA, PPa [Oct 2003] Genentech

Misc mAb Drugs:

 
Target: what human protein recognized by the mAb
Indication: why the mAb is prescribed and what it does in the body
Other notes
1. Synagis
(Palivizumab)
 RSV particles Binds and neutralized RSV in babies with comprimized lungs (McCaughey septuplets) [June 1998] Medimmune *
2. ReoPro
(Abciximab)
 GP IIb/IIIa receptors Platelet-aggregation inhibitor (GP IIb/IIIa inhibitor): prevents blood clot-related complications from balloon angioplasty, coronary intervention, unstable angina [Dec 1999] Centocor / Eli Lilly
3. Xolair
(Omalizumab)
IgE Decreases IgE in the blood and prevents binding to mast cells ass'd with allergy-related asthma [June 2003] Genentech
4. Lucentis
(Ranibzumab)
 VEGF overexpression
(tumor-derived GF)		 Wet (neovascular) age-related macular degeneration (AMD) - gain of 3 lines (15 letters) on the eye chart! [June 2006] Genentech
MOA - go here

June 30, 2006: Stock surge: Genentech has won approval for Lucentis, a drug to treat a common loss of vision known as wet age-related macular degeneration.

  • LUCENTIS inhibits the formation and leakage of new blood vessels in the back of the eye, the primary cause of central vision loss associated with AMD.
  • Nearly all patients (95 percent) treated with LUCENTIS maintained their vision in the Phase III clinical trials.
  • Vision improved by at least three lines (or 15 letters) on the study eye chart in up to 40 percent of these patients at one year.
  • Although not unexpected, positive sentiment surrounding the FDA's decision on 30 June sent the company’s stock soaring: on 6 July, it closed at almost US$86, up from $79 a week earlier.
  • Investor excitement was fueled by the company’s announcement that it will sell a single injection of the treatment ranibizumab for $1,950: thousands of elderly sufferers are expected to need it five or six times a year. (*gee, that's GREAT news?!?!*)

How is it different from Avastin?!?

  • Lucentis is a smaller derivative of Avastin (parent molecule) that Genentech developed specifically for injecting into the eye.
  • Avastin is approved for the treatment of metastatic colon cancer, is given intravenously, and has severe side-effects like hypertension, heart attacks, and strokes.
  • Avastin was thought too be too big to penetrate the retina.
    In 2005 a tiny amount of the drug injected directly in the eye (off label use) was effective at controlling the new blood vessels in wet AMD.
    September 27, 2005 Genentech in Competition With Itself on Eye Drug !

OBJECTIVES:

 

1. When making monoclonal antibodies, what is the purpose of: the immunized mouse (and its poor spleen)? What are myeloma cells and why are they so useful in this technique? What is a hybridoma? How are experimental conditions set up to select for hybridomas?

2 . What is meant by the terms chimaeric monoclonal antibody and humanized monoclonal antibody?

3. For each drug listed below, be sure to know: (1) the target antigen, (2) the indication, (3) what the mAb does in the body:

 

Transplantation: Orthoclone OKT3, Simulect , Zenapax
 Oncology: Rituxan, Zevalin, Bexxar, Herceptin, Avastin, Erbitux, Campath, Mylotarg
T-cell autoimmune: Enbrel, Remicade, Humira, Amevive, Raptiva   
Misc: Synagis, ReoPro, Xolair, Lucentis

 

Do you have to know the mysterious 'xyz'-mab names? Not for me, but you MAY need to know all of these someday soon!