Update on targeted cancer therapies, single or in combination, and their fine tuning for precision medicine

Background: Until recently, patients who have the same type and stage of cancer all receive the same treatment. It has been established, however, that individuals with the same disease respond differently to the same therapy. Further, each tumor undergoes genetic changes that cause cancer to grow and metastasize. The changes that occur in one person's cancer may not occur in others with the same cancer type. These differences also lead to different responses to treatment. Precision medicine, also known as personalized medicine, is a strategy that allows the selection of a treatment based on the patient's genetic makeup. In the case of cancer, the treatment is tailored to take into account the genetic changes that may occur in an individual's tumor. Precision medicine, therefore, could be defined in terms of the targets involved in targeted therapy. Methods: A literature search in electronic data bases using keywords “cancer targeted therapy, personalized medicine and cancer combination therapies” was conducted to include papers from 2010 to June 2019. Results: Recent developments in strategies of targeted cancer therapy were reported. Specifically, on the two types of targeted therapy; first, immune-based therapy such as the use of immune checkpoint inhibitors (ICIs), immune cytokines, tumor-targeted superantigens (TTS) and ligand targeted therapeutics (LTTs). The second strategy deals with enzyme/small molecules-based therapies, such as the use of a proteolysis targeting chimera (PROTAC), antibody-drug conjugates (ADC) and antibody-directed enzyme prodrug therapy (ADEPT). The precise targeting of the drug to the gene or protein under attack was also investigated, in other words, how precision medicine can be used to tailor treatments. Conclusion: The conventional therapeutic paradigm for cancer and other diseases has focused on a single type of intervention for all patients. However, a large literature in oncology supports the therapeutic benefits of a precision medicine approach to therapy as well as combination therapies

Novel SPEA Superantigen Peptide Agonists and Peptide Agonist-TGFαL3 Conjugate. In Vitro Study of Their Growth-Inhibitory Effects for Targeted Cancer Immunotherapy

Bacterial superantigens (SAgs) are effective T-cell stimulatory molecules that lead to massive cytokine production. Superantigens crosslink between MHC class II molecules on the Antigen Presenting Cells (APC) and TCR on T-cells. This enables them to activate up to 20% of resting T cells, whilst conventional antigen presentation results in the activation of 0.001–0.0001% of the T cell population. These biological properties of superantigens make them attractive for use in immunotherapy. Previous studies have established the effectiveness of superantigens as therapeutic agents. This, however, was achieved with severe side effects due to the high lethality of the native toxins. Our study aims to produce superantigen-based peptides with minimum or no lethality for safer cancer treatment. In previous work, we designed and synthesized twenty overlapping SPEA-based peptides and successfully mapped regions in SPEA superantigen, causing a vasodilatory response. We screened 20 overlapping SPEA-based peptides designed and synthesized to cover the whole SPEA molecule for T-cell activation and tumor-killing ability. In addition, we designed and synthesized tumor-targeted superantigen-based peptides by fusion of TGFαL3 either from the N′ or C′ terminal of selected SPEA-based peptides with an eight-amino acid flexible linker in between. Our study identified parts of SPEA capable of stimulating human T-cells and producing different cytokines. We also demonstrated that the SPEA-based peptide conjugate binds specifically to cancer cells and can kill this cancer. Peptides induce T-cell activation, and tumor killing might pave the way for safer tumor-targeted superantigens (TTS). We proposed the combination of our new superantigen-based peptide conjugates with other immunotherapy techniques for effective and safer cancer treatment.Scopu

Correction:Isolation and molecular characterization of novel glucarpidases: Enzymes to improve the antibody directed enzyme pro-drug therapy for cancer treatment

</p

In vitro studies on CNGRC-CPG2 fusion proteins for ligand-directed enzyme prodrug therapy for targeted cancer therapy

The sequence asparagine-glycine arginine (NGR), flanked by Cysteine (Cys) residues so as to form a disulfide-bridge (CNGRC), has previously been found to target and bind specifically to aminopeptidase N (APN), which is highly expressed on the surface of tumor cells. The goal of this study was to develop and evaluate the potential of fusion proteins carrying the CNGRC sequence linked to the enzyme carboxypeptidase G2 (CPG2) for targeted cancer therapy. We refer to this strategy as ligand-directed enzyme prodrug therapy (LDEPT). We constructed two forms of the CNGRC-CPG2 fusions, containing one or two copies of the cyclic NGR motif and designated CNGRC-CPG2 (X-CPG2) and CNGRC-CPG2-CNGRC (X-CPG2-X), respectively. binding assays of the purified constructs showed that both X-CPG2 and X-CPG2-X bound with high affinity to cancer cells expressing high levels of APN, compared to their binding to cells expressing low levels of APN. Further studies of the constructs to assess the therapeutic potential of LDEPT were carried out using cells expressing high and low levels of APN. Using methotrexate, it was demonstrated that cancer cell survival was significantly higher in the presence of the fusion proteins, due to the hydrolysis of this cytotoxic drug by CPG2. Conversely, when the prodrug ZD2767P was used, cancer cell killing was higher in the presence of the fused CPG2 constructs than in their absence, which is consistent with CPG2-mediated release of the cytotoxic drug from the prodrug. Furthermore, the doubly-fused CPG2 construct (X-CPG2-X) was significantly more effective than the singly-fused construct (X-CPG2)

Isolation and molecular characterization of novel glucarpidases:Enzymes to improve the antibody directed enzyme pro-drug therapy for cancer treatment

<div><p>Repeated cycles of antibody-directed enzyme pro-drug therapy (ADEPT) and the use of glucarpidase in the detoxification of cytotoxic methotrexate (MTX) are highly desirable during cancer therapy but are hampered by the induced human antibody response to glucarpidase. Novel variants of glucarpidase (formal name: carboxypeptidase G2, CPG2) with epitopes not recognized by the immune system are likely to allow repeated cycles of ADEPT for effective cancer therapy. Towards this aim, over two thousand soil samples were collected and screened for folate hydrolyzing bacteria using folate as the sole carbon source. The work led to the isolation and the characterization of three new glucarpidase producing strains, which were designated as: <i>Pseudomonas lubricans</i> strain SF168, <i>Stenotrophomonas</i> sp SA and <i>Xenophilus azovorans</i> SN213. The <i>CPG2</i> genes of <i>Xenophilus azovorans</i> SN213 (named <i>Xen CPG2</i>) and <i>Stenotrophomonas sp</i> SA (named <i>Sten CPG2</i>) were cloned and molecularly characterized. Both Xen CPG2 and Sten CPG2 share very close amino acid sequences (99%); we therefore, focused on the study of Xen CPG2. Finally, we demonstrated that a polyclonal antibody raised against our new CPG2, Xen CPG2, does not react with the CPG2 from <i>Pseudomonas sp</i>. strain RS-16 (Ps CPG2) that are currently in clinical use. The two enzymes, therefore could potentially be used consecutively in the ADEPT protocol to minimize the effect of the human antibody response that hampers current treatment with Ps CPG2. The identified novel CPG2 in this study will, therefore, pave the way for safer antibody directed enzyme pro-drug therapy for cancer treatment.</p></div

Studies on vascular response to full superantigens and superantigen derived peptides:Possible production of novel superantigen variants with less vasodilation effect for tolerable cancer immunotherapy

Superantigens (SAgs) are a class of antigens that cause non-specific activation of T-cells resulting in polyclonal T cell activation and massive cytokine release and causing symptoms similar to sepsis, e.g. hypotension and subsequent hyporeactivity. We investigated the direct effect of SAgs on vascular tone using two recombinant SAgs, SEA and SPEA. The roles of Nitric Oxide (NO) and potentially hyperpolarization, which is dependent on the K + channel activation, were also explored. The data show that SEA and SPEA have direct vasodilatory effects that were in part NO-dependent, but completely dependent on activation of K + channels. Our work also identified the functional regions of one of the superantigens, SPEA, that are involved in causing the vasodilation and possible hypotension. A series of 20 overlapping peptides, spanning the entire sequence of SPEA, were designed and synthesized. The vascular response of each peptide was measured, and the active peptides were identified. Our results implicate the regions, (61–100), (101–140) and (181–220) which cause the vasodilation and possible hypotension effects of SPEA. The data also shows that the peptide 181–220 exert the highest vasodilation effect. This work therefore, demonstrates the direct effect of SAgs on vascular tone and identify the active region causing this vasodilation. We propose that these three peptides could be effective novel antihypertensive drugs. We also overexpressed, in E.coli, four superantigens from codon optimized genes

Production of "biobetter" variants of glucarpidase with enhanced enzyme activity

Glucarpidase, also known as carboxypeptidase G(2), is a Food and Drug Administration-approved enzyme used in targeted cancer strategies such as antibody-directed enzyme prodrug therapy (ADEPT). It is also used in drug detoxification when cancer patients have excessive levels of the anti-cancer agent methotrexate. The application of glucarpidase is limited by its potential immunogenicity and limited catalytic efficiency. To overcome these pitfalls, mutagenesis was applied to the glucarpidase gene of Pseudomonas sp. strain RS-16 to isolate three novels "biobetter" variants with higher specific enzyme activity. DNA sequence analysis of the genes for the variants showed that each had a single point mutation, resulting in the amino acid substitutions: I100 T, G123S and T239 A. K-m, V-max and K-cat measurements confirmed that each variant had increased catalytic efficiency relative to wild type glucarpidase. Additionally, circular dichroism studies indicated that they had a higher alpha-helical content relative to the wild type enzyme. However, three different software packages predicted that they had reduced protein stability, which is consistent with having higher activities as a tradeoff. The novel glucarpidase variants presented in this work could pave the way for more efficient drug detoxification and might allow dose escalation during chemotherapy. They also have the potential to increase the efficiency of ADEPT and to reduce the number of treatment cycles, thereby reducing the risk that patients will develop antibodies to glucarpidase

Metformin and HER2-positive breast cancer: Mechanisms and therapeutic implications

Due to the strong association between diabetes and cancer incidents, several anti-diabetic drugs, including metformin, have been examined for their anticancer activity. Metformin is a biguanide antihyperglycemic agent used as a first-line drug for type II diabetes mellitus. It exhibits anticancer activity by impacting different molecular pathways, such as AMP-inducible protein kinase (AMPK)-dependent and AMPK-independent pathways. Additionally, Metformin indirectly inhibits IGF-1R signaling, which is highly activated in breast malignancy. On the other hand, breast cancer is one of the major causes of cancer-related morbidity and mortality worldwide, where the human epidermal growth factor receptor-positive (HER2-positive) subtype is one of the most aggressive ones with a high rate of lymph node metastasis. In this review, we summarize the association between diabetes and human cancer, listing recent evidence of metformin’s anticancer activity. A special focus is dedicated to HER2-positive breast cancer with regards to the interaction between HER2 and IGF-1R. Then, we discuss combination therapy strategies of metformin and other anti-diabetic drugs in HER2-positive breast cancer

Production of "biobetter" glucarpidase variants to improve drug detoxification and antibody directed enzyme prodrug therapy for cancer treatment

Recombinant glucarpidase (formerly: Carboxypeptidase G2, CPG2) is used in Antibody Directed Enzyme Prodrug Therapy (ADEPT) for the treatment of cancer. In common with many protein therapeutics, glucarpidase has a relatively short half-life in serum and, due to the need for the repeated cycles of the ADEPT, its bioavailability may be further diminished by neutralizing antibodies produced by patients. PEGylation and fusion with human serum albumin (HSA) are two approaches that are commonly employed to increase the residency time of protein therapeutics in blood, and also to increase the half-lives of the proteins in vivo. To address this stability and the immunogenicity problems, `biobetter' glucarpidase variants, mono-PEGylated glucarpidase, and HSA fused glucarpidase by genetic fusion with albumin, were produced. Biochemical and bioactivity analyses, including anti-proliferation, bioassays, circular dichroism, and in vitro stability using human blood serum and immunoassays, demonstrated that the functional activities of the designed glucarpidase conjugates were maintained. The immunotoxicity studies indicated that the PEGylated glucarpidase did not significantly induce T-cell proliferation, suggesting that glucarpidase epitopes were masked by the PEG moiety. However, free glucarpidase and HSA-glucarpidase significantly increased T-cell proliferation compared with the negative control. In the latter case, this might be due to the type of expression system used or due to trace impurities associated with the highly purified (99.99%) recombinant HSA-glucarpidase. Both PEGylated glucarpidase and HAS-glucarpidase exhibit more stability in human serum and were more resistant to key human proteases relative to native glucarpidase. To our knowledge, this study is the first to report stable and less immunogenic glucarpidase variants produced by PEGylation and fusion with HSA. The results suggest that they may have better efficacy in drug detoxification and ADEPT, thereby improving this cancer treatment strategy

CD spectra and high voltage of Xen CPG2 and Ps CPG2.

<p>A) Combined CD spectra data of Xen CPG2 and Ps CPG2 in molar ellipticity relative the wavelength in far UV region, the spectra obtained by dragging their spectral data over each other, where smooth 0 is molar ellipticity of Xen CPG2 and smooth 1 is for CD spectra of Ps CPG2. All Spectral data are corrected for the baseline buffer, B) represents the combined High voltage (HV) for both enzymes where average 0 is Xen CPG2 and average 1 is Ps CPG2. Also the table shows the calculated protein secondary structure of Xen CPG2 and Ps CPG2 by CDNN deconvolution analysis using their CD spectral data.</p