Chap 9Immunotherapy
I.Cancer Therapy
Introduction
Cancer therapy options:
Surgery, chemotherapy and radiation therapy.
Targeted therapy by
Small molecule drug
Ex: Imatinib (marketed by Novartis as Gleevec) is a tyrosine-kinase inhibitor used in the treatment of multiple cancers. Imatinib blocks a tyrosine kinase enzyme (BCR-Abl for phosphorylation essential in signal transduction in cancer cells) apoptosis of cancer cells. Because the BCR-Abl tyrosine kinase enzyme exists only in cancer cells and not in healthy cells, imatinib works as a form of targeted therapy—only cancer cells are killed through the drug's action. In this regard, imatinib was one of the first cancer therapies to show the potential for such targeted action, and is often cited as a paradigm for research in cancer therapeutics.
Monoclonal antibody (MAb)
Ex: trastuzumab (Herceptin®). Trastuzumab is a MAb that interferes with the HER2/neu receptor. Its main use is to treat certain breast cancers. The HER receptors are proteins that are embedded in the cell membrane and communicate molecular signals from outside the cell (molecules called EGFs) to inside the cell, and turn genes on and off. The HER proteins stimulate cell proliferation. In some cancers, notably certain types of breast cancer, HER2 is over-expressed, and causes cancer cells to reproduce uncontrollably.
Human tumors express a number of proteinantigens(tumor associated antigen, TAA) that can be recognized by the immune system,thus providing potential targets for cancerimmunotherapy.
However, the immune system ofpatients suffering from cancer is adversely affectedby tumorigenesis, even early in the course of disease.
Possible reasons for the resistance:
most T-cells capable of recognizingself-proteins are deleted from the repertoireduring T-cell differentiation in the thymus, tumor-specificself-antigens are generally weak immunogenically.
a natural selectionprocess leads to the selective enrichment of clones ofhighly aggressive tumor cells, which no longerexpress cell-specific molecules.
loss of MHC expression, downregulationof antigen processing machinery
expression oflocal inhibitory molecules
Mutation of epitopes at the TAA, leading to poor recognition by the immune system
Cancer vaccination attempts to activate immuneresponses against tumor antigens.
II.DC vaccination
DC are professional APCs that present Ag to T cells for activation of immune responses.
DC-based immunotherapy can induce antitumor immunity. However, clinical responses have been disappointing, with classic objective tumor response rates rarely exceeding 15%, but findings from emerging research indicate that DC-based vaccination might improve survival.
One particular concern with immunotherapy is the possibility of induction of autoimmunity. However, cancer vaccine strategies are rarely associated with severe immune-related toxicity.
First clinical trialinvolving DC reports that DC pulsedwith a tumor antigen could elicit specific tumor-reactiveT cells using theantigen expressed by non-Hodgkin’s lymphomas. In thistrial, 4 patients received three DC infusion per month followed bybooster injections of idiotype protein. No toxicitywas observed following infusion and all patients developed strong humoral and cellularimmune responses against TAA.
Clinical trials of DC vaccination are underway. These studies include a broad spectrumof tumors: melanoma (黑色素瘤), colorectal (結腸), lung and renal (腎) carcinoma, prostate cancerand multiple myeloma (骨髓瘤). These studies have shown that DC vaccination mayelicit tumor regression even in advanced cancerand have demonstrated the general safety of DC vaccinationin cancer patients.
Sources of DC
Circulating immature DC precursors
Representing less than 0.5% of peripheral bloodmononuclear cells (PBMC).
Can be isolated after 1–2days of in vitro culture in the absence of cytokinesusing density gradient centrifugation.
The most direct and the first method utilized in clinical trial of DC therapy.
Low yield of DC.
In vitro differentiation of DC from CD34+progenitors or blood monocytes
Culture of adherent PBMC with the cytokinesGM-CSF and IL-4 for 5–7 days leads to the generationof immature DC, which hasDC markers (e.g. CD1a)and maintain the ability to take up, digest andefficiently present protein antigen to T-cells.
Exposure of these immature DC to either cytokinecocktails or monocyte-conditionedmedia results in the generation of mature DC, including ‘veiled’ morphology,high-level expression of MHC, adhesionand co-stimulatory molecules, loss of CD14 andpowerful Tcell stimulatory capacity.
These techniques have been adapted for thegeneration of clinical grade DC.
The antigen presenting capacity and safety(adverse effects, autoimmunity) areimportant parameters of DC used for immunotherapy. In all recent vaccinationtrials mature DC are favored over immatureDC.
Choice of antigen
A common strategy for loading of DC withTAA explores pulsing with synthetic peptides(the immunodominant epitopesof TAA). This method requiresknowledge of the aminoacidsequence of relevant peptide.
Toavoid the generation of escape variantsin solidtumors, immune response against a broad spectrumof tumor-derived epitopes is highlydesirable.
Mixtures of peptides directed against differentepitopes.
TAA in the form of purifiedrecombinant proteins, mRNA, cDNAor virus vectors (a form of gene therapy) for antigen delivery.
Pulse the DC with entire tumorlysates, total tumor RNA, whole apoptoticor necrotic tumor cells. Such DC vaccinesmay have better antigen-presenting properties incomparison with the peptide pulse.
Two most important factors for choice of TAA:
- elicitation of both cellular and humoralimmune responsesTAA that are expressed on the surface oftumor cells are desired
- Specificityfor tumorwhentarget antigens are not tumor-specific, but arealso expressed in normal tissues severe autoimmune disease.
Commercial example: Provenge(FDA approval)
Consists of autologous blood APCs (mononuclear cells) pulsed with PA2024, a recombinant PAP-GM-CSF fusion protein produced using the baculovirus system. PAP (prostatic acid phosphatase) is the TAA while GM-CSF is an immune cell activator.
Provenge is designed to stimulate immune responses to the prostate cancer patients. This product contains a small fraction of DC.
Targets for DC immunization
It is assumed that DC vaccination regimens could workeffectively when the total tumor volume 100 cm3. Over this limit, DCvaccination may best be carried out as adjuvant treatmentfollowing primary surgery or chemotherapy.
The primary goal of DC vaccination will be to eradicatemetastatic tumor cells (which migrate from the original growth site to other locations in the body).
However, the pattern ofputative TAA expressed on the primary tumor may be different from that ofmetastasized tumor cells. The switch from localtumor growth to an invasive metastatic phenotyperequires a complex series of events and numerousalterations.
Route of DC delivery
Many clinical studies to date have utilizediv administration. The most successfulclinical trial for renal carcinoma exploited subcutaneous (皮下) application. DC vaccination trials formelanoma utilized a combination of subcutaneousand intradermal (皮內) application with iv boosting. It seems likely that iv applicationof DC may be effective for therapy ofhematopoetic tumors and solid tumors thatmetastasize through blood circulation or bone marrow.In all other cases, the route of delivery dependson localization of tumor. Since mature DC do notexpress chemokine receptors necessary for peripheralmigration, intratumoural or peritumouralroutes of vaccination are of interest in cases, where apreferentially local immune response should beelicited. Thus, intradermal and subcutaneousapplication may be advantageous for treatment ofmelanoma, whereas intranodal administration maybe beneficial to eliminate metastatic epithelialtumors.
Summary:
Since DC possess all necessary components for antigenpresentation, including the production of a varietyof cytokines and the expression of stimulatorysurface molecules, they have been termed ‘nature’sadjuvant’.
Although pilot clinical trials in variouscancers have yielded promising results, itappears likely that DC immunotherapy alone mayhave only limited success. Thus, the utilization of DC immunotherapy formanagement of minimal residual disease after resectionof primary tumor may be promising.
Two current trends: (1) developing next generation DC products with improved immunostimulatory activity; (2) Combination therapy (in combination with other therapy such as MAb)
Induction of humoral immunity seems to be indispensablefor treatment of many tumors. Since humoral immunityis effective only when the target structures areexpressed on the surface of tumor cells, the choiceof antigens for vaccination is crucial.
Other options for cancer therapy
Gene therapy with vectors carrying suicide genes.
Gene therapy with vectors carrying anti-angiogenesis genes.
Gene therapy with the replicating oncolytic virus.
Adoptive cell therapy (ACT) such as adoptive transfer of T cells that are genetically engineered to produce chimericantigen receptors (CARs)on the surface.
III. CAR T cells therapy
T cells contain receptors (T cell receptor, TCR) on the surface to recognize the cognate antigen. Autologous T cells are collected from the patient’s own blood. After collection, the T cells are genetically engineered to produce specialreceptors on their surface called CARs. CARs are proteins that allow the T cells to recognize a specific protein (antigen) on tumor cells.
Domains of CARs
Ectodomain
A signal peptide directs the nascent protein into the endoplasmic reticulum. This is essential if the receptor is to be glycosylated and anchored on the cell membrane.
The antigen recognition domain:single-chain variable fragment (scFv).
A spacer region links the antigen binding domain to the transmembrane domain. It should be flexible enough to allow the antigen binding domain to orient in different directions to facilitate antigen recognition.
The transmembrane domain is a hydrophobic -helix that spans the membrane. Different transmembrane domains result in different receptor stability. The CD28 transmembrane domain results in astable receptor.
Endodomain: After antigen recognition, receptors cluster and a signal is transmitted to the cell. The most commonly used endodomain component is CD3-zeta. This transmits an activation signal to the T cell after antigen is bound.
Production and use of CAR T cells:
Viral vectors are used to integrate the necessary genes into cells to deliver the genetic material needed to produce the T-cell receptors.
These engineered CAR T cells are then grown and expanded.
The expanded population of CAR T cells are then infused into the patient. After the infusion, the T cells can multiply in the patient’s body and, with guidance from their engineered receptor, recognize and kill cancer cells that harbor the antigen on cancer cell surface.
New generation of CAR T cells:
CD3-zeta may not provide a fully competent activation signal and additional co-stimulatory signaling is needed. For example, chimeric CD28 and OX40 can be used with CD3-zeta (CD3-)to transmit a proliferative/survival signal, or all three can be used together.
The second- and third-generation CAR typically consist of a piece of monoclonal antibody (scFv) that resides outside the T-cell membrane and is linked to stimulatory molecules (e.g. 4-1BB) inside the T cell.
The scFv portion guides the cell to its target antigen. Once the T cell binds to its target antigen, the stimulatory molecules provide the necessary signals for the T cell to become fully active. In this fully active state, the T cells can more effectivelyproliferate and attack cancer cells.
Researchers are still improving how CAR T cells are produced, including testing various delivery vectors and different stimulatory molecules to see which can help produce the most potent T cells.
Two phase 1/2a trials in 2014 show that 27 out of 30 patients with relapsed or refractory cancer experience complete remissions.
However, one side effect is the cytokine release syndrome (CRS, i.e. cytokine storm). CRS develops when the infused T cells proliferate as they attack a tumor, releasing a massive quantity of inflammatory cytokines (e.g. IFN-, IL-6), which can be life-threatening.
References:
[1]Goldman B, DeFrancesco L. The cancer vaccine roller coaster. Nat Biotech 2009; 27:129-39.
IV.Appendix
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