Immunotherapy, also known as antibody therapy, has emerged as the most effective treatment for solid and malignant tumors over the last 20 years. This treatment stimulates and targets cancer cells as antigenic elements by utilizing the innate immune system.

Radioimmunotherapy (RIT) is an innovative cancer treatment that effectively combines the principles of radiation therapy with the targeting capabilities of immunotherapy. At the heart of this approach are monoclonal antibodies, which play a crucial role in identifying and attacking cancer cells. By using monoclonal antibodies linked to radioactive isotopes, RIT targets and destroys cancer cells while minimizing damage to surrounding healthy tissue. This dual approach enhances the effectiveness of traditional cancer treatments and represents a significant advancement in oncology.

The use of a single antibody coupled to different radioactive elements enables the creation of a so-called theranostic strategy, combining diagnostic and therapeutic approaches. Cancer therapies can now been paired with imaging agents that allow doctors to visualize tumor responses, helping to tailor subsequent treatments based on how well a patient is responding. A first radiotracer (Zirconium 89 for instance) will so be used for imaging purposes, while a second active element (Lutetium 177 for instance) will be deployed to target and destroy localized cancer cells with an adjusted dose.

And so far, in cancer treatment, theranostic approaches have shown great promise. 

But how does radioimmunotherapy work?

Monoclonal antibodies are lab-produced proteins engineered to attach to specific antigens present on the surface of cancer cells. By binding to these antigens, the antibodies can identify and isolate cancerous cells. This specificity is crucial for improving treatment outcomes. But even at this stage, we are already facing several major problems.

Cancer cells often display a diverse range of antigens, making it difficult to create a universal antibody. Tumors can vary significantly between patients and even within the same tumor, complicating the targeting process.

You need to find the right target that is only expressed or significantly over-expressed by cancer cells, so as not to deplete healthy cells (avoiding the “off-target” effect).

The difficulty lies on raising effective antibodies against areas that are potentially difficult to access (steric hindrance), or for which no immune response is expected (proteins that are highly conserved between species or cancer antigens similar to normal cellular proteins, leading to immune tolerance), or antigens that may undergo modifications (Post-translational Modifications) that can alter their structure and make them less recognizable to the immune system.

Once produced and purified, these monoclonal antibodies are then labeled with a radioactive isotope. Once the monoclonal antibody binds to the cancer cell, the attached radioactive isotope emits radiation that directly targets the tumor.

Conjugation refers to the process of chemically linking two distinct molecules—in this case, antibodies and radioactive isotopes, creating a stable compound that can effectively target cancer cells.

Several methods of conjugation have been established.

This method employs chemical linkers to establish stable covalent bonds between antibodies and radioisotopes. The choice of linker can greatly influence the stability and functionality of the resulting conjugate. Common linkers include:

• N-hydroxysuccinimide (NHS) Esters: These linkers react with amino groups on the antibodies, allowing for a straightforward conjugation process. NHS esters are particularly effective due to their ability to form stable amide bonds, making them a popular choice for creating robust conjugates.
• Maleimide Linkers: These linkers specifically bond to thiol groups (cysteine residues) present on antibodies. Maleimide linkers are advantageous for their selective reactivity, which minimizes non-specific binding and enhances the specificity of the conjugation process.

In this sophisticated approach, antibodies are first biotinylated, which allows them to bind with high affinity to streptavidin. Streptavidin, a protein known for its strong binding to biotin, can then be conjugated to a radioactive element. This method is beneficial due to the strong and stable interaction between biotin and streptavidin, enabling the attachment of various payloads without compromising the antibody's structure or function.

This method involves the direct attachment of isotopes to antibodies, offering a simpler and faster approach. While this technique can streamline the labeling process, it may impact the stability and specificity of the antibody. Directly labeled antibodies may have altered binding properties, which can influence their efficacy in targeting and imaging.

Enzymes such as transglutaminase can facilitate the precise attachment of radioactive isotopes to specific sites on the antibody. This method offers a high degree of control over the labeling process, allowing for the modification of antibodies at predetermined sites. By ensuring that the isotope is attached in a targeted manner, enzymatic labeling can enhance the overall functionality and effectiveness of the antibody in imaging applications.

Each of these conjugation methods has its unique advantages and limitations, making them suitable for different applications in antibody-based therapies and imaging. The choice of method depends on factors such as the desired stability, specificity, and intended application of the radio-labeled antibodies. As research progresses, optimizing these conjugation techniques will be critical for improving the efficacy and safety of radiopharmaceuticals in clinical settings. While the conjugation of antibodies to radioactive isotopes holds great promise, several challenges must be addressed.

The conjugation process should not alter the antibody's ability to bind to its target antigen. Careful optimization of conjugation conditions is critical to preserving therapeutic potential.

Ensuring that the conjugated antibodies remain stable in circulation and maintain their binding ability is crucial for effective treatment delivery.

The presence of multiple attachment points can lead to a heterogeneous mixture of conjugates, complicating characterization and quality control.

The addition of radioactive isotopes or linkers may provoke an immune response, potentially diminishing treatment efficacy.

Because their radiotracers provide information on specific biological processes at the cellular and molecular levels, Positron Emission Tomography (PET) and Single-Photon Emission Computed Tomography (SPECT) are two common radionuclide-based imaging modalities. 

PET detects the energy released by the two gamma photons (511 keV) that are created when an atomic electron annihilates the positron released from the PET radionuclide. Among the myriad of PET probes, the most common ones are Gallium [68Ga], Fluorine [18F], and more recently Zirconium [89Zr]. 

Single gamma photons released directly from γ-emitting radiopharmaceuticals are measured by SPECT. SPECT is the nuclear imaging technique that is most frequently utilized in clinics since it is less costly than PET. The most frequent SPECT isotopes are Indium [111In], Iodine [123I] but above all, and now almost exclusively, Technetium [99mTc] due to its excellent physical properties.

Positron emission tomography (PET) using Zirconium-89-labeled antibodies holds significant promise for cancer tumor imaging. It offers critical insights into the pharmacokinetics and targeting efficiency of monoclonal antibodies, as well as helping to predict potential toxicity of antibody-drug conjugates. This technique facilitates precise dose planning for personalized radioimmunotherapy and enables early monitoring of responses to targeted therapies.

Zirconium-89 is particularly well-suited for Immuno-PET due to its advantageous properties. The emitted positron has relatively low energy, and the average β+ distance of 1.18 mm contributes to high-resolution imaging. These characteristics are comparable to those of Fluor-18 and Copper-64, which have mean β+ distances of 0.60 mm and 0.70 mm, respectively. Additionally, Zirconium-89’s half-life of 78.4 hours aligns well with the pharmacokinetics of monoclonal antibodies, which typically achieve an optimal tumor-to-blood ratio around three days after injection—a timeline that is not as favorable for Fluor-18 and Copper-64.

When it comes to using Zirconium-89 in PET imaging, Desferioxamine B (DFO) is the most frequently employed chelator. However, preclinical studies indicate that DFO may not be the best choice due to the instability of the [89Zr][Zr(DFO)] complex in vivo. This instability can lead to the release of radioactive zirconium, resulting in bone accumulation of radioactivity. Such accumulation not only complicates the accurate interpretation and quantification of bone uptake in PET images but also poses unnecessary radiation exposure to patients.

To enhance the efficacy of Zirconium-89 for immunoPET, there is a pressing need for new chelating agents that can form more stable complexes. Unfortunately, no current ligands for Zirconium-89 meet all the necessary criteria for optimal performance. An ideal radiotracer for PET immunoassays should be:

• Non-toxic for clinical use; ensuring patient safety is paramount.
• Maintaining pharmacokinetics; the antibody's behavior should remain unchanged when coupled with the ligand.
• Stable retention of the radiotracer; the ligand must not release its radioactive component during use.

The search for improved chelators for Zirconium-89 is critical for advancing PET imaging techniques and enhancing patient outcomes in cancer diagnostics.