Polymer conjugation is a clinically proven approach to generate long acting protein drugs with decreased immune responses. Although poly(ethylene glycol) (PEG) is one of the most commonly used conjugation partners due to its unstructured conformation, its therapeutic application is limited by its poor biodegradability, propensity to induce an anti-PEG immune response, and the resultant accelerated blood clearance (ABC) effect. Moreover, the prevailing preference of unstructured polymers for protein conjugation still lacks strong animal data support with appropriate control reagents. By using two biodegradable synthetic polypeptides with similar structural compositions ( l -P(EG 3 Glu) and dl -P(EG 3 Glu)) for site-specific protein modification, in the current study, we systematically investigate the effect of the polymer conformation on the in vivo pharmacological performances of the resulting conjugates. Our results reveal that the conjugate l 20K -IFN, interferon (IFN) modified with the helical polypeptide l -P(EG 3 Glu) shows improved binding affinity, in vitro antiproliferative activity, and in vivo efficacy compared to those modified with the unstructured polypeptide analogue dl -P(EG 3 Glu) or PEG. Moreover, l 20K -IFN triggered significantly less antidrug and antipolymer antibodies than the other two. Importantly, the unusual findings observed in the IFN series are reproduced in a human growth hormone (GH) conjugate series. Subcutaneously infused l 20K -GH, GH modified with l -P(EG 3 Glu), evokes considerably less anti-GH and antipolymer antibodies compared to those modified with dl -P(EG 3 Glu) or PEG ( dl 20K -GH or PEG 20K –GH). As a result, repeated injections of dl 20K -GH or PEG 20K -GH, but not l 20K -GH, result in a clear ABC effect and significantly diminished drug availability in the blood. Meanwhile, immature mouse bone marrow cells incubated with the helical l 20K -GH exhibit decreased drug uptake and secretion of proinflammatory cytokines compared to those treated with one of the other two GH conjugates bearing unstructured polymers. Taken together, the current study highlights an urgent necessity to systematically reassess the pros and cons of choosing unstructured polymers for protein conjugation. Furthermore, our results also lay the foundation for the development of next-generation biohybrid drugs based on helical synthetic polypeptides.
Discordance of estrogen receptor (ER), progesterone receptor (PR), and human epidermal growth factor receptor 2 (HER2) status between primary breast cancer, metastatic lesion and synchronous axillary lymph node metastasis has been reported in the series studies. Systemic treatment of primary invasive breast cancer patients with synchronous axillary metastasis is currently based on the biomarker characteristics of the primary tumor; however, hormone receptors and HER2 status may change throughout tumor progression from the primary tumor to the synchronous axillary metastasis. As local metastasis, the synchronous axillary lymph node metastasis may represent the potentially metastatic breast cancer cells much better than the primary tumor. Hence, the determination of hormone receptors and HER2 status should be routinely performed in synchronous axillary nodal metastasis, together with primary tumor, to guide therapy management and evaluate the recurrent risk of primary invasive breast cancer patients with synchronous axillary nodal metastasis, which may even change the postoperative risk categories (St. Gallen consensus) of breast cancer in these patients. This article will review the studies on the discordance and clinical significance of ER, PR, and HER2 receptor status between primary breast cancer and synchronous axillary lymph node metastasis.
Cyclization and polymer conjugation are two commonly used approaches for enhancing the pharmacological properties of protein drugs. However, cyclization of parental proteins often only affords a modest improvement in biochemical or cell-based in vitro assays. Moreover, very few studies have included a systematic pharmacological evaluation of cyclized protein-based therapeutics in live animals. On the other hand, polymer-conjugated proteins have longer circulation half-lives but usually show poor tumor penetration and suboptimal pharmacodynamics due to increased steric hindrance. We herein report the generation of a head-to-tail interferon-poly(α-amino acid) macrocycle conjugate circ-P(EGGlu)-IFN by combining the aforementioned two approaches. We then compared the antitumor pharmacological activity of this macrocycle conjugate against its linear counterparts, N-P(EGGlu)-IFN, C-IFN-P(EGGlu), and C-IFN-PEG. Our results found circ-P(EGGlu)-IFN to show considerably greater stability, binding affinity, and in vitro antiproliferative activity toward OVCAR3 cells than the three linear conjugates. More importantly, circ-P(EGGlu)-IFN exhibited longer circulation half-life, remarkably higher tumor retention, and deeper tumor penetration in vivo. As a result, administration of the macrocyclic conjugate could effectively inhibit tumor progression and extend survival in mice bearing established xenograft human OVCAR3 or SKOV3 tumors without causing severe paraneoplastic syndromes. Taken together, our study provided until now the most relevant experimental evidence in strong support of the in vivo benefit of macrocyclization of protein-polymer conjugates and for its application in next-generation therapeutics.
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