Overview of Tandem scFv-Toxin

Immunotoxins are fusion proteins composed of antibodies or their fragments and cytotoxic agents, which can specifically recognize and kill tumor cells, and are a promising strategy for targeted therapy. Traditional immunotoxins are usually composed of intact monoclonal antibodies or Fab fragments and plant or bacterial toxins (such as Pseudomonas exotoxin A chain, ricin A chain, etc.), but these immunotoxins have some drawbacks, such as large molecular weight, easy clearance by the immune system, and immunogenicity. To overcome these drawbacks, a new type of immunotoxin - Tandem scFv-Toxin was developed. Tandem scFv-Toxin is a recombinant immunotoxin composed of two or more single-chain variable fragments (scFv) and a toxin molecule, which has the following advantages: (1) smaller molecular weight, which can increase tissue penetration and tumor targeting; (2) flexible design of different scFv combinations by genetic engineering to enhance affinity and specificity; (3) reduced immunogenicity by humanization or chimerization; (4) improved expression level and stability by expression optimization or fusion of auxiliary factors.

Structural Features of Tandem scFv-Toxin

The structural features of Tandem scFv-Toxin depend mainly on the type, number and connection mode of scFv and toxin molecules. Generally, Tandem scFv-Toxin can be divided into the following types:

• Single-targeted Tandem scFv-Toxin: composed of two identical scFv and one toxin molecule, which can enhance the affinity and specificity for a single antigen, such as scFv-PE38KDEL targeting HER2.
• Dual-targeted Tandem scFv-Toxin: composed of two different scFv and one toxin molecule, which can simultaneously recognize two different antigens, increasing tumor targeting and selectivity, such as BL22 targeting CD22 and CD19.
• Multi-targeted Tandem scFv-Toxin: composed of three or more different scFv and one toxin molecule, which can simultaneously recognize multiple different antigens, further improving tumor targeting and selectivity, such as HA22-LR targeting CD22, CD19, and CD20.
• Bivalent Tandem scFv-Toxin: composed of one scFv and two toxin molecules, which can increase the amount of toxin carried by each scFv, improving killing efficiency, such as Ki-4(scFv)-ETA' targeting CD30.

Tandem scFv-Toxin usually connects scFv and toxin molecules with a peptide linker, which can be natural or artificially designed. Its function is to maintain the spatial distance and relative position between scFv and toxin molecules to avoid interference or inactivation. The length, sequence, and conformation of the linker will affect the stability, activity, and expression level of Tandem scFv-Toxin. In addition, some auxiliary factors can be fused in Tandem scFv-Toxin, such as signal peptide, transport peptide, polyethylene glycol (PEG), Fc fragment, etc., to improve its expression efficiency in the expression system, transport ability in vivo, half-life in blood, penetration in tumor tissue, etc.

Fig.1 The schematic diagram of the structure of Tandem scFv-Toxin (Creative Biolabs)

Clinical Data of Tandem scFv-Toxin

Tandem scFv-Toxin is a novel type of targeted therapy drug that has shown potential efficacy and safety in various tumors. Currently, there are two Tandem scFv-Toxin drugs that have been approved by the US Food and Drug Administration (FDA), namely scFv(FRP5)-ETA targeting ErbB2 (HER2) receptor and Blinatumomab targeting CD19 and CD3 dual targets.

In addition, there are some Tandem scFv-Toxin drugs in different stages of clinical trials, mainly targeting different tumor-related targets, such as CD22, CD33, CD123, EGFR, etc. Table 2 lists some of the main information about Tandem scFv-Toxin drugs in ongoing or completed clinical trials.

References

1. Movahedpour A, et al. Designing a Humanized Immunotoxin Based on HER2 Specific scFv and DFF40 Toxin Against Breast Cancer: An In-Silico Study. Int J Pept Res Ther. 2022;28:130.
2. Hornig N, et al. Production of Bispecific Antibodies: Diabodies and Tandem scFv. Methods Mol Biol. 2012;907:615-30.
3. Satheeshkumar PK. Expression of Single Chain Variable Fragment (scFv) Molecules in Plants: A Comprehensive Update. Mol Biotechnol. 2020;62(2):151-67.
4. von Minckwitz G, et al. Phase I clinical study of the recombinant antibody toxin scFv(FRP5)-ETA specific for the ErbB2/HER2 receptor in patients with advanced solid malignomas. Breast Cancer Res. 2005;7(4):R617-26.
5. Kantarjian H, et al. Blinatumomab versus Chemotherapy for Advanced Acute Lymphoblastic Leukemia. N Engl J Med. 2017;376(9):836-47.
6. Kreitman RJ, et al. Phase I Trial of Anti-CD22 Recombinant Immunotoxin Moxetumomab Pasudotox (CAT-8015 or HA22) in Patients With Hairy Cell Leukemia. J Clin Oncol. 2012;30(15):1822-8.
7. Jurcic JG, et al. Phase I Trial of Lintuzumab-Ac225 in Older Patients With Untreated Acute Myeloid Leukemia (AML). Blood. 2016;128(22):590.
8. Pemmaraju N, et al. Tagraxofusp in Blastic Plasmacytoid Dendritic-Cell Neoplasm. N Engl J Med. 2019;380(17):1628-37.
9. LoRusso PM, et al. A phase I clinical and pharmacokinetic study of the immunoconjugate nimotuzumab-saporin in patients with refractory epithelial cancers expressing EGFR. Cancer Chemother Pharmacol. 2018;82(6):1061-70.
10. Allahyari H, et al. Immunotoxins in cancer therapy: Review and update. Int J Cancer Manag . 2017a;10(8):e5779.
11. Harbeck N, et al. Breast cancer. The Lancet . 2019a;393(10174):1134–50.
12. Nagarajan D , McArdle PA . Surgical management of breast cancer. Clin Oncol . 2018;30(4):193–200.