# Ras Isoforms from Lab Benches to Lives--What Are We Missing and How Far Are We?
## 6 Future Recommendations for Devising Isoform-Specific Targeting
Indeed, such an approach was later employed by [PERSON] et al., where the catalytically active domain of TpeL was coupled to the binding domain of the anthrax toxin binding and translocation component PA (protective antigen) [197]. To increase the specificity for tumor cells, a CD46 interaction domain was engineered into the binding region, as CD46 is often overexpressed on cancer cells. In this in vitro study, human pancreatic cancer Capan-2 cells were shown to get successfully deleted. Such immunotoxins are elegant tools to specifically target and destroy cancer cells and several immunotoxins are in clinical or preclinical evaluation [201]. Three immunotoxins have already been approved by the FDA. The cell death-inducing diphthe toxin (DT) can be coupled to the cytokines IL-2 or IL-3 (DT-IL3, DT-IL3), respectively, and cells that express the cognate receptor take up the toxin and die due to the inhibition of protein translation by DT [202]. Lumoxiti makes use of the _Pseudomonas aeruginosa_ exotoxin A coupled to an anti-CD22 antibody that recognizes the phosphoglycoprotein CD22 expressed on B cells. However, some restrictions prevent the easy application of immunotoxins. Depending on the receptor that is recognized by the binding domain, the toxin might also get delivered into healthy cells causing equally efficient, but unwanted cell death. Additionally, protein-derived drugs will be recognized by the adaptive immune system through MHC antigen presentation, resulting in the production of protective antibodies. [PERSON] and [PERSON] also rightly pointed out that solid tumors are of heterogeneous nature so that immunotoxins would only target parts of the tumor, making it necessary to use immunotoxins in combination with conventional chemotherapeutics [201]. From the above discussion, it is clear that the 'one size fits all' approach cannot be adopted when designing inhibitors against Ras isoforms. From the localization in the plasma membrane and different organelle to their behavior on activation by the same or different receptors, multiple strata of functional complexity exist. In addition to this, the functioning of Ras in an oncogenic setup versus a normal cell is quite different. Our understanding of the effect of autonomous or synergistic/antagonistic signaling arising from various cellular compartments (mitochondria, Golgi, E.R, endosome, and nucleus) in deciding the signaling outcome is very limited. As mentioned in Section 5.5, few studies have shown activation of Ras isoforms by specific GEFs in these cellular compartments. However, a consolidated study of the distribution and activation of the RasGEFs-Ras isoforms-Ras effector's axis upon receptor stimulation is yet to be elucidated. Using SOS1 and Ras GRF1, [PERSON] et al. have identified in the catalytic domain of GEF, a 52 aa (aminoacid) segment that forms helices B and C as the key determinant of signaling specificity. On replacement of amino acids in SOS1 with analogous amino acids from Ras GRF1, SOS1 could activate the target GTPase of Ras GRF1 i.e., R-Ras. However, on replacement of Ras GRF1 sequences with that of SOS1, Ras GRF1 failed to activate R-Ras and H-Ras. This suggests that different Ras GEFs have different mechanisms to activate target GTPase [203]. Studies that show amino acid sequence specificity in Ras GEFs and their corresponding Ras isoforms targets under different signaling contexts would enable researchers a better understanding of divergence of signaling. Studies are pointing out possible new functions of Ras isoforms. RanGTPase is known to be a key regulator of nucleocytoplasmic transport of cargoes in and out of the nucleus. With studies showing the presence of H-Ras in the nucleus, many questions arise as to the function of Ras in the nucleus, regarding its mechanism of activation, nuclear proteins that may bind to H-Ras, and the signaling pathway. Whether H-Ras performs a similar function as RanGTPase remains to be elucidated.