HC14: Cancer immunity and immunotherapy

Immune responses in cancer

The incidence peak of cancer is around the 6^th decade of life. Therefore, it isn’t important in the evolution or in shaping the immune system → mainly occurs after reproductive age.

In the beginning of the 20^th century, a number of scientists proposed a theory of immune surveillance, which later was further confirmed:

1. If a mouse is injected with a carcinogen, a tumor starts developing
2. If the tumor is injected into another mouse, the tumor continues developing
3. If the tumor is first subjected to radiation and vaccinated into a mouse, the tumor stops growing

This shows that the immune system plays an important role in tumor growth.

Tumor-infiltrating immune cells

The presence of certain immune cells correlates with both improved and worse tumor behavior and prognosis.

T-cells:

T-cells are associated with both a good and bad prognosis, depending on the type:

• Presence of CD8 T-cells correlates with a good prognosis
• Presence of tertiary lymphoid structures are a good sign
• Presence of regulatory T-cells are associated with a worse prognosis → immune suppressive role

Macrophages:

Macrophages are often associated with immunosuppressive activity. They can produce cytokines like IL-10 or TGF-β, which counteract immune activity. There are 2 types of macrophages:

• M1: pro-inflammatory → better prognosis
• M2: immunosuppressive → worse prognosis

The majority of macrophages in tumors are M2 → make a bad prognosis.

Genetic instability and immune recognition

Immune recognition:

When a cell is targeted by carcinogens such as UV-radiation or cytotoxic drugs, the following happens:

1. Damage response pathways are activated, such as:
   • ATM
   • P53
2. When these proteins are activated, they induce the expression of a number of stress molecules which go to the surface of cells
3. The proteins are recognized by NK-cells or CD8 T-cells
4. The target tumor cell is eliminated

However, a lot of these pathways are shut down in tumors. This causes the immune system to not be able to recognize tumor cells through these mechanisms anymore.

Antigen processing and presentation:

HLA-II molecules present proteins which are cut into peptides to cells. Although the main function of B-cells is antibody production, they can also produce HLA-I and HLA-II. Antigen presenting cells are:

• B-cells
• Dendritic cells
  • Professional antigen presenting cells
• Macrophages

These cells have HLA molecules:

• HLA-I: presents to CD8 → kills the target cell
• HLA-II: presents to CD4 → activates B-cells to kill the pathogen

Both dendritic cells and macrophages have phagocytic capacity. Dendritic cells are the most effective APCs → provide the right co-stimulatory signal.

However, not all peptides/proteins are presented to T-cells. This depends on the HLA alleles present in the immune system, which differs per person. Furthermore, even if a peptide is presented, not all peptides can be recognized by T-cells → an individual must have a compatible T-cell receptor that can recognize a specific HLA/peptide complex.

Antigens

Antigens presented to immune cells by tumor cells are:

• Self-proteins: also present in healthy cells
  • Overexpressed antigens
  • Cancer testis antigens
    • The testis is a privileged site
  • Oncofetal proteins
    • Usually only are present during fetal development
• Non-self-proteins: not present in healthy cells
  • Neo-antigens
    • Derived from somatic mutations
  • Viral antigens
    • HPV in cervical cancer

Self-antigen therapy:

Self-antigens can all potentially be exploited in immunotherapy. These antigens all are normal → are expressed in malignant tissue, but can also be expressed in low levels in healthy tissues. This causes healthy tissue to also be affected during some cancer treatments. For instance, during colorectal cancer therapy, severe colitis can be a side effect.

Non-self-antigen therapy:

Non-self-antigen caused tumors are easier to treat, because they are not expressed in healthy tissues → non-self antigen-specific T-cells are not subjected to central tolerance.

Cancer immunity cycle

The whole process that generates an immune response against tumors can be demonstrated by the cancer immunity cycle:

1. Tumor cells die → release antigens and proteins
2. Antigens are picked up by antigen presenting cells
   • Most often dendritic cells
3. APCs travel to secondary lymphoid structures and present the antigens to T-cells
   • CD4 T-cells
   • CD8 T-cells
4. The T-cells enter the circulation, infiltrate the tumor tissue and engage with tumor cells
5. The T-cells kill tumor cells → release antigens and proteins
6. The cycle repeats itself until total elimination of the tumor

CD8 T-cells:

CD8 T-cells can kill tumor cells via 2 pathways:

• Produce cytolytic proteases which make holes in membranes → kill tumor cells
  • Most common route
• Fas-FasL interaction → apoptosis
  • Not the main mechanism

CD4 T-cells:

Th1 CD4 T-cells produce a number of pro-inflammatory cytokines which support the activity and killing by CD8 T-cells:

• IL-2
• IFN-γ

The production of IFN-γ not only supports CD8 T-cells, but also:

• Increases the activity of APCs → feeds the cancer immune cycle
• Tells tumor cells to upregulate MHC-I presentation
• Leads to arrest of tumor cell division and stimulates apoptosis
  • Thus, CD4 T-cells can also indirectly kill tumor cells

Other CD4 T-cells have different functions:

• Regulatory T-cells are not supportive of destroying cancer → accumulation correlates with a bad prognosis
• Th2 cells aren’t involved in tissue destruction
• Th17 cells aren’t relevant for now

Immunoediting

Right after transformation, there is an initial recognition of the tumor which sometimes eliminates the tumor. If this doesn’t happen, there is a generation of clonal heterogeneity of the tumor. If these clones start dominating, the immune cells aren’t able to recognize tumor cells anymore. A tumor micro-environment which doesn’t promote anti-tumor immune responses is created.

Examples of immune escape are:

• MHC-I expression loss → peptides aren’t presented anymore → T-cells fail to recognize tumor antigens
  • A severe defect for which the immune system is prepared → allows effector immune cells to recognize that a certain cell is a normal cell → is referred to as missing self
  • NK-cells should recognize that MHC-I is lost → become active
    • Usually, MHC-I cells tell NK-cells that they shouldn’t activate
    • Tumors can induce loss of NK-cell activation
• PD-L1 expression → transmits a negative signal to T-cells when bound to PD1 → T-cells cannot eliminate the tumor cell
• Through the production of TGF-β → functions are:
  • Suppressing cytotoxic T-cell activity
  • Suppressing NK-activity
  • Suppressingantigen presenting activity

Cancer immunotherapy

Cancer immunotherapy has 2 aims:

• Boost cancer immunity and activation mechanisms
• Target immunosuppressive pathways

Cancer immunotherapy uses existing features of the immune system to boost anti-tumor activity.

IL-2 therapy:

IL-2 therapy was one of the first immunotherapies that was applied to a large number of cases. IL-2 mediates T-responses. It was successful in:

• Metastatic melanoma
• Metastatic renal cell carcinoma

However, it was less successful in other tumor types. This is caused by IL-2 not only activating cancer specific T-cells, but also T-cells in general. This causes inflammation throughout the whole body → not ideal.

TIL therapy:

If tumor fragments are put in a culture grown with IL-2, outgrowth of T-cells present in the tumor will grow. These T-cells are tumor specific → recognize cancer cells. These cells are expanded in huge numbers and given to patients via TIL-infusion (tumor infiltrating lymphocyte infusion).

Advantages are:

• Specific
• Autologous cells

Disadvantages are:

• Personalized
• Highly dependent on in vitro manipulation
  • Requires time
  • Expensive

Adoptive T-cell transfer:

TIL-infusion T-cells may not be ideal, because they already are exhausted by the procedures they have undergone. A better option may be fusing cultured tumor cells with lymphocytes from the circulation of the patient → co-culture. Tumor specific lymphocytes are made, grown in large numbers and given back to the patient.

Antigen therapies:

Vaccinations against antigens present in tumors can be provided in case of viral related cancer. This especially proves to be successful in case of vulvar intraepithelial neoplasia.

Vaccinations for non-viral related cancers are based on a different type of antigens, such as antigens derived from a somatic mutation or neo-antigens. Patient-per-patient determination of mutations present in the tissue is necessary. This is a very specific therapy → the therapy only targets malignant cells. The treatment is very personalized and highly dependent on in vitro preparation.

Another option is antigen therapy based on tumor-associated proteins. Gp100, for instance, is overexpressed in melanomas and melanocytes. Vaccination with this antigen has been attempted, but remains unsuccessful. Explanations for this are:

• The antigen isn’t tumor specific
• The efficacy is limited
  • It’s very hard to stimulate the immune system to react

An advantage is that it isn’t necessary to vaccinate tumor-associated proteins in a personalized setting → sequencing isn’t necessary.

Checkpoint blockade:

Checkpoint blockade consists of components of antibodies which target the activation of T-cells. This was revolutionary for cancer treatment:

1. A T-cell receives a positive signal when the T-cell receptor recognizes a peptide presented by an MHC molecule
2. The activated T-cell starts expressing molecules such as PD1, which mediate inhibitory signals
3. If the PDL1 ligand is present, it binds to PD1 → T-cells cannot be activated
4. Tumor T-cells are often PD1 positive → susceptible to inhibition

Blocking of PD1 gives spectacular results. In case of advanced lung cancer, PD1 adds a significant increase of survival chance. Although immunotherapy doesn’t work on the majority of cancer patients, if it does work, it produces long lasting responses. This is the opposite of other therapies, which do cause an improved survival but eventually cannot prevent death. Therapeutic antibodies that target PD1 are most common, but therapeutic antibodies that target PD-L1 are also present.

PD-L1 expression is also used to identify cancer patients that may be susceptible to immunotherapy in the form of PD1 or PD-L1 blocker. This is mostly valued in lung cancer.

Checkpoint blocking has many side effects:

• T-cells in healthy tissue may become activated → healthy tissue is destroyed
• The level of increased inflammation can cause immune adverse events
• Some organs express PD-L1 to keep immunological activity away