Immuno-oncology: Leveraging Antibodies to Target Cancer

Immuno-Oncology harnesses, redirects, and boosts the power of the immune system to target cancer. Unlike traditional chemotherapy, which attacks all rapidly dividing cells whether they’re healthy or malignant, immunotherapy is more targeted, utilizing the body’s natural defenses to strategically attack cancer cells. Immunotherapy for cancer is a direct result of advances in technology for recombinant antibody generation, cell and gene therapy engineering, and increasing appreciation of the immune system’s role in cancer prevention and control. It has been successfully deployed in the clinic for hematological malignancies, such as Chronic Lymphocytic Leukemia (CLL), Non-Hodgkin Lymphoma (NHL), and Acute Lymphocytic Leukemia (ALL). While solid tumors have proven more challenging to target, immunotherapy is gaining traction for treatment in some cases. Once considered a last resort treatment, immunotherapy is often the first line of treatment for advanced and metastatic melanoma.

The immune system is involved at every stage of tumor suppression:

• Natural Killer (NK) cells, γδ T cells, and cytotoxic CD8 T cells promote apoptosis of tumor cells.
• Dendritic cells (DCs) prime CD4 and CD8 T cells against tumor-associated antigens (TAAs).
• Macrophages phagocytose dead and dying cells and promote inflammation.

However, during tumorigenesis and disease progression, the tumor actively suppresses this potent immune response by:

• Creating a hypoxic microenvironment, excluding cytotoxic T cells.
• Secreting TGF-beta (TGF-β) and adenosine.
• Promoting formation and recruitment of immunosuppressive cells: T regulatory cells (Tregs), Myeloid-derived suppressor cells (MDSCs), and tumor-associated macrophages (TAMs).
• Upregulating inhibitory ligands to prevent cell-mediated cytotoxicity.

In summary, the immune system has a potent anti-tumor response, and the tumor has a potent anti-immune response. Immunotherapy is what helps tip the balance in favor of the anti-tumor immune response.

Antibody-Mediated Immunotherapy

One of the primary strategies to boost the immune response to tumors leverages the specificity of antibodies. According to the Cancer Research Institute, there are three primary types of antibody-mediated therapies: Monoclonal antibodies (mAbs), Bispecific antibodies, and Antibody-Drug Conjugates (ADCs).

Note: More information about ADCs can be found at the Cancer Research Institute.

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Immune Checkpoint Blockade

The immune response is tightly regulated by checkpoints, achieving a careful balance between activation and inhibitory receptors. Inhibitory receptors on immune cells are critical to avoid unnecessary damage to the host. Innate cells, like NK cells and γδ T cells, constitutively express inhibitory receptors to prevent killing and phagocytosis of healthy host cells, while α/β T cells upregulate inhibitory receptors soon after activation to regulate expansion and effector function. Tumor cells, like those of non-small cell lung cancer (NSCLC), prostate cancer and melanoma, will upregulate inhibitory ligands to avoid destruction by the immune system.

In general, immune checkpoint blockade therapy seeks to disrupt the receptor-ligand interaction between the inhibitory receptor of the immune cells and the ligand expressed on the tumor cells. Checkpoint blockade therapy liberates the immune cells from this inhibition. Ex vivo or in vitro characterization of immune checkpoints is the first step towards development of effective immunotherapy.

In addition to flow cytometry, immunohistochemistry (IHC), and immunocytochemistry/immunofluorescence (ICC/IF), Bio-Techne has a large selection of highly validated immune checkpoint antibodies for blocking and neutralization.

Current and Emerging Targets for Immune Checkpoint Blockade

The first two immune checkpoint blockade therapies approved for use in humans target the CTLA-4/CD80-CD86 and PD-1/PD-L1 axes, both members of the B7-CD28 families of immune checkpoints. The B7 family proteins are expressed on surface of antigen presenting cells (APCs) and tumor cells and interact with receptors of the CD28 family of proteins on immune cells. Members of these families are required for both co-stimulatory and co-inhibitory functions of T cells. Many other members of B7-CD28 protein families are under investigation as additional targets for immunotherapy.

 B7 Family Immune Checkpoints

A 2018 study found that only about 43% of cancer patients are eligible for checkpoint blockade therapy, and the frequency of patients who respond is around 12%. Because such a small percentage of patients respond to current checkpoint blockade, additional checkpoints, beyond the B7 family, are of intense interest, including other inhibitors of cytotoxicity, like TIM-3 and TIGIT. Tumor cells can also upregulate ligands to inhibit phagocytosis by patrolling macrophages and myeloid cells. These ligands, such as CD24 and CD47, are expressed on all mammalian cells and are a “don’t eat me” signal to the innate immune system.

Learn about more immune checkpoint targets with Bio-Techne’s eBook, Current and Emerging Immune Checkpoint Targets for Immuno-Oncology Research.

Efficacy of immune checkpoint blockade is determined by several factors: expression of inhibitory receptors by tumors, penetration of therapeutic antibodies into solid tumors, and accessibility of the tumor to immune cells. Due to redundancy of inhibitor expression (e.g. both LAG-3 and PD-1 expressed on the same cell) and the complexity of the tumor microenvironment (TME), combination immune checkpoint blockade might be required to achieve optimal anti-tumor immunity. Combination checkpoint blockade could include two or more co-inhibitory receptors or a multipronged approach targeting an inhibitory receptor and phagocytosis inhibitor, like CD47. Combination blockade can be achieved with a cocktail of monoclonal antibodies or an engineered bispecific antibody.

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Bispecific Antibodies

Advances in antibody engineering have led to the development of bispecific antibodies. As indicated by their name, the variable regions of these antibodies recognize two different antigens.

For cancer therapy, bispecific antibodies are utilized in two ways:

1. Simultaneous blockade of two immune checkpoints.
2. Redirection of cytotoxic cells to interact with tumor cell.

Spotlight: Redirection of Cytotoxic Cells

Often, TAAs targeted by antibody-therapy are not unique to the tumor cells and are also expressed on healthy cells. However, TAAs are frequently overexpressed on tumor cells compared to healthy cells, providing an opportunity to exploit advanced antibody engineering technology. To limit the redirection of cytotoxic cells to healthy, non-malignant cells, bispecific antibodies provide a distinct advantage over traditional antibody therapies due to an engineering technique often referred to as affinity tuning. Affinity tuning involves selecting antibodies with certain binding properties, or affinity, for a particular antigen. During an immune response in vivo, antibodies often undergo a process called affinity maturation where high affinity antibodies are selected in the germinal center reaction. Researchers can simulate this process in vitro by introducing targeted mutations of the variable region and measuring antibody affinity via surface plasmon resonance (SPR).

An affinity-tuning approach allows for selection of bispecific antibodies with weak antibody binding (low affinity) to the activating receptor (CD3 or CD16) and strong binding (high affinity) to the TAA, such as EpCAM. This increases the likelihood that the antibody will be bound to a tumor cell before it activates the cytotoxic cell, enhancing efficacy and reducing non-specific activation.

As an additional challenge, antibodies directed against CD3 will indiscriminately target both CD8+ cytotoxic T cells and CD4+ T regulatory cells, which could promote an immunosuppressive TME. Though currently approved therapies target CD3 and TAA, researchers are also exploring bispecific antibodies targeting NK cell markers, like CD16A.

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Limitations and Future Directions of Antibody-Based Immunotherapy

With the implementation of antibody-based therapies, there have been significant advancements in treatment of high mortality malignancies such as metastatic NSCLC and melanoma. However, there are still significant limitations of antibody-mediated immunotherapy including:

• Limited penetration into solid tumors.
• Ig-like antibodies with Fc domains can be immunogenic.
• Checkpoint blockade is only effective for “hot” tumors with immune activity and expression of inhibitory ligands.

Nanobodies^® with single domain V_HH antibodies, like Bio-Techne’s LlaMABodies^TM, can target hard to reach epitopes and may have a role in future immunotherapies.