Molecular Oncology - The Novel Hallmarks of Cancer

CANCER IMMUNOTHERAPY

It includes compounds capable of reactivating immune system’s response, in order to

make it able again to recognize tumor cells

DOMANDA D’ESAME: Which are the features of immunologic (immunogenic) cell

death?

Immunogenic Cell Death (ICD) triggers an immune response by releasing danger

signals and tumor antigens that activate the immune system. Unlike other forms of cell

death, such as apoptosis or necrosis, which can be immunologically silent or even

suppressive, ICD is highly pro-inflammatory and enhances anti-tumor immunity.

This is done through the release of DAMP molecules (Damage-Associated Molecular

Patterns).

Key DAMPs include: →

- Calreticulin (CRT) A protein exposed on the cell surface that acts as an “eat-

me” signal for dendritic cells and macrophages. →

- ATP and HMGB1 (High-Mobility Group Box 1) They trigger the activation

of Toll-like receptors (TLR4) on dendritic cells, enhancing antigen presentation.

→

- Heat shock proteins (HSPs) They are translocated in stress conditions on the

plasma membrane, where they exhibit immunostimulatory effect, based on their

interaction with APC’ surface receptors CD91 and CD40; they also facilitate

cross presentation of tumour cells’ antigens on MHC class I molecule, leading

to the CD8+ T cell response.

DNA-damaging agents can

enhance the efficacy of immune

checkpoint inhibitors like anti-

PD1/PDL1 or anti-CTLA4,

enabling a sustained anti-tumor

immune response.

Immunogenic cell death bridges

both immunotherapy and

mutation-inducing therapies, as

therapies that induce DNA

damage can enhance immune

responses against cancer.

• Targeting cancer cells Cytokines

By combining a compound able to induce immunogenic cell death with cytokines,

which activate the immune system against the tumor, we can influence the early stages

of tumor-immune cell interactions (elimination).

In the lab, we have access to a wide range of cytokines, which they can be infused into

patients to stimulate the immune system, promoting T cell priming.

Furthermore, we can adapt specific cytokines or metabolites to target and enhance

specific stages of immune response. For instance, we can influence timing, recruitment

of immune cells like NK cells, or even checkpoint inhibitors (CPI). However, this

approach isn’t always the ideal, as tumors can take advantage of immune respone, for

example using VEGF to start migrating. Thus, while reactivating the immune response

with cytokines can be beneficial, it also enhances the probability to facilitate tumor

growth and proliferation. Cancer vaccines

Vaccines are potentially the most powerful tool we have, as they are proven to work.

Usually, vaccines are used for prevention rather than treatment, but research is

progressing in this area, and the development of peptidomics is a growing field in

cancer research.

Peptidomics involves identifying tumor antigens presented on MHC or HLA

molecules. This helps determine if specific peptides are associated with each cancer

type. By identifying and administering these antigens using personalized strategies, we

can enhance the immune response.

Adoptive cell transfer

It is one of the most classical applications, which includes isolating and expanding

immune cells ex vivo: we can take blood samples from patients, then isolate the

immune cells of interest (such as T cells), stimulate them with specific antigens (often

in combination with APCs), and re-infuse them into the deriving-patients to maximize

the anti-cancer response.

This can be done in different ways:

1) TIL therapy.

Isolate directly the tumor-infiltrating lymphocytes (TILs), which can be collected via

biopsy, then stimulated with specific antigens such as interleukin-2 (IL-2), which helps

support their growth and activation, and re-infused into the patient.

These kinds of ex-vivo approaches have shown significant results in certain cancers

like melanoma. So, even though they have the limitation of requiring time (typically 2

to 3 weeks), they allow the expansion of a robust immune cell population.

During this process, additional analyses, like exome sequencing, can identify tumor-

specific antigens.

Synthetic antigens can then be used to prime APCs, which, in combination with TILs,

create a potent immune response.

However, these protocols are complex, time-consuming, and costly. Indeed,

manipulating cells ex vivo requires specialized facilities, permissions, and skilled

personnel, often limiting such activities to specific labs or companies. Anyway, they

hold significant promise for advancing cancer immunotherapy.

TCR/CART therapy

By genetically engineering T cells to express chimeric antigen receptors (CARs), we

can target tumor antigens more effectively.

We also have the option to induce a cytokine response directly within the tumor, which

can help stimulate other immune cells and enhance the overall immunotherapeutic

effect.

There are different generations of CAR-T cells based on the activation pathways and

signalling mechanisms. The first generation provides limited activation, while the

second generation incorporates additional signalling domains that enhance cell

activation.

The protocol is the same:

‐ T cells are collected.

‐ They are manipulated, typically using viral vectors to express the CAR receptor

targeting the tumor.

‐ Then expanded in vitro and infused back into the patient.

CAR-NK cells are emerging as a promising alternative to CART, combining immune

checkpoint inhibitors with other immune approaches for enhanced results.

Tumor-specific mAbs

Antibodies play a critical role in cancer therapy, as they serve as an effective tool to

deliver targeted therapies against specific cells.

Immune checkpoint inhibitors are part of this group.

One of these inhibitory molecules is CTLA-4 (cytotoxic T-lymphocyte-associated

protein 4), which binds to the CD28’s ligands, CD80 and CD86 (B7).

When a T cell needs to be deactivated, it upregulates CTLA-4 expression; so, the shared

ligand B7, preferentially binds to CTLA-4 instead of CD28, resulting in a negative

signal that inhibits T cell activation. This mechanism ensures that the immune response

is appropriately regulated and prevents excessive immune activation.

Immune checkpoint inhibitors are designed to block the immunosuppressive phenotype

induced by tumor cells. Specifically, they use monoclonal antibodies to block negative

immune receptors, thereby preventing the deactivation of T cells. This strategy is

especially relevant in the tumor microenvironment, where not only tumor cells but also

other immune cells, such as macrophages, contribute to T cell suppression.

Thus, immune checkpoint inhibitors allow us to manipulate both positive and negative

co-stimulatory molecules.

While T cell activation is critical for eliminating pathogens like viruses or bacteria, it

is equally important to switch off this response at the appropriate time. Without proper

regulation, immune cells could potentially destroy all our cells, leading to a spread

damage.

This is particularly evident in systemic autoimmune diseases, where immune cells

slowly destroy various tissues throughout the body. However, in acute autoimmune

conditions, such as type 1 diabetes, the destruction of specific cells can occur rapidly,

sometimes within months. This destruction can result in conditions like hyperglycemia.

Immune checkpoint inhibitors have shown great results, especially in preclinical

models: early use of anti-PD-1 or anti-CTLA-4 antibodies resulted in the first examples

of tumor-free mice. Even after one year of treatment, these mice remained tumor-free,

demonstrating the durability of the response. This approach, which proved effective in

preclinical models, was successful also for human treatments, leading to positive

results in many patients.

So, it’s crucial to stratify patients based on tumor mutational burden (TMB): for

instance, melanoma and mismatch repair (MMR)-deficient colorectal cancer have high

mutational burdens and are responsive to these therapies, because they present many

neoantigens, which play a critical role in achieving a good therapeutic outcome.

On the contrary, tumors classified as cold, meaning that they lack sufficient

neoantigens, typically show poor responses to these treatments.

One important side effect of immune checkpoint inhibitors is immune system

hyperstimulation, which can lead to autoimmune diseases and inflammation. For

example, recent reports have linked these therapies to conditions such as diabetes

(classified as type 3 diabetes), arthritis, and even rare cases of leukemia.

Bispecific Abs

They bridge tumor cells and immune system, such as T cells or NK cells, to stimulate

cytotoxic effects against the tumor: by manipulating the antibody’s regions, we can

design molecules that enhance the interaction between immune cells and tumor

antigens, thereby promoting the formation of a cytolytic synapse. This facilitates the

activation of T cells or NK cells to destroy tumor cells.

These molecules are engineered antibodies that can bind two different targets

simultaneously.

Compared to more complex therapies like CAR-T cells, bispecific antibodies offer a

simpler and more accessible approach. They can be further modified by conjugating

them with other molecules to enhance their specificity and effectiveness. For example,

these antibodies can link T cells to tumor antigens or even target other immune cells to

amplify the immune response. Oncolytic viruses

These viruses can be genetically engineered to specifically target tumor cells: by

modifying their structure, we can direct these viruses to attack tumor-specific receptors

on the cell membrane.

Additionally, oncolytic viruses can be designed to deliver compounds that stimulate

immune cells or induce ICD in tumor cells. This process releases tumor antigens,

triggering an immune response and restoring immune surveillance.

Oncolytic viruses can also be combined with DAMPs and pathogen-associated

molecular patterns (PAMPs) in subsequent phases of treatment. This combination

amplifies the immune response, creating a multi-faceted attack to the tumor. While this

approach is highly specific and shows great potential, it involves complex protocols

and is still under investigation.

MODELS IN CANCER RESEARCH

Different models are used in cancer

research:

a. In vitro models → cell lines (usually)

b. In vivo models → usually rodents, but

also C.elegans, Drosophila or

Zebrafish

c. Ex vivo model → when

experimentation is done in or on

tissues obtained from an organism and maintained under optimum conditions,

mimicking the natural condition.

When choosing a model system, we should consider:

→

- Cost in vivo models are more expensive than in vitro model

→

- Complexity for example, to study the microenvironment (important in tumor

progression) we cannot use a sin