*5.2. E7-Specific Recombinant Antibodies*

The E7 protein of HR HPVs cooperates with E6 protein to drive oncogenesis mainly through deregulation of growth suppressors, which leads to uncontrolled cell proliferation as above detailed. Therefore, E7 is widely studied as a therapeutic target, and the now in-depth knowledge of its functions suggests that specific recombinant antibodies may be a useful tool to fulfil the anticancer purposes [79].

#### 5.2.1. ScFvs and mAbs

The first scFvs selected against 16E7 oncoprotein were constructed directly from murine spleen cells, and then provided with signals for subcellular localization by cloning [80]. When the scFv-expressing plasmids were transfected in HPV16-positive cells, the scFv with SEKDEL signal for localization in the endoplasmic reticulum (ER) was effective in decreasing E7 expression in a manner inversely related to the amount of plasmid used for cell transfection. Interestingly, when trying to generate cells stably expressing the anti-E7 scFvs, the researchers observed that stable expression of these antibodies was not compatible with clonal outgrowth of E7-expressing tumour cells. In fact, the expression of anti-16E7 scFvs, and of that with localization in the ER in particular, successfully and specifically inhibited the proliferation of HPV16-positive CaSki and SiHa cells (Figure 5). Wang-Johanning et al. concluded that the alteration obtained was due to the interaction between the scFv and E7 [80].

**Figure 5.** Schematic representation of the known effects of anti-E7 intrabodies expressed in HPVpositive cells. The effects of the intracellular expression of specific scFvs (pKDEL and 43M2SD) with localization in the ER are shown in the figure. pKDEL reduces the intracellular levels of E7, thus hampering its effect on cell proliferation. The binding of 43M2SD to E7 inhibits the translocation of the oncoprotein to the cell nucleus. This, in turn, hampers E7-mediated inactivation of Retinoblastoma (pRB), which regulates E2F activity on S-phase genes. The binding of 43M2SD and pKDEL can also inhibit the proteosomal pRB degradation mediated by the Cullin 2-RING ubiquitin ligase complex (CUL2). The effect of intracellular expression of the nB2 nanobody, which inhibits cell proliferation with a mechanism not yet investigated, is also shown in the figure.

More recently, our group selected from a Phage library of human recombinant antibodies, three different scFvs against the 16E7, provided them with signals for localization in the cell nucleus or ER by cloning in eukaryotic vectors, and evidenced their specific and significant antiproliferative effect in HPV16-positive cells in vitro [81]. We also characterized the scFv-binding regions by epitope mapping using immunoassays based on GST-tagged E7 proteins carrying deletions or aminoacid variations. This allowed deciphering E7 regions targeted by scFvs, and revealed that different regions known to be directly involved in transforming activities of E7 are bound by the scFvs. This suggested that different scFvs

may be used to target diverse E7 activities [82,83]. We were able to improve the half-life and thermal stability of the most reactive of the anti-16E7 scFvs, scFv43, by site-directed mutagenesis, confirming that small variations in the amino acid sequence can modify the antibody biophysical characteristics [84]. We then provided the modified scFv, namely scFv43M2, with the SEKDEL signal (SD) for localization in the ER and thereafter tested the resulting scFv43M2SD for its ability to counteract the 16E7 activity. In SiHa cells, the intrabody was able to subtract E7 from the usual localization and cause it to accumulate in the ER. In addition, the scFv43M2SD intracellular expression was able to inhibit significantly and specifically the proliferation of different HPV16-positive cell lines [85]. The scFv43M2 was then tested in vivo in mouse HPV tumour models, demonstrating the ability to counteract tumour progression both when administered to tumour cells before their injection into mice and when administered to already implanted tumours [72,85] (Figure 4).

The anti-tumour activity of the anti-16E7 TVG701Y mAb has been described together with the anti-16E6 C1P5 mAb in the previous paragraph, as the two mAbs were utilized in the same study [74] (Figure 4).

#### 5.2.2. Nanobodies

Li et al. selected four VHHs with high affinity for 16E7 from llama libraries by Phage display, and one of these was chosen for further analyses because of lacking Cysteine residues potentially able to form intra-molecular disulphide bonds. The nanobody was expressed in prokaryotic system as a protein, and its ability to bind to the recombinant E7 in vitro was confirmed in immunological assays. Furthermore, the nanobody was able to detect the endogenous E7 protein in Western blotting and, most importantly, induced a specific inhibition of the proliferation of HPV16-positive cells when these cells were transfected with a recombinant eukaryotic plasmid [86] (Figure 5).

#### *5.3. E5-Specific scFvs*

In light of the recognized tumourigenic role in the early phases of HPV-induced carcinogenesis and in immunoevasion, E5 protein can be also considered a suitable target for therapeutic purposes, possibly in combination with the main E6 and E7 oncoproteins. Nevertheless, the first and currently only scFv anti-HPV16 E5 (16E5) was developed with the purpose of investigating the E5 functions [87]. Monjarás-Ávila et al. selected this antibody by Phage display technology against the recombinant 16E5 fused to Maltosebinding protein to bypass difficulties due to the E5 hydrophobicity (Figure 6). They then tested this E5-specific scFv in W12 cells, with immortalized keratinocytes carrying up to a maximum of 1000 episomal copies of the HPV16 genome at a low number of passages [88]. The scFv was able to recognize E5 in W12 cells and to reveal its co-localization with EGFR. Therefore, it deserves further investigations to explore its possible application in the therapeutic field.

#### *5.4. E6 and E7-Specific Affibodies*

Although they are not properly recombinant antibodies, a mention is deserved by affibodies, a new class of single-domain protein scaffolds based on non-Ig Z domain derived from the staphylococcal protein A. Affibodies are very small molecules (6 kDa) that can be selected against any protein target, and are attracting the attention of the scientific community for biotechnological applications, in particular for in vivo imaging but also for anticancer therapy. Some anti-16E6, -16E7, -18E6, and -18-E7 affibodies were selected and tested successfully both in diagnostic and therapeutic applications [89–91], either as bs affibodies [92] or as fusion with toxins (affitoxins) [93,94].

**Figure 6.** Representation of binding of the H2-I intrabody to E5. The scFv H2-I, when expressed within HPV-positive cells, colocalizes with E5 and its target, the Epidermal Growth Factor Receptor (EGFR), able to activate the mitogen-activated protein kinase (MAPK) signalling cascade, leading to DNA synthesis and cell proliferation.

#### **6. Intracellular Delivery Methods for Recombinant Antibodies against HPV Oncoproteins**

In the various experimental contexts described above, recombinant antibodies showed to be effective in hindering the action of the HPV E6 and E7 oncoproteins, thus interfering with the main cancer hallmarks in which they are involved. Despite the safety and benefits of what would be a recombinant antibody-based therapy for HPV-associated lesions, still a low number of Nbs, scFvs, and mAbs against HPV oncoproteins have been developed, and none of them has reached the clinical stage so far. One of the reasons behind this essentially lies in the difficulty of identifying a delivery method that allows recombinant antibodies to cross biological barriers while maintaining biological activity, particularly when the targets are intracellular. In fact, when the target antigens are on the cell plasma membrane, the therapeutic antibodies diffuse in the extracellular environment from the bloodstream to the body tissues until they reach the target. In the case of intracellular targets, the delivery must be made first to the tumour cells, and secondly to the intracellular environment. Several studies are underway to address this criticality and permit translation to humans. In general, recombinant antibodies for intracellular targets can be either expressed within cells from DNA plasmids or delivered directly to cells as purified proteins. This is achievable by physical methods, transfection, electroporation, or fusion with a peptide transduction domain (PTD) or nanocarriers. Delivery as proteins guarantees high safety but implies the need for large quantities of Good Manufacturing Practices (GMP)-grade purified products. Without wishing to be exhaustive since the topic of "delivery" is addressed in more detail elsewhere [95], here we will mention potentially useful methods for the in vivo delivery of therapeutic antibodies against HPV E6, E7, and E5, some of which implement or are alternatives to those already explored for in vitro and in vivo use (Figure 7).

**Figure 7.** Schematic representation of delivery systems for recombinant antibodies. Some delivery systems already in use or potentially usable for the delivery to cells of mAbs and antibody fragments are illustrated. The mechanisms of cell entry are schematized for: (1) Electroporation; (2) Fusion with protein transduction domains (PTD)/Cell-penetrating peptides (CPP), shown with a red tail; (3) Exosome-based methods (entry by endocytosis is depicted as an example); (4) Viral vector-based methods; and (5). Ultrasound-based methods (sonoporation). MB, microbubbles.

#### *6.1. Electrotransfer/Electroporation*

EP applies voltage pulses to generate an electric field between two electrodes, which interrupts the integrity of cell membranes with the formation of pores allowing cell uptake of nucleic acids as well as proteins. EP is therefore a safe method for intracellular protein expression since it avoids insertional mutagenesis and immunogenicity problems inherent in other methods. As such, it can be exploited in a wide range of applications, particularly in immunotherapy [96]. One of the studies reported here used EP to achieve efficient expression of therapeutic scFvs injected as DNA plasmids in HPV-driven tumours [72]. Nevertheless, the methodology could even be used to deliver scFvs as proteins or mRNAs. Indeed, RNA electroporation of hematopoietic cells has been used successfully for two decades [97].

### *6.2. Fusion with Protein Transduction Domain*

PTDs or cell penetrating peptides (CPPs) are cationic and/or hydrophobic 10–30 amino acid long peptides that can be conjugated or fused to antibodies to make them able to penetrate the cell membrane via different mechanisms [98]. However, for effective translation in the clinic, the CPP-based delivery has some limitations to circumvent, mainly due to low in vivo stability and reduced binding capability.

#### *6.3. Exosome-Based Methods*

In our laboratories, an exosome-based strategy was recently investigated in vitro for the delivery of one anti-16E7 scFv previously studied, showing promise for translation to humans [99]. The approach relies on the property of a functional defective Nef protein of HIV-1 (Nefmut), acting as an exosome-anchoring protein for proteins fused to its Cterminus. The scFv43M2 delivered to HPV16-positive cells by engineered extracellular vesicles (EVs) carrying the Nefmut/43M2s chimeric product, was able to reproduce the

already observed antiproliferative effect of scFv43M2. The proliferation of HPV16-positive cells was hindered also when they were co-cultured in transwells with cells producing EVs uploading the Nefmut/43M2scFv fusion. This result confirmed the ability of therapeutic exosomes to be released and reach other cells, with interesting implications for in vivo translation. The established proof-of-concept that the EV-mediated delivery of scFvs can target intracellular antigens renders it feasible the development of this system for in vivo use. In addition, the possibility to obtain recombinant exosomes from the host following the administration of a genetic construct as a vaccine, suggests a feasible translation to humans of this delivery system for anti-E6 and E7 intrabodies [100]. This would also take advantage of the capacity of the recipient organism to produce the exosomes. Once the technology is optimized, the intramuscular injection of DNA plasmids expressing antibody constructs followed or not by electroporation, will permit the exosome-loaded antibodies to reach several body districts. As the antibodies are specific for the HPV oncoproteins, such broad distribution will not result in off-target effects while potentially affecting any metastatic cells derived from the primary tumour. Of course, further experiments are necessary to clarify the route followed by exosomes loaded with a therapeutic cargo in the recipient organism, and to establish dosages and timing of administration.

### *6.4. Viral Vector-Based Methods*

In the last 30 years, several clinical trials used viral vectors for gene transfer. Gammaretroviral and lentiviral vectors for haematological cancers; adenoviral vectors for prostate, ovarian and bladder cancer; and adenovirus-associated vectors for pathologies other than cancer were employed with more or less success, and are still the object of preclinical and clinical proof-of-concept studies [101]. Therefore, on the basis of the effective antibody expression achievable in vitro through the transduction of tumour cells with recombinant retroviruses [71,85], and of the advanced state of clinical studies, we believe that viral vectors can be considered a valid resource in addition to the non-viral systems for in vivo antibody delivery. Noteworthy, HPV-associated lesions have a confined localization that renders them accessible to topical therapy whatever the delivery system chosen. Furthermore, the expression of the target oncoproteins being limited to cancer cells represents an additional advantage for the safety of a therapy designed to inhibit protein–protein interactions such as that based on recombinant antibodies.

#### *6.5. Ultrasound-Based Methods*

The Ultrasound-mediated targeted delivery (UMTD) is a non-invasive method that is attracting increasing interest for many biochemical applications including immunotherapy of tumours. UMTD combined with microbubbles allows delivery of therapeutic molecules precisely in the tumour site. In fact, oscillation and cavitation of microbubbles under the influence of the acoustic beam causes the reversible formation of localized pores of about 100 nm in diameter in the cell membrane [102]. This phenomenon, known as sonoporation, allows the passive release of therapeutic molecules into target cells. The feasibility and specificity of sonoporation for anti-16E6 mAb delivery to cervical carcinoma cell lines were assessed in the in vitro study outlined above, although the effect obtained was transient and incomplete as it affected p53 levels but did not induce apoptosis [70]. However, the issue of delivery to nucleus, which probably underlies the observed partial efficacy, could be addressed using smaller antibody formats provided with NLS. Sonoporation is increasingly explored for both passive and active immunotherapy in vivo. For example, dendritic cells (DC) sonoporated with antigen mRNA and immunomodulating TriMix mRNA were successful in inhibiting tumour growth in mice [103]. Ultrasound in combination with microbubbles even allowed the Herceptin mAb (trastuzumab) to cross the blood-brain barrier in mice, thus opening up the possibility of treating brain metastases of breast cancer [104]. However, translation of the methodology to human therapy requires further investigation on the possible elicitation of immune response by microbubbles, the exact

mechanism of the therapeutic material release, the size-based microbubble capacity of penetrating cell membranes, and the excretion of microbubbles from the body.

#### **7. Conclusions and Perspectives**

Currently, recombinant antibodies for targeting antigens involved in the pathogenesis of a variety of diseases are obtainable by robust methodologies of immunization and in vitro screening. Nevertheless, their use as therapeutics may require optimization of crucial characteristics such as binding specificity and affinity, solubility, and pharmacokinetics, as well as setting up an appropriate delivery system. The possibility of designing bs antibodies that combine binding domains from different parental antibodies can expand the binding capacity of a single molecule. Bs antibodies could target at the same time multiple antigens such as E6 and E7, or multiple epitopes on the same antigen (such as DBD and E6AP binding domain on E6) but their solubility and stability may be affected and require corrections [105].

The implementation of therapeutic antibodies is an exciting challenge that can now make use of refined computational methods, allowing to design antibodies with the highest affinity towards antigens of interest [106], to predict the biochemical and biophysical characteristics of specific sequences, and to determine whether they conform well to antibodies that have already reached clinical stage [83]. Given that the global burden of HPV-associated cancers is unacceptably high, major efforts are required for the effective prevention and treatment of these tumours. A therapy for HPV-associated lesions relying on antibodies would present some advantages over more conventional systems of immunization such as, for example, those based on triggering tumour rejection. Specificity is among the main merits, due to the possibility of inhibiting the activity of oncoproteins that are expressed only in tumour cells. A further benefit is that such a therapy, not based on the need to elicit the host immune response, can also be effective in subjects immunosuppressed by natural or induced causes as co-infections or pharmacological treatments. The extraordinary potential of anti-HPV recombinant antibodies makes them key tools in the global strategy of fighting HPV-associated cancers.

**Author Contributions:** Conceptualization, L.A. and C.A.; methodology, C.A. and L.A.; validation, L.A., C.A., P.D.B. and M.G.D.; formal analysis, C.A.; data curation, L.A., C.A.; writing—original draft preparation, L.A.; writing—review and editing, L.A., C.A., M.G.D., P.D.B., M.V.C.; visualization, C.A.; supervision, L.A. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research received no external funding.

**Conflicts of Interest:** The authors declare no conflict of interest.

#### **References**

