The uncontrolled growth and spread of abnormal cells is a characteristic displayed by cancer. Healthy cells usually undergo a cell cycle and grow, divide and die. However, in cancer, the cell cycle often becomes deregulated, dividing and growing uncontrollably, forming a mass of abnormal cells called a tumor. Molecules displayed directly on the surface of cancer cells (which are not expressed or expressed to a lesser extent by healthy cells) offer themselves as biomarkers and therapeutic targets, as well as abnormal levels of peptides and enzymes in the biofluids, which can be quantified. However, it is often not possible to give a definite cancer diagnosis using the quantification of such peptide and enzyme biomarkers, so these tests are considered alongside biopsies and imaging techniques, such as X-rays, CT scans, MRI scans, or PET scans.

From immunoassays and tumor-homing peptides to nanobodies, enzyme biomarkers and peptidomimetics, this blog will briefly explore the leading role that peptides, antibodies and enzymes play in many aspects of cancer diagnosis.

Firstly what exactly are diagnostic peptides, antibodies and enzymes?

Diagnostic Antibodies

The natural immune mechanism, including its antibody components, can be harnessed by scientists for both therapeutic and diagnostic purposes. Antibodies can be artificially manufactured with specificity to any desired disease pathogen or molecule in a patient’s body. When labelled with a detectable label such as a fluorescent or enzymatic tag, the disease marker can be identified in the event of antibody binding. One common diagnostic application of antibodies is in immunoassays.

Antibodies can also be used in point-of-care diagnostic tests, such as lateral flow assays, which are simple and rapid tests that can be performed outside of a laboratory setting. In these tests, the antibody is embedded on a test strip, and a sample (such as blood or saliva) is applied to the strip. If the antigen of interest is present in the sample, it will bind to the antibody, causing a visible color change or other signal.

Diagnostic Peptides

Peptides have a strong presence in the diagnostic industry due to a handful of characteristics. Peptides can be naturally degraded and cleared by the human body, penetrate cells and tissues without inducing an immune response, target and bind to specific targets within the body and be easily synthesized and modified to display desired and improved properties. Furthermore, their strong specificity and high sensitivity can be exploited to accurately detect minimal concentrations of target molecules, meaning they are used in many diagnostic tests to enable the early diagnosis and monitoring of certain conditions. For example, diagnostic peptides can be used to detect the presence of specific antibodies or antigens in the blood or to identify the presence of specific proteins in cancer cells.

Diagnostic Enzymes

Like peptides, enzymes have rightfully earned themselves a place in the diagnostic field. Enzymes have highly beneficial characteristics, such as substrate specificity, and their activity can be quantified. Tissue damage may occur in disease states and initiate the release of specific enzymes in response to the damage or disease in a particular organ or tissue. As a result, there is an elevation in certain types of enzymes, and this activity can be measured. This is usually done by taking patients' serum, plasma or other types of biofluid samples.

Of equal importance are enzyme substrates, which, through their interaction with enzyme counterparts, can generate bioluminescent, chemiluminescent, fluorogenic and chromogenic signals, a vital asset in diagnostic testing kits, staining and probing applications.

Tumor-homing peptides

With the ability to penetrate tumor cells and target tumors with specific lineages, tumor-homing peptides play a central role in in vivo tumor imaging applications. These novel peptides can be chemically bound to labels such as fluorescein dyes or near-infrared dyes, namely 5-aminolevulinic acids and indocyanine green (FI10558), thus allowing an operator to visualize a tumor in real-time directly. Because tumor-homing peptides can be incorporated rapidly into their target tumor, they can be a real asset in precision medicine and be used in developing novel photodynamic diagnosis. During surgery, photodynamic diagnosis enables clinicians to visualize and determine the size and metastasis of the cancer, which may lead to improved and more informed treatment decisions.

Biosynth’s custom synthesis and GMP facilities can design and modify high-quality tumor homing peptides, supporting this beneficial line of cancer diagnostics. Not only can we custom synthesize your desired peptide for cancer diagnostics, but we also have research products readily available to purchase in our product catalog, such as:

· iRGD (H-[Cys-Arg-Gly-Asp-Lys-Gly-Pro-Asp-Cys]-NH2) (RGD-3761-PI): A ‘tumour homing peptide’ with the ability to penetrate tumor cells as well as endothelial cells, and also inhibit cancer metastasis in vivo.

· H-CREKA-OH (VAA-41742): Contains the tumor homing peptide sequence: CREKA which targets the fibrin‐fibronectin complex, tumor vessels and stroma tissue.

· H-CGNKRTRGC-OH (VAA-49423): Contains the tumor homing peptide sequence: CGNKRTRGC, known as LyP‐1 which targets the NRP receptor and Glioma cells.

Nanobodies

One pioneering antibody is the single monomeric variable antibody domain, termed a nanobody. Despite lacking a variable domain, nanobodies have retained their ability to bind to specific antigens through complementarity-determining regions (CDR). Using their CDR3 loop, nanobodies can have stronger interactions with antigens and form finger-like projections, allowing the binding of epitopes to be initially inaccessible.

Due to their tumor penetrating properties, shorter serum half-life and generation of the high tumor to background ratio, nanobodies are highly suitable for use in molecule imaging. These are diagnostic techniques using a tumour antigen-binding molecular probe. During SPECT imaging techniques, nanobodies are labeled with gamma-emitting nucleotides and PET is labeled with positron-emitting nuclides. Nanobodies also have the potential to be used in magnetic resonance imaging (MRI), optical imaging and ultrasound. In optical imaging, they are given a fluorescent label and provide a flexible, safe, simple and more cost-effective technique.

Many nanobody probes targeting human epidermal growth factors are being researched. During clinical trials, it was shown that a 68Ga-HER2 nanobody detected primary and metastatic tumors. Similarly, a nanobody combining HER and carbonic anhydrase IX (CAIX), HER2-CAIX, could detect lung metastases. Other nanobody probes showing success in enhancing tumor-to-background signal ratios are 99mTc-EGFR-cartilage oligomeric matrix protein (COMP), 99mTc-dipeptidyl-peptidase-like protein 6 (DPP6), 99mTc-EGFR, 99mTc-dipeptidyl-peptidase-like protein 6 (DPP6), 99mTc-mesothelin, and 131I-HER2.

Nanobodies can also be used to visualize tumor angiogenesis. This can be done through developing nanobodies to target metastasis markers such as cell adhesion molecule-1 (VCAM-1). The use of anti-VCAM-1 nanobody microbubbles in ultrasound imaging is being investigated.

Monitoring immune infiltration through nanobody probes targeting antigen-presenting cells and the antigen profile of a tumor can be very useful in predicting a patient’s therapeutic response. Interestingly, an anti-ADP-ribosyltransferase-2 (ART-2) nanobody can investigate T-cell infiltration while exhibiting therapeutic properties through inhibition of ART-2.

Enzyme Biomarkers

The level of enzymes in the body can also be a significant cancer biomarker, as demonstrated by the following enzyme biomarkers.

Glucose-6-phosphate 1-dehydrogenase (JAA00140) (G6PD)

· Is naturally involved in membrane integrity maintenance.

· The overexpression of G6PD may be linked to gastric cancer progression.

· In acute non-lymphocytic leukemia (ANLL) levels of G6PD are found to be reduced whereas in chronic myeloid leukemia levels are increased.

Acid phosphatase (JAA00177) (ACP)

           · Can be used within an ACP assay to monitor the metastases of prostate cancer as high levels of ACP exist in the male prostate gland.

Cystein capthepsins B, L and H

· They are involved in catalyzing the hydrolysis of proteins in lysosomes.

· Have elevated expressed in human tumors such as melanoma, ovary, lung, cervix, brain, neck, head, breast and gastrointestinal.

· Cathepsin B is upregulated in premalignant lesions in the prostate, breasts, thyroid, colon and brain.

· Cysteine cathepsins may also be diagnostic and prognostic markers in inflammatory diseases and cancer.

Cyclooxygenase-2

· It is involved in tumor progression and is expressed in the early cancer stage.

· As it is not present in healthy tissues, it can be used as a molecular target to detect and prevent cancers.

Lactate dehydrogenase (LDH)

· It is a successful and validated prognostic marker for lymphoma, colon cancer, leukemia, and testis with germ cell malignancies.

· Depending on the level of LDH activity, the most appropriate treatment plans can be devised.

Peptide Biomarkers

Peptide biomarkers have further illustrated their dominance in the diagnostic field through their ability to contribute to cancer diagnostics. One example is the breast cancer biomarker mammaglobin, for which an ELISA was developed to detect mammaglobin in breast cancer patient’s blood.

Another instance is the use of Parathyroid hormone-related peptide (PTHrP) (PTH-4215-V) as a biomarker for prostate and breast cancer due to its relation to hypercalcemia and bone metastasis found in these cancers. One lab investigated the value of PTHrPs in diagnosing aggressive prostate cancer by using an ultrasensitive microfluidic assay for PTHrPs, where PTHrBs were bound to a secondary antibody labelled with polyhorseradish peroxidase. It was found that the levels of PTHrP could determine aggressive and indolent prostate cancers.

Peptidomimetics

Peptidomimetics are synthetic peptides designed to mimic natural peptides' structure and biological function. These peptidomimetics are paramount in medicinal chemistry as they help increase the stability and selectivity of a peptide, which was lacking from its natural counterpart. As a result, they play a significant role in diagnostics and drug discovery.

In cancer diagnosis, peptidomimetics can be designed to target receptors that are overexpressed in tumors. One such example is the development of antagonists of the integrins: α5β1, α6β4, αvβ3, α4β1, αvβ6, αvβ8 and αvβ5. In particular, αvβ3 is being investigated, and peptidomimetics are being produced that copy guanidine and carboxylate pharmacophores within RGD peptides. Two forms of imaging can be used to detect αvβ3, nanoparticle-case and radionuclide. For radionuclides, radiotracers 99mTc-NC100692, 18F-Galacto-RGD, and 18F-fluciclatide can be used in the non-invasive imaging processes single-photon emission computed tomography (SPECT) and positron emission tomography (PET). The peptidomimetic αvβ3 integrin antagonist (IAC) has also been shown to be an asset to PET and SPECT tumor imaging.

It is evident that the use of peptides, antibodies and enzymes is extensive in cancer diagnostics and research. Biosynth are experts in the custom synthesis of peptides, antibodies and enzymes designed especially for diagnostic applications. We even offer large-scale and GMP manufacture of peptides. As part of our catalog of over a million research products we have a range of research kits, reagents, buffers and diluents and other assay components for use in immunoassays. Contact a member of our team today at sales@biosynth.com or click the below links for more information on how Biosynth can assist in your diagnostic project.

   Diagnostic Peptides        Custom Antibodies       

Custom Enzymes        Raw Materials for IVD

References

Dhanapala, L., Joseph, S., Jones, A. L. Moghaddam, S., Lee, N., Kremer, R. B., Rusling, J. F. (2022). Immunoarray Measurements of Parathyroid Hormone-Related Peptides Combined with Other Biomarkers to Diagnose Aggressive Prostate Cancer. Analytical Chemistry, 94(37): 12788-12797.

Gomari, M. M., Abkhiz, S., Pour, T. G., Lotfi, E., Rostami, N., Monfared, F. N., Ghobari, B., Mosavi, M., Alipour, B., Dokholyan, N. V. (2022). Peptidomimetics in cancer targeting. Molecular Medicine, 28(1), 146.

Ruvo, S., Malviya, G., Dvorakova, M. C. (2021). Role of Peptides in Diagnostics. International Journal of Molecular Sciences, 22(16), 8828.

Singh, R. S., Singh, T., Singh, A. K. (2019). Enzymes as Diagnostic Tools. Advances in Enzyme Technology. 9.

Y. Yang, E. Y., Shah, K. (2020) Nanobodies: Next Generation of Cancer Diagnostics and Therapeutics. Frontiers in Oncology, 10