5Immunotherapy Non-specific immunotherapy Specific immunotherapy BCGCytokinesSpecific immunotherapyActive immunotherapyAntibody therapyAdoptive transfer of T cellsVaccine-based immunotherapyTumour-based vaccinesVirus-based vaccinesPeptide-based vaccinesothers
6Key immune cells in cancer The main immune cells that play a role in the protection against tumours and their rejection areCytotoxic T-lymphocytes (CTLs)MHC restricted - recognize only small endogenously processed protein fragments (peptides) that must be presented in a surface protein called the major histocompatibilty complex (MHC)Natural Killer (NK) cells:toxicity without prior sensitization and without MHC restriction. Usually activated when T cells cannot be activatedMacrophages:usually activated by bacterial / viral infections leading to tumour cell deathStimulates secretion of the tumour necrosis factor (TNF)CTLs - recognize only small endogenously processed protein fragments (peptides) that must be presented in a surface protein called the major histocompatibilty complex (MHC) to be recognized. Therefore, T cells are ‘MHC restricted’ Cytotoxic T lymphocytes (CTL) recognize surface markers on other cells in the body that label those cells for destruction. In this way, CTLs help to keep virus-infected or malignant cells in check. Here, a smaller CTL (arrow) is attacking and killing a much larger influenza virus-infected target. The sequence represents 30 minutes elapsed time.
7Cytotoxic T-lymphocytes (CTLs) Macrophage engulfing a bacillus
8class I MHC antigen processing pathway The class I MHC antigen processing pathway acting as an internal surveillance mechanism to detect any abnormal or foreign protein synthesizedin the cell. Tumor antigens encoded in the endogenous DNA of the tumor cell, or encoded in a DNA plasmid or viral vector vaccine taken up byan APC, are synthesized and cleaved by the 26S proteasome into fragments that are transported by TAP, the transporter associated with antigenprocessing, into the endoplasmic reticulum, where they are loaded onto newly synthesized class I MHC molecules that transport them to the cellsurface for recognition by the T cell receptor.The Journal of Clinical Investigation Vol 113 Number 11 June 2004 pp 1515
9How tumour cells avoid immunosurveillance Altering Their Characteristics : Generate variants lacking antigens normally detected by CTL, NK cells and antibodies. Tumour cells may also lack co-stimulatory molecules, which activate T cells, and signaling molecules needed to respond to cytokines, such as gamma-interferon, that promote tumour cell killing by immune mechanisms.Suppressing the Immune Response : Tumour cells inappropriate or ineffective signals to CTL, or secreting TGF-beta etcHiding from the Immune Response : Immunoprivileged sitesExploiting the Immune System's Ignorance: Growth without eliciting any immune response.Outpacing the Immune Response: Tumour cells can simply proliferate so quickly that the immune response is not fast enough to keep their growth in checkCTLs - recognize only small endogenously processed protein fragments (peptides) that must be presented in a surface protein called the major histocompatibilty complex (MHC) to be recognized. Therefore, T cells are ‘MHC restricted’
10Immune escape mechanisms of tumour cells Figure 1 Immune escape mechanisms of tumour cells. Recognition of tumour cells by T cells requires a tumour-associated peptide (pep) to be presentedby a major histocompatibility complex (MHC) molecule and recognition of this peptide–MHCcomplex by the T-cell receptor (TCR) and ligation withCD4/CD8 molecules. The TCR consists of an ab chain, and recognition leads to a signal through the CD3 complex (g, Z (two), d and x (two) chains).To activate the T cell, the signal needs to reach the nucleus through downstream molecules such as p56 lck, p59 fyn and zeta-associated protein of 70 kDa(ZAP-70). Downregulation or inactivation of any of the molecules involved in this cascade will prevent T-cell activation and lead to subsequent killing of the tumour cell. Inability to release peptides from a protein, or to load the peptide on the MHC molecule, will prevent recognition of the tumour cell.Finally, expression of Fas ligand on tumour cells may induce apoptosis (programmed cell death) in the specific T cell, which prevents the tumour cellfrom being eradicated.Downregulation or inactivation of any of the signalling cascade molecules leads to tumour cell death.However, tumour cell will not be recognised ifPeptides are not released from a protein orPeptides not loaded onto the MHC moleculeExpression of Fas ligand on tumour cells may induce apoptosis in the specific T cell
12Activating the Immune System Non-specific approach WB Coley observed tumour regression after bacterial infectionsBCG vaccine to treat bladder carcinoma’s – cytokinesincludes interferons, interleukins and tumor necrosis factor (TNF)Limited success
13Specific approach – The promise of antibody-based therapy Search for tumour specific antigensDevelopment of monoclonal antibodies1975 Milstein and Kohler developed hybridoma technologyantibody-producing cells could be made to survive indefinitely if they were fused with cancer cells
14The problems of antibody-based therapy virtually all these antigens are also found on normal cells!Ab therapy may still be used becausethe antigen in normal tissues may not be accessible to blood-borne antibodiesthe cancer cells may express more antigen than normal cells doantibody-induced injury of normal cells may be reversible.
15Clinical trials – Ab-based therapy A33, a 43k glycoprotein with selective expression in normal and malignant epithelium of the (gastrointestinal tract)G250, a glycoprotein expressed by a high percentage of renal cancers;LewisY (LeY), an oligosaccharide epitope expressed on glycolipids and glycoproteins by a wide range of epithelial cancers;GD3, a ganglioside with high expression in melanoma and other neuroectodermal tumors;FAP-alpha, a 95 k glycoprotein strongly expressed in the stromal fibroblasts of epithelial cancers;Truncated EGF receptor, a 140 k form of the EGF receptor (deleted in exons 2-7), which is expressed by a proportion of brain cancers and other tumour types.antibodies have been genetically modified to provide chimeric (G250, GD3) or humanized (A33, LeY, F19) constructs
16Adoptive immunotherapy stimulating T cells by exposing them to tumour cells or antigens in the laboratory and then injecting expanded populations of the treated cells into patientsPatient is both donor and recepient
17Adoptive immunotherapy The Journal of Clinical Investigation Vol 113 Number 11 June 2004 pp 1515Generation of antitumor DC vaccines from peripheral blood monocytes. Elutriated monocytes from a leukapheresis are cultured with GM-CSF andIL-4 to produce DCs, which are then matured with CD40 ligand (CD40L) or other agents, pulsed with peptide or tumor lysate, or transduced withan expression vector and then injected into the patient as an autologous DC vaccine to induce a T cell immune response against the tumor.Generation of dendritic cell vaccines from peripheral blood monocytes:Monocytes cultures with GM-CSF +IL-4 to produce DCsMatured with CD40 ligandPulsed with peptide or tumour lysateRe-injected as vaccine to induce T-cell immune response against tumour
18Vaccine-based immunotherapy ’s, tumour immunogeneity seen in micethe tumors bore antigens that could immunize other mice of the same strain against transplants of the tumors.T lymphocytes from immunized animals could transfer immunity against tumours to healthy animals of the same strain.T cells from the immunized mice could kill tumour cells in vitro.Antibodies failed to transfer immunitychemicals or viruses induced tumors in mice, the tumors bore antigens that could immunize other mice of the same strain against transplants of the tumors. Subsequent studies showed that immune system cells known as T lymphocytes taken from immunized animals could transfer immunity against tumors to healthy animals of the same strain. And workers devised techniques to show that the T cells from the immunized mice could kill tumor cells grown in test tubes as well. In contrast, antibodies elicited by the tumor cells generally failed to transfer immunity or kill tumor cells.
19Vaccinesadministration of some form of antigen to induce a specific antitumour immune response.Tumour-based vaccinesUse whole cell/crude extracts of tumoursVirus-based vaccinesUse of viral oncolysate e.g. Vaccinia viruses expressing carcinoembryonic antigen (CEA)Peptide-based vaccinestumour-associated antigens (TAAs) epitopes bound directly to MHC on the cell surface can activate CTLsOthers:humoral responses e.g. Her2-neu, CEA, TP53, gangliosidesTumour example: colonic cancer recurrence risk reduced by 61% when injected with tumour-BCG follog operative surgery
20Approaches to antitumor vaccination Approaches to antitumor vaccination. (A) Irradiated tumor cells transduced with a viral gene transfer vector encoding a cytokine such as GM-CSFattract APCs (DCs) that acquire, process, and present tumor-associated antigens (TAAs) encoded by the vector in the context of MHC. (B) DCscan be directly loaded by incubation with tumor protein lysates or peptides with sequences based on expressed tumor antigens, or by viral genetransfer vectors expressing TAAs. (C) TAAs can be locally supplied to DCs by the direct injection of peptides, viral gene expression vectors, ornaked DNA expression plasmids. DCs migrate to secondary lymphoid tissues where they present the antigen epitopes to T cells to generate anantitumor cytolytic T cell response.The Journal of Clinical Investigation Vol 113 ( 11) June 2004 pp 1515APC – antigen presenting cell TAA – tumour associated antigenDC – dendritic cell MHC – major histocompatibility complex
21Immunoconjugates RADIOACTIVE ISOTOPES: I131 or yttrium 99 TOXINS: Use of antibodies to deliver toxins to a tumor site. E.g. ricin (made from castor beans), which inhibits protein synthesis and thwarts tumor growth.CHEMOTHERAPEUTIC DRUGS: Reach tumours in larger and lethal doses when delivered by an antibody.ENZYMES: convert "prodrugs" into cytotoxins will home to tumors when attached to antibodiesGENETIC DRUGS: e.g. antisense DNA can be linked to antibodies directly or packaged into viral particles engineered to have targeting antibody on their surface.INFLAMMATORY MOLECULES: tumour necrosis factor (TNF) and other messenger molecules of the immune system as well as certain microbial products, can bring about an inflammatory reaction that destroys tissues at the tumour site.
24Hormone therapyHormone sensitive cancers (Breast cancer in females and prostate cancer in males) are susceptible to deprivation of the corresponding mitogenic hormone.Treatment of either involvesdirect inhibition of steroid synthesis : E.g. using either LHRH superagonists or aromatase inhibitors in breast cancerblocking their effects at the target cell level through the receptors: Steroid receptor antagonists block receptor activity. E.g. tamoxifen is an oestrogen receptor antagonist.Problems with hormone therapy include sexual dysfunction (e.g.ovulation), secondary cancers etc
26References Immunotherapy for cancer by L.J Old Scientific American (Sept 1996) pg 102Tumours: ImmunotherapyMP Rubinstein and D J ColeProgress on new vaccine strategies for the immunotherapy and prevention of cancerJay A. Berzofsky, et al The Journal of Clinical Investigation Volume 113 Number 11 June