How tumor antigens are obtained
To create an effective antitumor vaccine, the first step is to obtain the antigen itself—the molecular target that the patient's immune system will react against. Modern laboratory practice distinguishes two main methodological approaches to solving this problem.
Biochemical approach
This method is based on the direct physical isolation of peptides already complexed with MHC-I membrane molecules on the surface of a malignant cell. The process includes several sequential steps:
- Tissue collection. Extraction of living cells directly from the tumor tissue.
- Acid elution. Exposure to an acidic environment to disrupt the bond between the peptide and the MHC-I molecule, resulting in the release of protein fragments.
- Purification. Isolation of the obtained peptides from the total cell mass.
- Sequencing. Precise determination of the amino acid sequence of the isolated fragment.
- Artificial synthesis. Laboratory reproduction of the identical peptide on an industrial scale.
Main disadvantage of the method: MHC molecules on the cell surface retain not only specific mutant tumor proteins, but also a huge number of completely normal, endogenous (autologous) peptides of the body. Technically, it is extremely difficult to separate the target oncoantigen from this background "noise".
Molecular biological approach
Today, this is the most sought-after and widespread approach. Instead of "stripping" ready-made proteins from the membrane, researchers work at the genetic level by cloning the genes encoding the desired antigens.
- A cDNA library (complementary DNA) is created based on the genetic material of tumor cells.
- The cloned genes are inserted into a special vector plasmid. A carrier virus is often used as a delivery system.
- Transfection is performed—the introduction of the plasmid into a target cell culture. For example, COS cells (derived from monkey kidney tissue) can be used for this purpose utilizing the SV40 large-T antigen.
- Inside the transfected cell, the plasmid begins to actively replicate (multiply).
- The translation process is triggered: the factory cell synthesizes the target tumor protein in large quantities, which is then harvested to create the vaccine.
Dendritic cell loading: presentation pathways
Obtaining a tumor-associated peptide is only half the battle. For the vaccine to work, the antigen must be "presented" to the immune system. To do this, dendritic cells (DCs)—the body's main antigen-presenting cells—are used. The way the antigen is loaded into DCs strictly determines what type of immune response will ultimately develop.
Endocytic pathway (via MHC-II molecules)
- Mechanism: The antigen preparation is simply added to the extracellular nutrient medium. The dendritic cell naturally engulfs it via endocytosis, digests it inside vesicles, and displays the fragments on its membrane in association with MHC-II molecules.
- Indication/Use: This is the classical pathway for creating vaccines against allergies and autoimmune conditions. In the context of oncology, this pathway is also used because it allows the activation of tumor-specific Th1 cells (type 1 T helper cells), which secrete cytokines and support the overall inflammatory response.
Cytosolic pathway (via MHC-I molecules)
- Mechanism: Unlike simple uptake from the medium, here researchers must artificially create an antigen delivery pathway bypassing endosomes, directly into the cytosol of the dendritic cell. Only in this case will the protein be processed by intracellular enzymes and displayed on the surface in a complex with MHC-I molecules.
- Indication/Use: This is a critically important condition when developing cancer vaccines. Antigen presentation via MHC-I is the only way to induce cytotoxic T lymphocytes. It is these killer cells that have the ability to recognize and physically destroy tumor tissue.
Briefly: answers to topical questions
Which classes of tumor antigens are used in the development of antitumor vaccines?
Various classes of tumor antigens, obtained by biochemical or molecular biological methods, are used in vaccine development.
- Viral — antigens of causative viruses (EBNA, E6, E7, HHV-8).
- Mutant (unique) — products of mutant genes (p53, Cdk4, Cas8, β-catenin).
- Cancer-testis — expressed in embryos and gonads (MAGE, BAGE, GAGE, NY-ESO-1, SSX2 series).
- Differentiation — normal differentiation antigens (Melan A/MART-1, tyrosinase, gp100, PSA).
- Amplified — overexpressed antigens (HER-2/neu, BIRC, cyclins B1 and D1, Bcr/Abl).
- Products of aberrant processing — antigens with excessive glycosylation (MUC-1 — MUC-7).
Which specific cytokines do Th1 cells secrete to support the antitumor immune response?
To support the antitumor immune response, mature Th1 cells secrete a specific spectrum of cytokines.
- Interferon gamma (IFNγ) — activates macrophages, suppresses angiogenesis, and induces chemokine production.
- Interleukin-2 (IL-2) — stimulates the maturation and proliferation of tumor-specific cytotoxic T lymphocytes.
- Tumor necrosis factor alpha (TNFα) — participates in macrophage activation and ensures bactericidal activity.
Th1 lymphocytes also produce IL-3, TNFβ, and GM-CSF.
What methods are used for the artificial delivery of the antigen directly into the cytosol of the dendritic cell?
Artificial delivery of the antigen directly into the cell cytosol is carried out using specialized adjuvants.
- Immunostimulatory complexes (ISCOMs) — vesicles in which viral antigens are associated with Quil A glycoside micelles.
This method ensures transmembrane penetration of the vaccine antigen directly into the cytosol. Subsequently, the antigen is degraded by the proteasome, and its peptide fragments are integrated into MHC-I molecules to induce a cytotoxic T-cell response.
Which viral vectors are used for gene delivery in the molecular biological approach to vaccine creation?
In the molecular biological approach, carrier viruses are used for gene delivery.
- Vaccinia virus — a classic example of a neutral carrier virus into which a recombinant plasmid containing the protective antigen gene is inserted to create a recombinant vaccine.
To avoid confusing the presentation pathways, remember the rule: "Cytosol — Unit — Killer". If the antigen enters directly into the cytosol, it is presented on MHC-I and activates cytotoxic T lymphocytes, which directly kill the tumor.
Class and exam questions
What is the weak point of the biochemical approach to antigen production?
The problem lies in the non-specificity of harvesting. During acid elution from MHC-I molecules, not only tumor peptides are stripped off, but also numerous normal body proteins, from which it is incredibly difficult to separate the target antigen.
Why is it necessary to activate type 1 T-helper cells in tumor treatment?
Activation of Th1 cells occurs upon antigen presentation via MHC-II (endocytic pathway). These cells are essential for the general support of the immune response and the secretion of cytokines that stimulate the fight against the tumor.
Which pathway of dendritic cell loading is critically important for cancer vaccines and why?
The cytosolic pathway (presentation via MHC-I) is critically important. Only it allows the induction of cytotoxic T lymphocytes capable of directly destroying malignant cells.
What else to study on this topic
- Mechanism of acid elution of peptides from the MHC-I complex
- Features of creating cDNA libraries from tumor material
- Application of COS cells and SV40 large-T antigen in biotechnology
- Methods of artificial antigen delivery directly into the cytosol
- Interaction of Th1 cells and cytotoxic T lymphocytes in the response to a tumor
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