**6.1 Immunobead-based EV capture**

The immunocapture-based methods generally use beads coated with selected Abs for EV pulldown. In the simplest approach, beads coated with Abs are added directly to plasma diluted in phosphate-buffered saline (PBS) with the expectation that all EVs bearing the target Ag on the surface will bind to the Ab coated beads. This strategy for capture may not be and usually is not very effective, because EVs in plasma carry a variety of soluble plasma proteins on the surface, such as albumin, immunoglobulins (Igs) and other "contaminating" plasma proteins. These plasma proteins form a protein "corona" associated with the sEV surface membrane which is likely to block the access of capture Abs to targeted Ags, leading to an incomplete pulldown or even lack of pulldown. Also, if the target Ag is present in soluble form in plasma, it might compete with the counterpart carried on the EV membrane, binding to the Ab coated beads either specifically as a soluble protein or as a "contaminating" EV

surface-associated protein. The soluble target Ag present in plasma (especially when its abundance is high) could non-specifically associate with proteins/glycoproteins decorating EV surfaces. As a result, the target Ag could mediate the pulldown of EVs that do not constitutively express the Ag. The result will be a pulldown of EVs carrying a soluble Ag in addition to EVs genuinely endowed with the target Ag embedded in the EV membrane. Such capture will not distinguish between these two types of EVs, and thus the strategy is useless for selective capture of EVs carrying the targeted tumor-specific Ag. This capture strategy has been also used with EVs isolated from plasma by ExoQuick, which concentrates rather than "purifies" EVs, with the same unsatisfactory results [21].

Perhaps a good example of this strategy is immune capture from cancer patients' plasma of EVs carrying PD-L1 as recently reported [22]. This protein, commonly carried by TEX in most cancers, is also present in the plasma of cancer patients both as a soluble protein derived from malignant and various non-malignant cells and as the integral membrane protein of non-TEX released by macrophages or other immune cells [23]. Therefore, beads coated with anti-PD-L1 Abs cannot be used for selective capture of PD-L1-positive TEX, because such beads will capture soluble PD-L1, non-TEX carrying PD-L1 as well as TEX carrying PD-L1, thus making it impossible to distinguish which EV subset delivers inhibitory signals to PD-1-positive recipient cells. The contribution of soluble PD-L1 to negative signaling by the captured vesicles may not be disregarded, because in addition to its specific binding to Ab-coated beads, soluble PD-L1 might non-specifically "associate" with all EVs in plasma, similar to albumin or other plasma proteins. Data in the literature [24] and the protein content of the ExoCarta data base [25] confirm that EVs isolated from plasma carry numerous non-specific plasma-derived proteins and suggest that the discrimination of "true" EV proteins from plasma "contaminants" is a major challenge in the field.

### **6.2 Selection of abs for immune capture of TEX**

The selection of Abs for TEX immune capture depends on the convincingly demonstrated ability of such Abs to selectively bind to tumor cells expressing the target Ag on the cell surface, with the exclusion of any binding to non-malignant cells which do not express the Ag. This is a rigorous requirement and one that may be difficult to implement, because few tumor-specific antigens are known, except for mutated epitopes in cancer cells. To emphasize, the capture Abs selected for TEX capture must be specific for an antigen (or an epitope) present only in parent cells and in EVs these parent cells produce but not in any other cells or tissues. Even if such tumor Ag- specific Ab is available, it is necessary to ensure that its binding affinity for the target Ag is high and that the target antigen is not present in soluble forms in body fluids. Low-affinity Abs will not be effective in pulldowns, and the presence of a target Ag in soluble forms might not only interfere with Ab binding to TEX but upon its non-specific association with EVs, as described above, will lead to the capture of EVs derived from non-malignant cells, thus interfering with selective TEX capture. As most Ags expressed on the surface of cancer cells are enzymatically cleaved and are present in plasma, this requirement may not be readily addressed. Importantly, adding Ab coated beads directly to plasma without prior attention to these restrictions will jeopardize the selectivity and efficiency of TEX immune capture.

Clearly, the use of the best capture Abs is by far the most critical aspect of TEX immune capture from plasma. In the absence of such Abs, immune capture using a mix of Abs specific for Ags highly overexpressed on cancer cells relative to

#### *Diversity of Extracellular Vesicles (EV) in Plasma of Cancer Patients DOI: http://dx.doi.org/10.5772/intechopen.101760*

non-malignant cells and on the EVs these cells produce could be utilized for immune capture, and this approach has been successful [26]. It is possible to perform immune capture of TEX with a cocktail of Abs carefully selected for specificity to proteins overexpressed on tumor cells and weakly expressed on non-malignant cells. The complexity of immune capture increases with the use of Ab cocktails largely due to `Ab titration requirements and the need for extensive controls as well as the limitations imposed by the presence in plasma of the Ags recognized by the Abs used for immune capture, in soluble form.
