Treatment with antibody drug conjugates targeting receptors over-expressed on cancer cells is well established for clinical use in several types of cancer, however, resistance often occurs motivating the development of novel drugs. We have recently investigated a drug conjugate consisting of an affibody molecule targeting the human epidermal growth factor receptor 2 (HER2), fused to an albumin-binding domain (ABD) for half-life extension, loaded with the cytotoxic maytansine derivative DM1. In this study, we investigated the impact of the cytotoxic payload on binding properties, cytotoxicity and biodistribution by comparing DM1 with the auristatins MMAE and MMAF, as part of the drug conjugate. All constructs had specific and high affinity binding to HER2, human and mouse albumins with values in the low- to sub-nM range. Z_HER2-ABD-mcMMAF demonstrated the most potent cytotoxic effect on several HER2-over-expressing cell lines. In an experimental therapy study, the MMAF-based conjugate provided complete tumor regression in 50% of BALB/c nu/nu mice bearing HER2-over-expressing SKOV3 tumors at a 2.9 mg/kg dose, while the same dose of Z_HER2-ABD-mcDM1 provided only a moderate anti-tumor effect. A comparison with the non-targeting Z_Taq-ABD-mcMMAF control demonstrated HER2-targeting specificity. In conclusion, a combination of potent cytotoxicity in vitro, with minimal uptake in normal organs in vivo, and efficient delivery to tumors provided a superior anti-tumor effect of Z_HER2-ABD-mcMMAF, while maintaining a favorable toxicity profile with no observed adverse effects.

Human epidermal growth factor receptor 2 (HER2) is a clinically validated target for breast cancer therapy. Previously, a drug-fused HER2-targeting affinity protein construct successfully extended the survival of mice bearing HER2-expressing xenografts. The aim of this study was to evaluate the influence of the number and positioning of the protein domains in the drug conjugate. Seven HER2-targeting affibody-based constructs, including one or two affibody molecules (Z) with or without an albumin-binding domain (ABD), namely Z, Z-ABD, ABD-Z, Z-Z, Z-Z-ABD, Z-ABD-Z, and ABD-Z-Z, were evaluated on their effects on cell growth, in vivo targeting, and biodistribution. The biodistribution study demonstrated that the monomeric constructs had longer blood retention and lower hepatic uptake than the dimeric ones. A dimeric construct, specifically ABD-Z-Z, could stimulate the proliferation of HER2 expressing SKOV-3 cells in vitro and the growth of tumors in vivo, whereas the monomeric construct Z-ABD could not. These two constructs demonstrated a therapeutic effect when coupled to mcDM1; however, the effect was more pronounced for the non-stimulating Z-ABD. The median survival of the mice treated with Z-ABD-mcDM1 was 63 days compared to the 37 days for those treated with ABD-Z-Z-mcDM1 or for the control animals. Domain permutation of an ABD-fused HER2-targeting affibody-based drug conjugate significantly influences tumor cell proliferation and therapy efficacy. The monomeric conjugate Z-ABD is the most promising format for targeted delivery of the cytotoxic drug DM1.

Increasing evidence suggests that therapy targeting the human epidermal growth factor receptor 3 (HER3) could be a viable route for targeted cancer therapy. Here, we studied a novel drug conjugate, Z_HER3-ABD-mcDM1, consisting of a HER3-targeting affibody molecule, coupled to the cytotoxic tubulin polymerization inhibitor DM1, and an albumin-binding domain for in vivo half-life extension. Z_HER3-ABD-mcDM1 showed a strong affinity to the extracellular domain of HER3 (K_D 6 nM), and an even stronger affinity (K_D 0.2 nM) to the HER3-overexpressing pancreatic carcinoma cell line, BxPC-3. The drug conjugate showed a potent cytotoxic effect on BxPC-3 cells with an IC₅₀ value of 7 nM. Evaluation of a radiolabeled version, [^99mTc]Tc-Z_HER3-ABD-mcDM1, showed a relatively high rate of internalization, with a 27% internalized fraction after 8 h. Further in vivo evaluation showed that it could target BxPC-3 (pancreatic carcinoma) and DU145 (prostate carcinoma) xenografts in mice, with an uptake peaking at 6.3 ± 0.4% IA/g at 6 h post-injection for the BxPC-3 xenografts. The general biodistribution showed uptake in the liver, lung, salivary gland, stomach, and small intestine, organs known to express murine ErbB3 naturally. The results from the study show that Z_HER3-ABD-mcDM1 is a highly potent and selective drug conjugate with the ability to specifically target HER3 overexpressing cells. Further pre-clinical and clinical development is discussed.

The human epidermal growth factor receptor 2 (HER2) is a clinically validated target for cancer therapy, and targeted therapies are often used in regimens for patients with a high HER2 expression level. Despite the success of current drugs, a number of patients succumb to their disease, which motivates development of novel drugs with other modes of action. We have previously shown that an albumin binding domain-derived affinity protein with specific affinity for HER2, ADAPT₆, can be used to deliver the highly cytotoxic protein domain PE25, a derivative of Pseudomonas exotoxin A, to HER2 overexpressing malignant cells, leading to potent and specific cell killing. In this study we expanded the investigation for an optimal targeting domain and constructed two fusion toxins where a HER2-binding affibody molecule, Z_HER2:2891, or the dual-HER2-binding hybrid Z_HER2:2891-ADAPT₆ were used for cancer cell targeting. We found that both targeting domains conferred strong binding to HER2; both to the purified extracellular domain and to the HER2 overexpressing cell line SKOV3. This resulted in fusion toxins with high cytotoxic potency toward cell lines with high expression levels of HER2, with EC₅₀ values between 10 and 100 pM. For extension of the plasma half-life, an albumin binding domain was also included. Intravenous injection of the fusion toxins into mice showed a profound influence of the targeting domain on biodistribution. Compared to previous results, with ADAPT₆ as targeting domain, Z_HER2:2891 gave rise to further extension of the plasma half-life and also shifted the clearance route of the fusion toxin from the liver to the kidneys. Collectively, the results show that the targeting domain has a major impact on uptake of PE25-based fusion toxins in different organs. The results also show that PE25-based fusion toxins with high affinity to HER2 do not necessarily increase the cytotoxicity beyond a certain point in affinity. In conclusion, Z_HER2:2891 has the most favorable characteristics as targeting domain for PE25.

Multidrug-resistant bacterial infections pose a severe global health threat, highlighting the urgent need for innovative therapeutic options beyond traditional antibiotics. Phage therapy, which employs bacteriophages to infect and eradicate pathogenic bacteria, specifically offers a promising solution. However, the lack of well-characterized therapeutic phages has limited their broader clinical use. A critical aspect of activating the lytic potential of dormant prophages involves the strategic manipulation of transcription factor binding sites (TFBS), which function as pivotal regulatory nodes governing the transition between lysogenic dormancy and lytic activation. Our platform utilizes advanced bioinformatics tools to accurately identify and analyze TFBS, facilitating the targeted redesign or replacement of these sites to disrupt host-mediated repression. By systematically simulating modifications of these regulatory ‘switches,’ our platform computationally predicts reduced repressor activity, suggesting the potential for prophage activation and bacterial cell lysis. This novel methodology not only broadens the spectrum of therapeutic bacteriophages but also establishes a basis for individualized phage-based therapies, presenting a robust strategy to address the escalating challenge of antibiotic-resistant infections. By enabling the precise identification and engineering of TFBS, our platform signifies a transformative advancement in phage biology, effectively bridging the divide between computational analysis and therapeutic application.