advantages and disadvantages. Therefore, finding a suitable vector to deliver therapeutic genetic material has become a challenge to make gene therapy a viable and better treatment option than conventional methods. Part of the challenge is therapeutic DNA's inability to pass through the cell membrane because of its large size and negative charge. Also, the therapeutic DNA needs to escape the cellular endonucleases and renal clearance. An ideal vector should have enough space to transport large therapeutic genes, high transduction efficiency, and the ability to provide long-term and stable expression, as well as target specific cells, avoid random insertion of the therapeutic gene into the host genome, and infect mitotic as well as postmitotic cells. It should not be immunogenic or pathogenic, should not cause inflammation and should possess the ability to be manufactured on a large scale. Research on developing novel viral vectors is advancing steadily with a special focus on substituting pathogenic genes with therapeutic DNA (66). In fact, non-pathogenic, replication-defective, and humanfriendly viral vectors are being used in more than 70% of the ongoing gene therapy clinical trials worldwide (67). One particularly popular use of viral vectors, such as adenovirus, Seneca Valley virus, poliovirus, vaccinia virus, herpes simplex virus, reovirus, Coxsackievirus, parvovirus, Newcastle disease virus, vesicular stomatitis virus, and measles virus, is in the form of oncolytic viruses (OV). In 2016 alone, more than 40 clinical trials using OV were conducted (68). OV destroy malignant cancer cells by specifically replicating in those cells to effectively lyse them as well as induce a robust antitumor immune response. OV selectively replicate in tumor cells through a variety of methods such as virus-specific receptors on the cells. They can be used to deliver anti-angiogenesis genes, suicide genes, immunostimulatory genes, and DNA encoding small nucleic acids. Apart from carrying immunostimulatory genes, OV can induce an immune response by releasing cell debris and viral antigens (68). Many other innovative approaches are being developed to use viral vectors for treating diseases and disorders. Since Edward Tatum's initial proposal to repurpose viruses for therapeutic gene delivery in 1966, gene therapy has come a long way from the construction of many types of viral vectors to their use in more than 3,000 clinical trials to date (32, 69, 70). However, during this incredible journey with obscure regulations, gene therapy has experienced a few undesired clinical outcomes due to off-target effects, cytotoxicity, viral transmissibility, impurity, and an immune response to the viral vector itself (68). Nonetheless, diseases for which a cure has been attempted include β-thalassemia, Xlinked severe combined immunodeficiency (X-SCID), adenosine deaminase deficiency (ADA-SCID), cystic fibrosis, hemophilia, liver enzyme ornithine transcarbamylase (OTC) deficiency, head and neck cancer, metastatic melanoma, HIV, Leber's congenital amaurosis, Wiskott-Aldrich syndrome (WAS), metachromatic leukodystrophy (MLD), and severe lipoprotein lipase deficiency disorder (LPLD) (52, 71). In fact, the possibilities for gene therapy-mediated treatments are endless because virtually every cell in the human body is a potential target for genetic manipulation. Viruses display specificity in infecting cell types; therefore, viral vectors can be selected based on the type of cell that needs gene delivery. Here, we describe some widely used viral vectors in gene therapy.

### ADENOVIRUS (AV)

AV was the first viral vector developed for gene therapy and was approved for clinical trials in 1990. It was isolated from human adenoid tissue-derived cell cultures for the first time in 1953, hence the term adenovirus, and included in a diverse family of non-enveloped double-stranded DNA (dsDNA) viruses called Adenoviridae (72). According to the Centers for Disease Control and Prevention (CDC), AV rarely causes serious illness and death in healthy individuals but immuno-compromised individuals may develop a wide range of illnesses including the common cold, sore throat, bronchitis, pneumonia, diarrhea, conjunctivitis, fever, and neurologic disease. As of today, there are 57 human AV serotypes isolated and classified into seven categories based on their properties of agglutination (73, 74). AV carries a linear dsDNA ranging from 26 to 45 kb in a medium sized (∼100 nm) non-enveloped icosahedral viral particle composed of penton and hexon subunits. While the hexon subunits form a major part of the viral capsid coat and carry antigenic motifs, the penton subunits constitute fiber and knob domains required for infection (75). The fiber knob domain initiates AV infection by binding to a variety of proteinssuch as MHC-1 α2 subunit, CD46, sialic acid saccharides on glycoproteins, coxsackievirus, and AV receptor (CAR) expressed on cell surface (76). The interaction between arginine-glycine-aspartic acid (RGD) sequence of the fiber penton subunit and αν integrins on the cell surface drives endocytosis of viral particle and completion of viral infection (77–79). This creates broad tissue tropism and a nodal for AV transduction efficiency, giving an opportunity to manipulate binding sites for CAR and other ligands to de-target AV infection, an essential feature of popular viral vectors used in gene therapy. Therefore, since its discovery, AV has been repurposed through the deletion of its pathogenic genes.

Based on the expression of AV genes during infection and multiplication, its genome is organized into early (E1, E2a, E2b, E3, and E4), intermediate (IVA2 and IX), and late genes (L1, L2, L3, L4, and L5). Also, its genome carries non-coding inverted terminal repeat (ITR) sequences, ψ packaging sequences, and many viral RNAs (75, 80, 81). The genome of AV has been manipulated many times to develop safe and efficient vectors for gene therapy applications. The first-generation vectors with a partial deletion of E1 or E3 genes do not replicate or display oncogenicity but can deliver less than an 8 kb gene and display leaky expression of viral proteins, strong immune response, and contamination with replication-competent virus (82). To circumvent this, second-generation vectors were created by deleting E2A, E2B, and E4 from the genome of the firstgeneration AV vectors. However, their production has become complicated, and they do not prevent leaky expression of viral proteins and rapid loss of therapeutic gene expression, and thus have lost enthusiasm for their widespread use in gene therapy (83, 84). The third-generation vectors, otherwise known as gutless or helper-dependent AV vectors, lack all viral genes except the ψ and ITR sequences. They have received special attention because of their capacity to carry larger therapeutic genes (up to 37 kb in size), their ability to display long-term transgene expression, and lesser contamination with replicating virus particles. They are also less immunogenic than first- and second-generation vectors (85). The third-generation vectors were successfully used to express transgenes for about 2 years in animals with no adverse effects (86, 87). Co-transduction of these vectors with Sleeping Beauty transposon along with FLP recombinase was used to insert a FIX gene in the chromosome of dogs suffering from hemophilia B and expressed for up to 960 days (88, 89). Recently, they were successfully used for the long-term expression of a gene encoding an alanine-glyoxylate aminotransferase (AGT) in patients with primary hyperoxaluria type 1 (PH1), a rare kidney disorder that causes recurrent kidney stones (90).

Since AV vectors allow episomal or stable insertion of therapeutic genes, they carry advantages over vectors that integrate into cellular DNA. This provides clinicians an opportunity to offer appropriate treatment for patients with different diseases or disorders. For instance, AV is suitable for treating cancers and offering bioscavenger-mediated short-term protection against nerve gases and other chemical weapons. As depicted in **Figure 2**, we have demonstrated an AV-mediated episomal insertion of PON1, BChE, and PD bioscavenger genes in the liver to express and secrete proteins to detoxify the circulating lethal nerve gases for 10–15 days in mice (13, 20– 22, 36).  For example cancer cells can go undetected by reducing the expression of tumor antigens on their surfaces, inducing immune cell inactivation, and releasing substances in the microenvironment to promote their growth and survival. Therefore, new oncolytic adenoviruses that effectively induce immune response, and specifically target and lyse tumor cells are being created by replacing their native E1A promoter with tumor-specific promoters and genetically modified CAR, a highly expressed AV receptor in tumor cells (68, 91). For example, the CV706 and OBP-30 AV vectors carry the viral E1A gene under prostate cancer-specific antigen promoter and telomerase reverse transcriptase promoter, respectively (92). Other engineered oncolytic adenoviruses target the components of tumor cells and their microenvironment and inhibit their proliferation by expressing antibodies, relaxin, hyaluronidase, and inhibitors of metalloproteinases to hinder angiogenesis and proper function of the extracellular matrix (91, 93). Oncolytic adenoviral vectors that induce autophagy-related immunogenic cell death were also developed to treat cancer (94). A novel oncolytic AV vector expressing an interfering long non-coding RNA (lncRNA) to inhibit 12 oncogenic miRNAs has been constructed in order to perform selective killing of tumor cells (95). AV vectors carrying complementary sequences of liver-specific miRNA-122a incorporated into 3'-UTR of E2A, E4, or pIX to reduce the leaky expression of viral genes and hepatotoxicity were developed (96). In addition, AV vectors with E1A carrying mutations complementary to retinoblastoma (RB) or p53 gene mutations

FIGURE 2 | Mechanism of adenovirus-mediated delivery of a therapeutic DNA. Upon infection, adenovirus delivers the encapsulated therapeutic DNA into the cytoplasm of the target cells. Various stages of viral gene delivery, viz cell attachment, internalization, endocytosis, uncoating, transcription and translation of the therapeutic protein, are shown.

in tumor cells that can specifically replicate and lyse tumor cells were created (92).

Despite many technological improvements made in vector design and production, there are still certain issues that have to be addressed for better clinical outcome. For example, infecting target cells with the optimal amount of highly purified AV particles is critical for the successful insertion of a therapeutic gene. Recently, it was shown that 1010-1012 AV particles per patient are required for a successful Ebola vaccination (97). Production of such high titer virus with no or minimal empty vector contamination is still a formidable challenge. Also, high prevalence of anti-AV vector immunity in the human population and differential expression of CAR and other receptor proteins on target cells have been serious issues in clinical trials (98, 99). For example, the transduction efficiency of the widely used AV serotype 5 in gene therapy is dampened by the prevalence of neutralizing antibodies in the human population (100, 101). An estimated 80% of the human population is believed to carry antibodies against AV serotype 5, resulting in a significant transduction deficiency and stimulation of inflammatory shock (102). There has been a positive correlation between body fat and the presence of circulating antibodies against AV serotype 36 in humans (103). In addition, during systemic administration, the tendency of AV vectors to undergo sequestration in the liver has prevented efficient transgene transduction and displayed severe hepatotoxicity, even causing the death of a clinical trial participant (104, 105). This was due to the binding of blood coagulation factor FV and FX to the hyper variable region (HPV) of AV hexon subunit (106, 107). Therefore, mutating the HPV site in such a way that it neither activates complementary pathway nor interacts with FX could be an ideal way to resolve the liver sequestration issue. Attempts are being made to improve the safety of AV vectors by treating with chemicals and developing chimeric and hybrid vectors to minimize inflammation and immunogenicity (108, 109). For example, the chimeric AV serotype 5 vector carrying receptor-binding epitopes derived from other human AV serotype 3, serotype 35, and serotype 43 displayed low seroprevalence and low affinity for CAR (110, 111). Similarly, the chimeric human AV serotype 5/3, consisting of receptor epitopes derived from serotype 3 and 5, showed high binding affinity for CD46, an AV receptor commonly expressed on many solid tumors. It was thus found to be particularly useful in targeting solid tumors (112, 113). Another CD46-targeted chimeric AV vector derived from human serotype 5 and 35 has been shown to be suitable for transducing vascular smooth muscle cells, treating colorectal cancers, and ischemic wounds as well as manipulating T-cells (114–117). Novel chimeric AV vectors developed from AV serotypes 5 and 11 and 3 and 11 were found very effective in exclusively targeting glioma and colon cancer cells, respectively (118, 119). Other types of chimeric vectors were also derived entirely from low prevalent human and non-human AV serotypes such as human AV serotype 26, canine AV serotype 2, and chimpanzee serotype 3. For example, the chimeric AV vector developed from human AV serotype 26 and chimpanzee AV serotype 5 has been used successfully for Ebola vaccination in animal models (120, 121). A novel hybrid vector developed from AV serotype 5 and alpha virus was found very useful for the expression of transgenes in malignant hematopoietic cells (122). Many laboratories have developed a library of AV vectors that carry random-peptides on their fiber knobs in order to overcome the paucity of cancerspecific ligands (123–125). This resulted in the generation of many AV vectors that are specific to prostate and pancreatic cancer as well as glioma (123, 125–128). One such vector carrying pancreatic cancer-targeting ligand has shown strong oncolytic effect in primary pancreatic neuroendocrine tumors and appears promising as a next-generation therapy (129). Given the advancements made in developing safe and efficient AV vectors, their choice for delivering therapeutic genes has become apparent in clinical trials.

#### ADENO-ASSOCIATED VIRUS (AAV)

AAV is yet another popular viral vector used in gene therapy. This small microbe was first isolated as a contaminant in the simian adenovirus preparation and then named adeno-associated virus (AAV) by the Bob Atchison group at Pittsburgh University and the Wallace Rowe group at the NIH (130, 131) and later found in a wide range of animal samples including human, nonhuman primates, avian, bovine, reptiles, pigs, sea lions, bats, and caprine samples. The 4.7-kb-long single-stranded DNA (ssDNA) packed inside a non-enveloped viral particle carries p5, p19, and p40 promoters as well as rep and cap genes flanked by two 145 nucleotide-long inverted terminal repeats (ITR) and no polymerase gene (132, 133). While ITRs having palindromic sequences base pair to allow synthesis of cDNA, both rep and cap genes undergo alternate splicing to express replication proteins (Rep78, Rep68, Rep52, and Rep40), capsid or virion proteins (VP1, VP2, and VP3), and an assembly activating protein (AAP), respectively (134). Being a non-structural protein, AAP assists virion proteins in capsid formation (135). VP1, VP2, and VP3 expressed from p40 promoter at a ratio of 1:1:10 form the outer capsid of the virion. These capsid proteins carry phospholipase domain to protect virions from the onslaught of intracellular protease system (136). Unlike other viruses, AAV requires a few other helper proteins, agents or viruses such as AV, herpes simplex virus type I/II, pseudorabies virus, cytomegalovirus, genotoxic agents, UV radiation, or hydroxyurea to infect cells and complete replication (137). AAV can also be generated by providing the missing genes E1a, E1b, E2a, E4orf6, and VA that are needed for viral infection. These genes are often cloned in pXX6 helper plasmid and used to co-transfect HEK293 cells along with AAV expression plasmid (rep-cap plasmid) to produce AAV (134, 138). Therapeutic genes are cloned in the AAV expression plasmid carrying ITR sequences, and their size can be increased by cotransfecting another plasmid carrying repcap genes or by generating virus in rep-cap stable cells. Since the formation of dsDNA from its ssDNA is the rate-limiting step of viral infection, gene delivery, and expression in the target cells, a self-complementary viral dsDNA (scAAV) is developed; however, it reduces the capacity of AAV vectors to deliver a therapeutic gene (139, 140). AAV inserts a therapeutic gene in the genome of target cells to provide long-term transgene expression. For instance, the gene expressing FIX blood coagulation factor in one individual of a cohort persisted for more than 10 years during a clinical trial (141). AAV inserts a therapeutic gene in the host genome at a specific location on the q arm of chromosome 19 (142, 143). Despite having no large homology regions, more than 70% of the transgene integration events occur within this site; however, the underlying mechanism remains unknown. But AAV lacking its rep-cap genes can deliver a therapeutic gene in the episomal form without inserting into the genome of the target cells. The therapeutic gene in the episomal form can develop into a chromatin-like structure and remain quiescent in cells for months to years without damaging the patient's body. Recently, we have used AAV vectors to make episomal insertion and expression of a bioscavenger gene in the liver cells for about 6 months (unpublished results). This allows clinicians to apply AAV-mediated gene therapy to treat a wide variety of diseases or disorders.

AAV displays broad tropism but requires the expression of heparin sulfate proteoglycan, αvβ5 integrin, α5β1 integrin, fibroblast growth factor receptor 1, platelet-derived growth factor receptor, hepatocyte growth factor receptor, epidermal growth factor receptor, laminin receptor, and sialic acid moieties on the surface of target cells for efficient transduction and delivery of a therapeutic gene. Recently, AAVR has been identified as a universal host cell receptor for AAV infections (144). Although every serotype has the ability to infect cells, transport to nucleus, uncoat, and insert its genome into the host's chromosome or leave in the episomal form, not all 13 AAV serotypes isolated to date use the same receptor repertoire on host cell surface for infection (145, 146). This makes AAV a very useful system for a specific cell or tissue type transduction. For example, AAV1 displays high transduction efficiency of muscles, neurons, heart, and retinal pigment epithelium. AAV2 has been shown to infect many types of cancer cells, neurons, kidney, retinal pigment epithelium, and photoreceptor cells. AAV2 is the only serotype that can infect and delivery a therapeutic gene to kidney. AAV4 and AAV5 serotypes infect retina and retinal pigment epithelium, respectively. While AAV6 displays strong tropism for heart, AAV7 has some bias for liver (147). AAV6 is also effective in infecting airway epithelial cells (148). AAV8 and AAV9 have displayed successful infection of lymphoma and HPV tumors, respectively (149). AAV8 is the only serotype that infects pancreas, and it was extensively used to express a therapeutic FIX in the liver to treat hemophilia in clinical trials (150). AAV tropism was further refined by mixing the capsid proteins of one serotype with the genome of another serotype. For example, AAV2/5 serotype, which transduces neurons more efficiently than the parental AAV2, was generated by packaging AAV2 genome in AAV5 capsid proteins. Another example, the pseudotyped AAV8 and AAV9, can cross the endothelial barrier of blood vessels to target muscles (66). For increasing transduction efficiency, hybrid AAV serotypes were also generated by mixing the capsid proteins of multiple serotypes with the genome of another serotype. For example, AAV-DJ serotype that consists of a hybrid capsid is generated by mixing the capsid proteins of eight different AAV serotypes. This made AAV-DJ to display higher transduction efficiency than any other wild type serotype in vitro and high infectivity of a broad range of tissue in vivo. Its mutant, AAV-DJ8 serotype, displays high infectivity of brain. AAVHSC, a new class of genetic vector isolated from hematopoietic stem cells, has been shown to be ideal for manipulating stem cells (151). Since more than 50% of the adult human population carries AAV neutralizing antibodies, a wide range of mosaic or hybrid vectors were generated by engineering and de novo shuffling of capsid proteins (152, 153). For example, the AAV2.5 hybrid vector generated by combining the muscle tropism determinants of AAV1 with parental AAV2 displays immune evasion of their neutralizing antibodies (154). The other hybrid vectors AAV6.2, AAV2i8, AAVrh10, and AAVrh32.33 were found beneficial for intravenous delivery, reduction of liver sequestration, and T-cell response in the clinic, respectively (138, 155–157). Since CpG motifs are responsible for immune response and failure of many clinical trials, AAV vectors were further refined by deleting CpG motifs, known ligands of Toll-like receptor 9 (TLR9), to reduce immune response for maximal expression of a transgene in clinical trials (158). Cre-recombination-based AAV variants are also developed to allow efficient transgene expression in the central nervous system, muscle, and liver (159, 160). Also, the AAV-CRISPR/Cas9 system has been developed to perform in vivo genome editing and broaden therapeutic horizons (161).

### HERPES SIMPLEX VIRUS (HSV)

Herpes simplex viruses are believed to have tremendous potential as a preventative and therapeutic vaccine against cancer and other diseases because of their ability to evade the immune system and circulating anti-viral drugs, deliver multiple genes, infect a wide variety of cells, pose low risk of adverse health effects, and multiply specifically in tumor cells. They are large enveloped viruses that carry a linear dsDNA of 120–240 kb and infect reptiles, birds, fish, amphibians, and mammals. There are eight known human herpesviruses: herpes simplex virus-1 (HSV-1), herpes simplex virus-2 (HSV-2), human cytomegalovirus (HCMV), varicella-zoster virus (VZV), Epstein-Barr virus (EBV), Kaposi's sarcoma-associated herpesvirus (KSHV), and human herpesviruses 6 and 7 grouped under alpha, beta, and gamma genera. Though they share common virion structure and replication cycle, and many other biological properties, there is a significant difference in their tropism, infection, and clinical manifestations. Some of their genes show homology with regions of human chromosomes. Here, we delve into HSV that infects ∼60% of the human population worldwide and mainly transmits through contact, especially oral-oral contact (162). After infecting oral or genital epithelial cells, HSV enters neurons to establish lifelong latent infection and reactivates periodically causing fever, blisters, cold sores, genital herpes stromal keratitis, blindness, cancer, and encephalitis. Both HSV-1 and HSV-2 carry envelope and sub-envelope structures called tegument and a regular icosahedral capsid consisting of a relatively large dsDNA of 153 kb encoding ∼200 proteins (163). Nearly half of the total 84 genes present in the HSV genome encode proteins required for virus replication, and many were found unnecessary for delivering therapeutic genes. Several genes involved in virulence and immune evasion, and those considered non-essential for viral life cycle in vivo were also identified. HSV-1 is relatively less pathogenic than HSV-2 and is, therefore, ideal for vector development and gene therapy (164). HSV infects cells by using its envelope glycoprotein B, glycoprotein C, and glycoprotein D to bind cell surface particles and transmembrane receptors such as heparin sulfate, herpesvirus entry mediator (HVEM), nectin-1, and 3-O sulfated heparin sulfate. While the nectin receptors provide a strong point of viral interaction with the host cell, the other viral envelope proteins, especially glycoprotein B, glycoprotein D, glycoprotein H, glycoprotein L, and HVEM, create an entry pore for the viral capsid. The viral capsid enters through capsid pore and travels through the cytoplasm to the nucleus in order to inject its DNA content. HSV evades the immune system by secreting its immediate-early protein, ICP47, and inducing a transporter associated antigen processing (TAP) protein to block MHC class I antigen presentation on the cell surface. HSV-1 infects many types of mitotic and post-mitotic cells including neurons (36). After infection, HSV induces the expression of the virion host shutoff protein (VHS or UL41) to inhibit protein synthesis by degrading the host mRNA. This makes way for viral replication and lysis of the host cells.

The HSV genome carries immediate-early, early, and late genes for replication and allows creation of replicationcompetent, replication-incompetent, and helper-dependent vectors, or amplicon vectors, for preclinical and clinical studies. The replication-competent vectors have the capacity to deliver transgenes up to 50 kb in size or the entire locus since treatment of certain diseases requires huge therapeutic gene cassettes carrying complex regulatory elements. These vectors can replicate selectively in cancer cells and have less virulence because of deleted genes. They do not insert transgenes in the host chromosomes and are therefore used primarily as oncolytic viruses to treat glioma, melanoma and ovarian cancers and to stimulate an immune response (66). They are further refined by using tumor specific promoters to express viral genes and target tumor-specific receptors. These vectors with robust replication capacity are believed to enhance intramural vector distribution and lyse tumor cells very effectively. These vectors are generally constructed by homologous recombination in eukaryotic cells by co-transfecting the viral genome and a plasmid carrying the therapeutic gene flanked by the sequences homologous to the target locus on viral genome to undergo recombination. The replication-incompetent vectors are created by either mutating or deleting several immediate early genes including ICP4 and ICP27 that are essential for replication and, therefore, can grow Goswami et al. Gene Therapy in Modern Medicine

only in specifically designed cell lines complemented transiently. For example, Vero-7b cell line is capable of providing in trans the proteins encoded by deleted viral genes (165). They are safe and non-inflammatory advanced vector platforms known to persist and express in the nerve cells for life and therefore used to treat neuropathic, inflammatory, and cancer-associated pain (166–168). The helper-dependent HSV vectors, or amplicon vectors, carry deletions in one or more non-essential genes and retain the ability to replicate in vitro but are compromised in vivo in a context-dependent manner (169, 170). These viruses are the same as wild type HSV, with plasmids containing a packaging signal and the gene of interest. Amplicons have the ability to accommodate a very large therapeutic sequence up to 100 kb in size but have production and stability issues. The replication-incompetent vectors and amplicons have been used to express genes in the nervous system, muscle, heart, and liver.

 Unlike all other RNA viruses, these viruses are capable of reverse transcribing their genetic blueprint of positive, single-stranded RNA into dsDNA, and inserting it into the host cell genome. RVs carry a non-covalently linked dimers (two copies) of RNA genetic material probably as a fail-safe mechanism for producing genomic DNA and increasing viral RNA diversity due to interstrand recombination (171). Thus, RNA dimerization has been viewed as a prerequisite for RV genome encapsidation and life cycle. With restricted vertebrate hosts, these viruses are divided into exogenous retroviruses (XRV) and endogenous retroviruses (ERV). While the XRVs transmit horizontally among hosts, the ERVs inherit vertically in the genome of their hosts (172). By scattering all over chromosomes and comprising nearly eight percent of the human genome, the ERVs are thought to be relics of ancient retroviral germline infections and believed to play a role of friend or foe in human life (173–175). The two most common types of retroviruses are gammaretrovirus and lentivirus, which are derived from MLV and HIV-1, respectively. The genome of gammaretrovirus has three essential genes, gag, pol and env, and is flanked on both sides by long terminal repeats (LTRs). Gag inserts viral genome mRNA into virions when assembling, pol is the reverse transcriptase, integrase, and protease encoding gene, and env encodes the surface and the transmembrane glycoprotein. Also, RV genome carries a cis-acting ψ packaging element that involves in regulating the essential process of packaging the RV mRNA into viral capsid during replication. In addition, RV genome carries RNA dimerization signal element. With U3, R, U5 elements, the LTRs display promoter/enhancer activity for gene transcriptional regulation. RVs use their envelope proteins to bind a variety of receptor molecules such as murine cationic amino acid transporter (mCAT), a sodium-dependent Pi transporter (PiT2), xenotropic and polytropic retrovirus receptor 1 (XPR1), CD4, CD46, CD150, and the RD114-and-D-typeretrovirus/alanine-serine-cysteine transporter 2 (RDR/ASCT2) expressed on different cell surfaces to initiate infection, a critical step in determining the target cell tropism of the virus. This leads to a conformational change in the envelope proteins, leading to the entry of virus into the cytoplasm via fusion or endocytosis. With the help of the host cell proteins, the endosome travels through cytoplasm to eject its RNA. After the RNA reverse transcription takes place, viral DNA is integrated into the host cell chromatin, transcribed into RNA with 5' Cap and 3' poly(A) tail, and translated into viral proteins that assemble and bud from the plasma membrane to complete the life cycle with extracellular maturation (171). The matured RVs can infect a wide variety of somatic cells including embryonic stem cells, hematopoietic and neural stem cells. With active nuclear elements, these vectors can transduce therapeutic genes into proliferating cells only and are, therefore, ideal for targeting specifically cancer cells. A downside to gammaretrovirus is that it has broad species specificity, leaving the possibility of transducing undesired cells, faulty reverse transcription, intracellular restriction factors, and risk of insertional mutagenesis. The major difference between gammaretrovirus and lentivirus is that lentivirus can infect postmitotic cells. It requires four plasmids for production: the gag and pol plasmid, the rev plasmid to transport mRNA into the cytoplasm, VSV-G for membrane fusion and the gene of interest. Other retroviruses require three plasmids: the gag and pol plasmid, the VSV-G plasmid and the gene of interest. Transient or stable co-expression of all these plasmids in HEK293T packaging cell lines produces RV vector particles carrying no replication-competent virions that are essential for research and therapeutic purposes. Using these cell lines, methods to produce clinical grade RV particles at a concentration of 106 to107 /ml are optimized (176). As gag/pol and env expression constructs carry no ψ packaging and RNA dimerization element, viral structural proteins only recognize the ψ-containing RV vector construct resulting in a preferential packaging of RV vector genomes into infectious particles. After entry of the particle into the host cell, only the RNA of the RV vector construct is reverse transcribed and stably integrated into the host genome. This method prevents generation of replication competent retroviral vector progeny during therapeutic viral particles production. Lentivirus has been used to treat X-SCID, cancers and monogenic diseases. For example, self-inactivating lentiviral vectors can engineer T cells with receptors to better target tumors when treating cancer. Recently, we have successfully used lentiviral vectors to deliver an anti-angiogenic Kininogen gene to budding blood vessels (177, 178). There have been no reports of significant adverse effects from the lentivirus (37).

Some advantages of using retroviruses are that they can accommodate a 9–12-kb-large insert size for the gene of interest and produce high titers. The most significant disadvantages are lack of cell specificity and the possibility of insertional mutagenesis (18). The enzyme "integrase" inserts copies of the retroviral genome into the host cell chromosomes but there is a risk of inserting the genome copy into an unfavorable location such as a tumor suppressor gene or an oncogene, which would lead to uncontrolled cell division (36). It is critical to evaluate the risk of insertional mutagenesis for each retroviral vector. Gammaretroviral vectors have a tendency to integrate near gene regulatory regions, which can pose a significant risk. For example, patients in a cohort of 20 died due to leucosis development in a clinical trial (179). On the other hand, lentiviral vectors tend to integrate into the body of genes, leading to lower risk of genotoxicity (52). A possible step to address this issue would be to use self-inactivating retroviral vectors that are transcriptionally inactive. Since mature T cells are relatively resistant to oncogenic transformation by RV, developing T cellbased therapeutic approaches to treat cancer and other diseases would be another approach to avoid insertional mutagenesis. Recently, a non-integrating RV-based CRISPR/Cas9 vectors have been created for targeted gene knockout (180). Creating such vectors to target specific genes would help developing therapeutic approaches without insertional mutagenesis issue. Renal fibrosis was treated by using high-fidelity RV-based CRISPR/Cas9 vectors (181). Development of similar vectors would not only address insertional mutagenesis issue but also radically transform basic and applied biomedical research. Also, using AAV vector which inserts a therapeutic gene selectively into known chromosome 19 sequences would be another possibility. Using zinc finger nucleases or including certain sequences such as the β-globin locus control region to direct the site of integration to specific chromosomal sites is yet another way to minimize the risks. However, further studies are needed to address this issue by designing specific vectors and understanding the frequency of insertional mutagenesis, and role of other factors involved. Insertional mutagenesis is an issue that will likely be solved in the coming years. Until then, the use of retroviruses remains a concern. Nonetheless, over 500 gene therapy clinical trials have been conducted using retrovirus to date.

#### GENE THERAPY DRUGS IN THE MARKET

Despite many technological challenges and barriers, more than a dozen gene therapy-based drugs have entered the world pharmaceutical market to date (**Figure 3**). The first gene therapy drug, GendicineTM, was developed by Shenzhen SiBiono GeneTech for the treatment of patients with tumors carrying a mutated p53 gene, a common cause for more than 50% of all types of human cancers. The State Food Drug Administration of China approved GendicineTM for the treatment of head and neck squamous cell carcinoma on October 16, 2003 (182, 183). However, the USFDA has turned down Introgen's Advexin, another AV-based viral drug that uses p53 due to concerns about the safety of the AV vectors after Jesse Gelsinger died in 1999 while participating in a clinical trial but no information is available about the submission of GendicineTM clinical data for approval from the USFDA to date. GendicineTM, a replication defective AV loaded with wild-type p53 gene, is given to patients by less invasive intramural injections and or intracavity infusions. According to the manufacturer, a single dose of this viral drug, costing less than US \$400, is given to patients once a week for 8 weeks as a cure. After injection, the therapeutic activity of p53 activated by target tissue cellular stress induces cellcycle arrest, DNA repair, apoptosis, senescence, and autophagy to cause tumor growth regression. GendicineTM has been given to more than 30,000 cancer patients, and it has displayed an exemplary safety record with no significant side effects to date (14). According to the manufacturer, GendicineTM has shown a higher response rate when combined with chemotherapy and radiotherapy in comparison with standard therapies alone. Because GendicineTM is injected directly into tumors and becomes useless for treating tumors neither detectable nor accessible, other advanced replication-defective AV-based drugs, such as Advexin and SCH-58500, that carry wild type p53 gene were developed to target all tumors in the patient's body in an intravenous injection; however, neither Advexin and SCH-58500 has entered the pharmaceutical market to date. However, OncorineTM, another replication defective AVbased drug that carries p53 gene to cure head and neck cancer, made it to the Chinese pharmaceutical market in 2005. According to the manufacturer, Shanghai Sunway Biotech Co., the curative effective of OncorineTM combined with chemotherapy is superior to chemotherapy alone with a good safety profile. Since low transduction is a major issue with these approved replication defective AV drugs, more advanced tumorspecific p53-expressing conditionally replicating AV vectors such as ONYX 015, AdDelta24-p53, SG600-p53, H101, and OBP-702 have been developed but none of them is approved for cancer treatment to date. As many clinicians prefer cancer management rather than a cure due to the complex nature of the disease, the future of oncolytic viral therapy demands further advancement in vector design and discovery of appropriate therapeutic genes for better treatment.

The next advanced gene therapy drug, Rexin-GTM, a chimeric retrovector loaded with a cytocidal dominant negative cyclin G1 gene to target and kill solid tumors, was approved by the Philippines FDA in 2005. Rexin-GTM developed by Epeius Biotechnologies Corporation was designated by the US FDA as an orphan drug for pancreatic cancer. After intravenous injection, this viral drug carrying a motif derived from von Willebrand coagulation factor (vWF) on its surface selectively binds receptors and collagenous proteins exposed heavily in tumor microenvironment in order to fuse, enter, uncoat, and insert its genetic material randomly in the chromosomes of the actively dividing tumor cells only (184). Recent clinical studies confirmed its safety, anti-tumor activity, and potential to increase survival time and survival rate of patients. Recently, another retrovirus-based drug, StrimvelisTM, was approved in Europe to treat an ultra-rare immunodeficiency syndrome, ADA-SCID, or Bubble Boy Syndrome, a fatal and life-threatening disease due to lymphopenia, and recurrent and opportunistic infections. A bone marrow transplant from a young child donor with matched leukocyte antigen is the recommended treatment for ADA-SCID patients, but the availability of a suitable donor is challenging. Therefore, StrimvelisTM is designed and developed to offer ex vivo gene therapy and involves use of RV to insert copies of the ADA gene into the chromosomes of stem cells extracted from the

bone marrow of patients. The stem cells carrying the ADA gene are then reintroduced into the patients whose bodies can express protein to repair their immune system on their own. This drug, with a list price of \$714,000, is available for ADA-SCID patients without a donor that has matched human leukocyte antigen (HLA). Clinical studies revealed a 100% remission rate for StrimvelisTM (**Table 2**). Nonetheless, there is now a push toward using self-inactivating retroviral vectors that have less risk of insertional mutagenesis, especially self-inactivating HIV-1-based lentiviral vectors (185). A few months ago, the FDA approved KymriahTM, a lentivirus-based chimeric antigen receptor T cell (CAR-T) therapy for acute lymphoblastic leukemia (186). The underlying mechanism of this cancer type disease development still remains unknown, but patients carry abnormal lymphocytes in many of their body parts. KymriahTM was developed by Novartis in collaboration with the University of Pennsylvania to treat patients with non-Hodgkin lymphoma (NHL) and Bcell acute lymphoblastic leukemia (ALL). KymriahTM is a novel immunocellular therapy that uses a patient's own reprogrammed T cells with a transgene encoding CAR to identify and eliminate CD19-expressing malignant and non-malignant cells; overall remission rate with the therapy is 83% (**Table 2**). The autologous peripheral blood T cells are reprogrammed to carry intracellular 4-1BB and CD3-zeta costimulatory domains fused with a murine single-chain antibody fragment in its CAR to recognize CD19 increase, cellular expansion, and persistence. YescartaTM is another retrovirus-based CAR-T cell immunotherapy developed by Kite, a Gilead company, and approved by the FDA in 2017. This breakthrough hematologic cancer drug is a customized treatment generated using an NHL patient's own T-cells to help fight lymphoma. The patient's T-cells are collected and genetically modified using a RV to generate a CAR consisting of anti-CD19 CAR-T cells linked to CD28 and CD3-zeta costimulatory domains.  According to the manufacturer, ∼7,500 patients with refractory DLBCL are qualified to receive Yescarta treatment in the USA alone. With a list price of \$373,000 in the USA, Yescarta is believed to get approval for the European market in the near future. Zalmoxis is another T-cell based medicine designated an orphan drug and approved by the EMA for treating certain leukaemias and lymphomas. This is used as an add-on treatment in patients who received hematopoietic a stem cell transplant (HSCT) from a partially matched donor to restore the immune system. This is a somatic cell therapy product consisting of T-cells that are genetically modified using a RV to express 1NGFR and HSV-TK Mut2 suicide genes. This drug sometimes attacks the patient's body by causing graft-vs.-host disease, but the suicide gene makes these T cells become susceptible to ganciclovir or valganciclovir medicine commonly given to treat and prevent further disease development.

NeovasculgenTM, a non-viral first-in-class gene therapy drug developed by the Russian Human Stem Cells Institute, has been available since 2012 for the treatment of atherosclerotic peripheral arterial disease (PAD) including critical limb ischemia. Intramuscular injection of a single dose of this drug, costing less than \$50, delivers a plasmid DNA-carrying VEGF gene cloned under a CMV promoter and stimulates angiogenesis and blood supply to decrease the risk of amputation and death in patients suffering from PAD. A recent post-marketing surveillance study revealed a significant increase in pain-free walking distance by PAD patients and confirmed the therapeutic efficacy of this drug (56, 57). Recently, Spinraza R has become the first approved treatment for the rare and often fatal disease spinal muscular atrophy (SMA). SMA patients suffer muscle strength affecting their ability to sit, stand, and breathe. SMA is caused by widespread splicing defects due to mutations in survival motor neuron 1 (SMN1), a ubiquitously expressed cytoplasmic and nuclear protein involved in transcriptional regulation, biogenesis of small ribonucleoproteins, telomerase regeneration, and intracellular trafficking. Although the SMA patients carry its paralog SMN2, low-level expression due to alternative pre-mRNA splicing appears responsible for this disease development. Therefore, Spinraza carrying SMN2 directed antisense oligonucleotides is designed and developed to resurrect normal SMN2 protein expression in SMA patients. This non-viral drug developed by Biogen Inc. has received orphan drug status and was approved for treating all types of SMA in the USA, Canada, Japan, the European Union, Switzerland, Australia, South Korea, Chile, and Brazil. Spinraza solution upon intravenous and or intrathecal administration enters many cells in the body and induces SMN2 protein expression. According to the manufacturer, this medicine, with a list price of \$125,000 per injection, costs \$750,000 per year for the first year and hundreds of thousands of dollars every year for the rest of patient's life. An AAV-mediated drug designed to express SMN1 protein in patients was developed by a Novartis company, AveXis Inc., and may become available for the treatment of SMA in the near future.

The first AAV1-based drug, Alipogene tiparvovec, or GlyberaTM, was approved by the EMA to treat LPLD, a rare monogenic genetic disorder characterized by accumulation of triglycerides in plasma due to mutations in LPL. GlyberaTM carrying correct copies of LPL was developed by UniQure Inc., and widely heralded as the "the first gene therapy" in the Western world (**Figure 3**). However, only one or two people in every one million are estimated to carry LPLD, and despite Glybera' s demonstrated potential in curing LPLD, it was withdrawn from the market due to low patient demand. Recently, another AAV-based drug has entered the pharmaceutical market to treat Leber congenital amaurosis, an inherited visual dysfunction characterized by pigmented retina, wandering nystagmus, and amaurotic pupils and caused by a mutation in the RPE65 (187). Upon completion of the late-stage clinical trials, this AAV2 based voretigene neparvovec, LuxturnaTM, has been designated by the FDA as a breakthrough therapy and an orphan drug for the treatment of choroideremia. Clinical trials revealed a remarkable improvement in the patients' ability to see in dim light (188). According to the manufacturer, Spark Therapeutics, Inc., Philadelphia, USA, LuxturnaTM has successfully cured one blind America's Got Talent semifinalist, Christian Guardino. Recently, LuxturnaTM has become the first viral-based drug approved by the FDA to treat blindness. LuxturnaTM, loaded with wildtype RPE65, will be given to patients with confirmed biallelic RPE65 mutation-associated retinal dystrophy to restore their vision within a few months. Since LuxturnaTM comes with a record sticker price, the manufacturing company offers an outcome-based rebate arrangement with a long-term durability measure and payment option over multiple years. Another AAVbased drug is poised to enter the pharmaceutical market in the near future to treat choroideremia, an X-linked inherited retinal dystrophy that causes night blindness and a constricted visual field.

Recently, the USFDA approved an HSV-based drug called T-VEC (ImlygicTM) Talimogene Laherparepvec, developed by BioVex Inc., and now acquired by Amgen for melanoma treatment. T-VEC directly kills metastatic melanoma cells and enhances the immune response against them. According to the manufacturer, this advanced oncolytic virus replicates in the tumor cells and synthesizes granulocyte-macrophage colony stimulating factor (GM-CSF), resulting in tumor-lysis and release of tumor antigen, which can then trigger an immune response. The target areas include cutaneous, subcutaneous and nodal lesions. ImlygicTM also serves as an in-situ vaccine (189). The T-VEC treatment course involves a series of HSV injections into the melanoma lesions for 6 months for a complete cure. T-VEC was approved also in Europe and Australia for melanoma treatment. G471 or DS-1647 is a third generation oncolytic HSV developed by Daiichi-Sankyo Ltd., Japan, and Professor Tomoki Todo at the University of Tokyo for the treatment of malignant glioma. This has shown excellent safety and efficacy in treating glioma in preclinical and clinical studies and has been designated as an orphan drug and "Sakigake," or ahead of the world, by the Ministry of Health, Labor and Welfare of Japan (190). However, this drug is not available for the treatment of cancer patients to date. In addition, a few more drugs are available in the market for treating different diseases (**Table 2**).

### GENE THERAPY DRUGS IN CLINICAL TRIALS

The world's first gene therapy clinical study was conducted to test a viral-mediated drug at the NIH in 1989, and now 3704 gene therapy studies from 204 countries are listed in the US Government's clinical trials database to date (**Figure 4A**). More than 50% of them are being conducted in the USA alone. Recently, the US government has removed NIH special oversight rules on gene therapy studies, and the USFDA has decided to consider gene therapy drugs like other medications for approval in order to make gene therapy a therapeutic reality for patients. These clinical studies are testing both viral and non-viral gene therapy drugs to find cures for a wide variety of human diseases, disorders, and infectious diseases. While the majority of these clinical studies are focused on treating cancer, and immune and digestive diseases, skin diseases are yet to receive momentum (**Figure 4B**). These ongoing gene therapy clinical studies are delivering a wide variety of therapeutic cytotoxic/suicide, tumor suppressor, vaccine antigen, cytokine, receptor, replication inhibitor, and anti-angiogenic genes. Some of the therapeutic genes, vectors, targeted diseases, and their manufacturers are mentioned in **Table 1**. A large number of nonviral vectors are being used to deliver these therapeutic genes, but viruses dominate as successful vectors in the ongoing clinical studies. The most popular viral vectors being used in clinical studies are AV, AAV, HSV, and RV.

#### AV-MEDIATED GENE THERAPIES IN CLINICAL TRIALS

Both AV and RV vectors are being used in more than 50% of the ongoing viral-mediated clinical studies (**Figure 4C**). The main focus of these are on vaccination and oncolytic therapies. For example, an AV-mediated Theragene (Ad5 yCD/mutTKSR39rep-ADP) delivers a double suicide gene to target stage III pancreatic cancer. AV is being used to deliver the p53 gene in phase II trials to treat recurrent ovarian epithelial, fallopian tube, and primary peritoneal cancer as well as hepatocellular carcinoma (NCT02435186). Also, AV vectors are being used to deliver anti-angiogenic and immunostimulatory genes to treat prostate cancer and malignant pleural mesothelioma (NCT02555397 and NCT01119664). A significant antitumor activity has been demonstrated in phase I-III clinical trials when an AV-based Onyx-015 that undergoes replication selectively in tumors was applied in combination with chemotherapy (14, 191). Vaccination using AV, along with other viruses such as the modified vaccinia Ankara virus (MVA), retrovirus, Sendai virus, and vaccinia virus, is being tested in many clinical trials. AV vectors are also being tested in delivering therapeutic genes for treating malaria, anthrax, HIV, influenza, hepatitis B and C, and severe hemophilia, as well as cardiovascular and many more diseases. AV vectors carrying site-specific endonucleases are being used to edit the CCR5 gene in hematopoietic stem or progenitor cells in AIDS clinical trials (192). The lack of functional dendritic cells in the brain has been attributed to the growth of one of the most aggressive and malignant tumors called gliomas. AV vectors are being used to empower the immune system by expressing the HSV-1 derived thymidine kinase (HSV-1 TK) and cytokine fms-like tyrosine kinase 3 ligand (Flt3L) in the brain. While HSV-1 TK converts ganciclovir into phospho-ganciclovir, a toxic compound to dividing glioma cells, Flt3L differentiates precursors into dendritic cells and acts as a chemokine for dendritic cells resulting into killing of glioma cells and release of tumor antigens in the tumor microenvironment. This follows release of HMGB1, a TLR2 agonist that activates dendritic cells and stimulates dendritic cells loaded with glioma antigens to migrate to the cervical lymph nodes to prime a systemic CD8+ T cytotoxic killing of glioma cells without causing brain toxicity and autoimmunity (193). The median survival of glioma patient is under 2 years and the ongoing clinical trials with DNX-2401, a replication-competent oncolytic AV capable of infecting and killing glioma cells by stimulating an anti-tumor immune response revealed favorable safety profile and prolonged survival of glioma patients (194, 195). Enadenotucirev, a non-natural chimeric oncolytic AV that can retain anti-tumor activity despite intravenous delivery, showed a predictable and manageable safety profile in several advanced cancer patients in phase I clinical studies (196). With encouraging clinical outcome being observed in a large number of ongoing clinical trials, especially in treating cancer, AV-mediated gene therapy is anticipated to make a significant impact on eradicating cancer in the near future.

Although the AV-mediated gene therapy carries a unique advantage over other systems, several concerns must be addressed to offer treatment without side effects. For instance, further improvement in vector development technologies is essential to avoid activation of the endogenous signal transduction pathways and production of cytokines due to antivector immune responses that can potentially complicate the clinical outcomes. The necessity of integrin and CAR protein expression on the surface of target cells or tissue to allow efficient infection of AV limits the prospects of treating many diseases. Therefore, generation of novel AV vectors that can infect and transduce target cell or tissue with high specificity, express transgenes up to the therapeutic requirement, induce low organ toxicity and inflammation, and can be detected easily in vivo is the need of the hour. Understanding the disease-specific biomarkers, designing and engineering novel AV capsids carrying cell or tissue-specific receptor binding epitopes can reduce the occurrence of unwanted clinical outcome. Since the presence of AV-neutralizing antibodies varies from patient to patient, designing and developing personalized patient-specific capsids can be a promising approach to cure diseases in the future. Development of AV particles that resist inactivation by serum proteins is necessary to promote intravenous administration of therapeutic particles during treatment. Development of strategies to avoid dose-associated toxicity is needed. In addition, contamination with replicationcompetent virus still remains a serious issue in large scale production of AV preparation for therapeutic purposes (197). Therefore, further advancement in the production of purified AV and AV-based gene delivery technologies is required for using gene therapy to its full potential.

#### AAV-MEDIATED GENE THERAPIES IN CLINICAL TRIALS

AAV vectors are being used in more than 200 ongoing clinical studies to treat a wide variety of diseases and disorders worldwide. After the approval of the AAV-based drugs Gendicine and Luxturna, another AAV-based drug is poised to enter the

SNUBH, Seoul National University Bundang Hospital; BDHCMU, Beijing Ditan Hospital of Capital Medical University; BCCA, Vancouver Cancer Centre Vancouver, British Columbia Canada; CCF, Cancer Center of Florida; MUMC, McMaster University Medical Center; SUSM, Stanford University School of Medicine; WCMC, Weill Medical College of Cornell University; CCVTM, Centre for Clinical Vaccinology and Tropical Medicine; MEH, Moorfield's Eye Hospital; AADC, Aromatic L-amino Acid Decarboxylase Deficiency; SCRC, Surrey Clinical Research Centre; NCH, Nationwide Children's Hospital; BCH, Boston Children's Hospital; TCR, T cell receptor; FHCRC, Fred Hutchinson Cancer Research Center; FA, Fanconi Anemia; UA, University of Alabama; Oriengene Bio, Oriengene Biotechnology Ltd; PENK, Preproenkephalin.

pharmaceutical market in the near future to treat Choroideremia, an X-linked inherited retinal dystrophy that causes night blindness and a constricted visual field. Mutations in REP1 encoding Rab escort protein 1, a protein involved in lipid modification of Rab proteins, have been implicated in the development of Choroideremia. Patients that received AAV-REP1 therapy showed a significant increase in their visual acuity (198). The product of CNGB3 provides instructions for making the β-subunit of the cone photoreceptor cyclic nucleotidegated (CNG) channel, but mutations lead to a defective photoreceptor, decreased visual acuity, and total color blindness, or achromatopsia. In a phase I/II clinical trial sponsored by Applied Genetic Technologies Corporation, AAV was used to deliver CNGB3 for the successful treatment of achromatopsia (187). AAV is being tested to cure another eye disease, Leber's hereditary optic neuropathy (LHON), a maternally transmitted common mitochondrial disorder caused by point mutations in mitochondrial DNA and impairment of ATP generation. The LHON disease is characterized by apoplectic, bilateral, and severe visual loss. In an ongoing phase I interventional clinical trial, scAAV2 is being used to deliver the P1ND4 gene to rescue visual loss in five legally blind patients (NCT02161380). P1ND4 is a synthetic nuclear encoding gene involved in mitochondrial oxidative phosphorylation. The initial results obtained from this study have showed an improved acuity in two of five patients with no serious adverse events (199). Since treating diseases of the central nervous system is challenging due to the blood brain barrier (BBB), many AAV vectors, especially AAV1, AAV2, AAV5, AAV8, and AAV9, are found to be very useful in transducing neurons (200), and therefore, many AAVmediated treatments are being tested to cure lysosomal storage disorders, Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis (ALS), epilepsy, spinal muscular atrophy type 1, metachromatic leukodystrophy, aromatic L-amino acid

CAR-T, Chimeric antigen receptor T- cell; ALL, Acute lymphoblastic leukemia; LPL, Lipoprotein lipase; LPLD, Lipoprotein lipase deficiency; ADA, SCID—Adenosine deaminase severe combined immunodeficiency; HSC, Hematopoietic stem cell; VEGF, Vascular endothelial growth factor; PAD, Peripheral arterial disease; CLI, Critical limb ischemia; ICP34.5, Infected cell protein 34.5; GM, CSF-Granulocyte-macrophage colony stimulating factor.

decarboxylase (AADC) deficiency, and Batten disease. Like AV, AAV is yet another useful viral vector for cancer gene therapy. Several AAV vectors are being used to test the expression of antiangiogenic, cytotoxic, cytokine, and tumor suppressor genes, small RNAs, antigens, and antibodies for cancer cures. A large number of preclinical studies revealed successful treatment with AAV-mediated gene therapy for improved tumor regression (201–206). AAV is considered a powerful vector in targeting the liver for treating hematological diseases. Complete treatment of severe hemophilia B by delivering FIX in patients was described as the "holy grail" of gene therapy clinical application (207). In ongoing phase I/II clinical trials, FVIII and FIX are being delivered to hemophilia A and B patients, respectively (NCT03003533, NCT02484092).

Although AAV vectors are non-pathogenic and safe, and found among the commonly used platforms for gene delivery in preclinical and clinical studies, their potential application in gene therapy is limited by the inability to deliver a therapeutic gene more than 5.0 kb in size, immunogenicity of capsid proteins, difficulty in producing a large supply, requirement of large doses of highly purified vectors, broad tropism, and presence of an extensive anti-AAV immunity in human populations (208–211). Adding empty vectors of AAV to the final vector preparations to serve as a decoy and developing new vectors with high transduction and gene expression potential as well as better understanding of Tcell response to all AAV serotypes in clinical settings would reduce inflammation, immune response, and other viral particleassociated side effects because capsid is the primary interface with the target cell that defines pharmacological, immunological, and molecular properties (150, 207, 212, 213). Therefore, designing and developing more chimeric capsid proteins are critical to generate disease- and cell- or tissue-specific viral particles. For example, substitution of tyrosine to phenylalanine in the AAV capsid protein has enhanced the transduction efficiency with reduced toxicity (214). Better understanding of the underlying mechanisms of intracellular transportation of AAV particles in a disease-specific setting will help developing strategies to improve gene delivery efficacy. AAV vectors are commonly delivered to patients by systemic, intramural, central nervous system, cardiac, and pulmonary delivery but certain sites of the human body elicit no immune response to injection of antigens or viral particles because the BBB prevents the entry of antibodies or resting lymphocytes and the absence of traditional antigen-presenting cells. Therefore, applying AAV particles to patients through immune-privileged sites, such as the central nervous system, mucosal surfaces, eye, placenta, fetus, testicles, and articular cartilage, could be a better option to avoid T-cell response. For example, AAV vectors injected intraparenchymally into the central nervous system to treat Batten's, Canavan, and Parkinson's diseases showed little or no adaptive immune response in many clinical trials (155, 215– 218). Monitoring T-cell response in patients by using advanced tools especially multicolor flow cytometry, mass cytometry, and enzyme-linked immunospot (ELISpot) assay will minimize the risk of the unexpected clinical outcome (209). Also, for reduced T-cell response and optimal expression of a therapeutic gene, intramuscular instead of systematic injection of AAV particles is recommended because healthy muscles express only low levels of MHC class I antigens (209, 219). Use of immunosuppressive drugs was found safe to maintain therapeutic gene expression in many clinical trials (150, 220, 221), and their use could be an option for better clinical outcome, but maintenance of transgene expression remains unpredictable. Although AAV offers the expression of a therapeutic gene for nearly 1 year without integrating into the host's chromosomes, applying CRISPR/Cas9 technology would resolve long-term expression and mutagenesis issues. Production of high titers of purified AAV particles by employing ionic iodixanol gradients and ion exchange chromatography instead of using the toxic CsCl is also important for the success of gene therapy (222, 223). Recent developments in the production of high quality AAV particles from transfection efficient HEK293 cell suspensions in shaker flasks and WAVE bioreactors free of all animal and human products will certainly improve the success of gene therapy application (224). This system was further improved by changing the NaCl concentration in the medium and optimizing conditions for Expi293F cell infection by helper herpes simplex virus (HSV) (225). However, contamination of the final AAV particle preparation with HSV cannot be ruled out. The AAV particles generated from the baculovirus expression system carry low levels of VP1 capsid protein, so high doses are used in clinical trials to increase transduction efficiency at the expense of immune response (226). No disease caused by AAV infection has been reported to date but repression of PPP1R12C gene promoter in host cells by the rep proteins of AAV2 is clearly a concern (227). Therefore, more efforts are necessary to smooth out the landscape surrounding AAV for its more pronounced clinical benefits in gene therapy.

### HSV-MEDIATED GENE THERAPIES IN CLINICAL TRIALS

More than 90 gene therapy clinical trials have been conducted using HSV as a vector to deliver therapeutic genes for curing various diseases to date. They have been extensively used for tumor therapy and vaccine development. After the advent of HSV-based T-VEC drug for melanoma treatment, many HSV vectors are being used to deliver suicide genes to treat anaplastic thyroid cancer (228). Since immunotherapy is currently a hot topic in cancer research and gaining more attention; oncolytic viruses are often combined with immune checkpoint blockades such as T-VEC combined with anti-PD1 Pembrolizumab, anti-CTLA-4 Ipilimumab, and neoadjuvant to increase their therapeutic potential (68). Also, the oncolytic HSV-1 carrying four copies of miR-145 target sites combined with radiation has been shown to be more effective than radiation alone (122, 229).  HSV is also used as a transneuronal tracer defining connections among neurons by virtue of traversing synapses (230). HSV has much potential in treating problems of the urinary system. A recent study reports HSV-1 as a vector for delivering poreless TRPV1 channels or protein phosphatase 1α to reduce bladder over-activity in rats (231). HSV-mediated treatment also alleviated bladder pain. These have the potential to offer treatment to cases of overactive bladder (OAB) and interstitial cystitis/bladder pain syndrome (IC/BPS). However, infectivity of solid tumors, leakage, off target viral replication, sequestration, and delivery methods are still hampering the progress of HSV-mediated oncolytic viral therapy. Although the nervous system is the natural site for HSV latency, the full potential of HSV-mediated gene therapy in treating nerve diseases is yet to be discovered. Several studies treating chronic pain were successful in animal models but very few have reached clinical trials to date. HSV vectors have certainly promising perspectives in clinic trials but detailed understanding of virushost interaction will minimize cytotoxicity and biohazards generation. Recently, strategies to develop transduction efficient, alternate vector entry and transcriptionally retargeted oncolytic HSV viruses were reviewed (232–234). The therapeutic potential of amplicons is still undermined by production and stability issues; therefore, focus needs to be on improving vector design, construction, and production technology. Developing new HSV vectors carrying genes that enhance tumor cell lysis will increase oncolytic therapeutic efficacy. While gliomas do not express miR-124, it is highly expressed in normal brain, and designing HSV vectors carrying the same could be a promising approach to treat glioma. The full potential of expression libraries created by using HSV vectors in regenerative medicine is yet to be seen in curing human diseases (235). Since oncolytic virus therapy is considered a major breakthrough in treating cancer after the success of radiation and immunotherapies, development of safe and tumorselective new HSV vectors is necessary for its promising future. Optimizing vector delivery methods especially to solid tumors and in immune-compromised patients will certainly improve oncolytic viral therapy. Exploring their roles in gene editing and repair will expand the horizons of gene therapy.

#### RV-MEDIATED GENE THERAPIES IN CLINICAL TRIALS

RV vectors can be applied to cure a wide variety of diseases and disorders such as cancer, HIV, ADA-SCID, melanoma, WAS, and many others. Though the majority of retinal gene therapy trials use AAV, some use lentivirus because of its larger gene capacity. For example, Usher syndrome causes hearing loss, less vestibular function, and a pigmented retina (187). Currently, a phase II trial is underway to use lentivirus to deliver a 5.0 kb MYO7A. Additionally, a phase II trial that is projected to deliver ABCA4 by lentivirus to treat Stargadt disease, an inherited macular degeneration that causes cell degeneration, is underway (187). Furthermore, lentivirus is a favorable vector to treat sickle cell anemia because of the advantages it offers, including a large transgene capacity, stable long-term expression, and safer integration (236). A single base substitution in the β-globin gene causes the erythrocyte sickling characteristic of sickle cell anemia. Treatments for sickle cell anemia are transitioning into self-inactivating lentivirus with a deletion in the U3 region of the 3' LTR, which has a safer integration profile (236). A clinical trial sponsored by Bluebird Bio used LentiGlobin BB305, which delivered β-globin T87Q. Clinical results showed 24% anti-sickling (NCT03207009). For treating immunodeficiency, there have been adverse effects reported in the past by gammaretroviral vectors. In the treatment of Xlinked SCID, CD34+ hematopoietic stem cells were transduced with murine gammaretroviral vector, which led to an increase in immune function, but 5 patients developed T cell leukemia because of insertional mutagenesis into oncogenes (185). In the treatment of WAS, a gammaretroviral vector expressing WAS transgene delivered to patients caused 7 out of 10 to develop leukemia (185). Recently, self-inactivating lentivirus was used to treat five patients with X-linked SCID. Two patients had restoration of immune function even 2–3 years after treatment (237). A current phase I/II clinical trial is using a selfinactivating gammaretrovirus to treat SCID-X1 (NCT01129544). Other current clinical trials include a phase II trial using a retroviral vector to transfer ADA into hematopoietic stem cells to treat ADA-SCID (NCT00598481). A replicating Toca 511 RV vector is being used in a phase I trial to treat recurrent high-grade glioma (NCT02598011). RV is being used in a phase I/II trial to transduce white blood cells with the CAR-T cell receptor to target mesothelin for patients with metastatic cancer (NCT01583686). Donor T cells are being transduced with RV to express the caspase-9 suicide gene in a phase I trial to treat cancer (NCT01494103). Duchenne muscular dystrophy occurs when a lack of dystrophin expression causes muscle degeneration. In a proof-of-concept study, the full-length sequence of dystrophin was spread over two co-packaged RNA copies and delivered via a lentiviral vector. The vector integrated and gave longterm expression of dystrophin (238). Additionally, a RV vector expressing MazF endoribonuclease is being used to transduce CD4+ T cells to treat HIV in a phase I trial (NCT01787994). AIDS-related non-Hodgkin lymphoma is being treated in a phase I clinical trial that transduces stem cells with genes for HIV RNA using lentivirus in order to evoke an immune response (NCT01961063).

Since immunity is the primary barrier for the success of viral gene therapy, it is critical to design viral vectors that can subvert the complement system. The LTRs of RV serve as promoters, enhancers, binding sites for various nuclear proteins, chromatin modulators, and polyadenylation signals. Therefore, applying genetic engineering and CRISPR technology will avoid exacerbating the insertional mutagenesis issue. This issue can also be avoided by using non-integrating RV vectors or integrase inhibitors during treatment. The RV-mediated gene therapy will immensely benefit from developing technologies to guide and monitor transgene insertion in the host cell chromatin. Although RV vectors can deliver a transgene up to 10 kb in size, production of high titer virus, chromatin structure, and epigenetic modification near the insertion site still remain issues in clinical applications. Thus, better RV vectors are needed for future gene therapy applications. Since viral infection depends on the expression of target cell surface receptors and viral envelope protein, designing and constructing to produce efficient and cell-, tissue- and disease-specific recombinant RV vectors are necessary to obtain the expected clinical outcome. New RV vectors with optimized LTRs, created by replacing promoter/enhancer elements with cell- and tissue-specific promoters and enhancer sequences, will boost their use in curing many diseases with fewer or no side effects. Novel RV vectors are needed to transduce heart and other body organs for their wide spread use in gene therapy. Introduction of miRNA binding sites in the viral RNA has been suggested to control posttranscriptional regulation of disease-causing genes (239). The use of advanced RV vectors carrying the woodchuck posttranscriptional regulatory element (wPRE) to increase transgene mRNA stability, export, and translatability will help to accomplish better clinical outcome (240). As delivering genetic information in the form of RNA is an increasingly popular method, RVs carrying no RT or integrase are poised to play a significant role in a gene editing, vaccination, tumor therapy, gene therapy, transdifferentiation, reprogramming, and other biotechnological applications in the near future (241).

#### RISKS ASSOCIATED WITH VIRAL VECTORS

Since an estimated 1031 virus-like particles exist on the Earth and they are present in the blood, nose, mouth, lung, vagina, gastrointestinal tract, conjunctiva, skin, and the mammalian genome, viruses appear to play a major role in human life (242, 243). The general concerns with viral vectors are the risks of an immune response, off-target effects, inflammation, and insertional mutagenesis. An immune response could make a viral treatment less efficient, or the resulting creation of antibodies could preclude a second dosage of the same virus (244–248). Inflammation was seen as a worst-case scenario in the 1999 death of Jesse Gelsinger caused by a very high dosage of adenovirus (249). Tailoring the viral dose to the patient, however, can better control this risk. Also, insertional mutagenesis is a major obstacle that the gene therapy field must overcome. The risk of inserting a gene into a tumor suppressor gene or activating an oncogene is present for the vectors that integrate into the unwanted locations of the genome, such as retrovirus. To counter this, vectors can be used that do not integrate readily into the genome. Additionally, self-inactivating vectors can be manufactured that do not contain their own promoter; rather, another internal promoter in the cell is used. This leads to less genotoxicity and is a safer alternative to traditional integrating vectors (52). Other concerns are that viral vectors are only relevant for monogenic disorders because of their limited DNA-carrying capability. However, HSV-1 is an example of a virus that has enough carrying capacity for multiple genes. Additionally, dual vector systems, such as dual vector adeno-associated virus, have larger transgene capacities. Also, finding the appropriate virus to infect the desired cells is often difficult, and there is the risk that the virus could cross the Weismann barrier and infect germ line cells. Furthermore, viruses are generally susceptible to genetic variations. Integration into undesirable sites such as regulatory, oncogenes or tumorsuppressor genes would be undesirable. Deletion of virulence genes may affect their ability to infect or integrate with the host chromosome, thus compromising their effectiveness as vectors. Additionally, a social stigma is associated with viral therapy. Most patients would be concerned about being infected by a live virus—a concern also held about viral vaccines. Since their ubiquitous presence is a reality, why shouldn't humankind start accepting them as wonderful molecular biological tools with which to build novel and powerful medicine?

#### CHALLENGES AND THE WAY FORWARD

Since its birth in the 1960s, gene therapy has come quite a long way by providing an alternate one-time treatment option for cancer, metabolic disorders, and neuronal, immune, and infectious diseases. Notably, it has been able to treat beta thalassemia, Leber's congenital amaurosis, severe immunodeficiency diseases such as ADA-SCID, and more. However, the full potential of gene therapy is yet to be witnessed in regenerative medicine, a branch of translational medicine where engineering or regenerating human cells, tissue or organs enables restoration or establishment of normal function. With recent impressive results observed in vaginal gene therapy in preclinical trials, gene therapy is poised to enter the clinical phase for treating infectious diseases in the near future (250). Both viral and non-viral vectors can be used to deliver DNA, each of which has its own advantages and disadvantages. Additionally, genome-editing technology is an up-and-coming method of delivering DNA to specific parts of the genome. With all of these breakthroughs have come hurdles, such as the death of Jesse Gelsinger in 1999 and the development of leukemia in patients who have been treated for WAS and ADA-SCID. The ethical concerns of patients must be heeded as well. However, these challenges do not reflect a flaw in the concept. Simply, more research is needed to avoid technical issues such as the production of viral particles in large scale, formulations for long-term storage stability, immune responses, and insertional mutagenesis. Loading of viral particles with a therapeutic gene during production is mostly done by transient transfections, a rate-limiting step in large scale production of viral particles. Alternate approaches such as stable cell lines expressing capsid proteins and insect cells based baculovirus expression systems would be useful for mass production of viral particles. This underdeveloped modern medicine needs discovery and engineering of better viral vectors to deliver therapeutic genes precisely to the target diseased cells or tissue.

Gene therapy is a rapidly expanding field, and it seems that scientists have only scratched the surface of its potential. The more that is discovered about how to optimize gene delivery vectors, the closer this field gets to delivering widescale solutions to modern medicine. The future of gene therapy moves toward engineering safer and more efficient vectors, combining multiple existing strategies such as viral vectors with genetic engineering technologies, and personalizing all characteristics of gene therapy treatments to the patient, as it has been shown that host genetic variants affect the efficacy of vector-mediated gene delivery (251). This includes understanding of the repertoire of receptors on a target cell in diseased conditions to help in designing appropriate capsid proteins for viral particles. Although the full panoply of gene therapy's might is yet be witnessed, it has enormous potential to shed light on human afflictions, add value to patients' lives, and contribute to future economic growth. Although gene therapy currently shares less than one percent of the total \$1.2 trillion world annual pharmaceutical market, it is expected to create approximately a \$12 billion market in the next 10 years. According to a market research and advisory company, Allied Market Research, cancer gene therapy alone has created a \$289 million market in 2016 but it is expected to reach \$2,082 million by 2023. Gaining popularity among the global medicinal community, gene therapy has become an attractive market for companies and investors. However, the ethical acceptance and advancement in the technology to avoid unwanted clinical outcomes are critical for driving its market growth. Also, the translation of laboratory studies to animal studies and then to clinical trials is a long, tedious, and expensive process to ensure the safety of patients. As a result, if the USFDA, with its patchy regulations, continues its approval rate, providing gene therapies for all the genetic diseases will take many years to come. Therefore, a new perspective on creating a conducive atmosphere for improving this modern cutting-edge gene therapy technology is necessary to transform the lives of patients with severe genetic illnesses, infectious diseases, and disorders. As mentioned elsewhere, knowledge has no boundaries, and there exist unlimited methods to develop a novel invention; every bump in the investigating path can be considered an inspiration and source of energy to advance research, a never-ending learning process.

#### REFERENCES

#### AUTHOR CONTRIBUTIONS

VB and RG conceived the concept and wrote the manuscript, and the other authors listed made substantial, direct intellectual contributions to the work, and approved it for publication.

#### FUNDING

Funding for this work was provided by Joint Science and Technology Office (JSTO), Defense Threat Reduction Agency (DTRA), Department of the Army, Department of Defense, United States of America.

#### ACKNOWLEDGMENTS

Authors thank Mr. Abraham A. James for his artwork and Ms. Neehar Mishra for her comments, and Dr. Manubhai Patel and the late Dr. Douglas M. Cerasoli for their unconditional support. Authors apologize to the scientists whose contributions to gene therapy could not be acknowledged because of space limitations.

immunogenicity of recombinant human butyrylcholinesterase. Chem Biol Interact. (2008) 175:255–60. doi: 10.1016/j.cbi.2008.05.020

a translational optimized AAV vector. Mol Ther. (2012) 20:443–55. doi: 10.1038/mt.2011.237

pathway. Protein Cell. (2017) 8:165–8. doi: 10.1007/s13238–016- 0334-x

**Disclaimer:** The views expressed in this review article are those of the author(s) and do not reflect the official policy of the Department of Army, Department of Defense, or the U.S. Government.

**Conflict of Interest Statement:** The authors declare that that they have no affiliations with or involvement in any organization or entity with any financial interest (such as honoraria; educational grants; participation in speakers' bureaus; membership, employment, consultancies, stock ownership, or other equity interest; and expert testimony or patent-licensing arrangements), or non-financial interest (such as personal or professional relationships, affiliations, knowledge or beliefs) in the subject matter or materials discussed in this manuscript.

Copyright © 2019 Goswami, Subramanian, Silayeva, Newkirk, Doctor, Chawla, Chattopadhyay, Chandra, Chilukuri and Betapudi. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

# The Analysis of Key Factors Related to ADCs Structural Design

Haichao Tang1,2, Yan Liu3 , Zhaojin Yu1,2, Mingli Sun1,2, Lu Lin1,2, Wensi Liu1,2 , Qiang Han1,2, Minjie Wei1,2 \* and Ying Jin3 \*

1 Department of Pharmacology, School of Pharmacy, China Medical University, Shenyang, China, 2 Liaoning Engineering Technology Research Center for the Research, Development and Industrialization of Innovative Peptide Drugs, China Medical University, Shenyang, China, 3 Liaoning Research Institute of Family Planning, Shenyang, China

Antibody–drug conjugates (ADCs) have developed rapidly in recent decades. However, it is complicated to map out a perfect ADC that requires optimization of multiple parameters including antigens, antibodies, linkers, payloads, and the payload-linker linkage. The therapeutic targets of the ADCs are expected to express only on the surface of the corresponding target tumor cells. On the contrary, many antigens usually express on normal tissues to some extent, which could disturb the specificity of ADCs and limit their clinical application, not to mention the antibody is also difficult to choose. It requires to not only target and have affinity with the corresponding antigen, but it also needs to have a linkage site with the linker to load the payloads. In addition, the linker and payload are indispensable in the efficacy of ADCs. The linker is required to stabilize the ADC in the circulatory system and is brittle to release free payload while the antibody combines with antigen. Also, it is a premise that the dose of ADCs will not kill normal tissues and the released payloads are able to fulfill the killing potency in tumor cells at the same time. In this review, we mainly focus on the latest development of key factors affecting ADCs progress, including the selection of antibodies and antigens, the optimization of payload, the modification of linker, payload-linker linkage, and some other relevant parameters of ADCs.

Keywords: antibody–drug conjugates, precision choice antibody and antigen, elaborate modification linkers, proper payloads, optimized linker-payload linkage

## INTRODUCTION

In traditional tumor treatment, chemotherapy is one of the main treatment strategies. However, the toxicity from non-specific accumulation in normal tissues, narrow therapeutic window and low tolerance all limit chemotherapy drug development in the tumor treatment process (Atkins and Gershell, 2002; Alley et al., 2010; Ashley et al., 2011). In recent decades, scientists have gained an in-depth understanding of cancer biology, taking advantage of some unique features

#### Edited by:

Zhe-Sheng Chen, St. John's University, United States

#### Reviewed by:

Xiaolan Deng, Beckman Research Institute of City of Hope, United States Liwu Fu, Sun Yat-sen University, China Zhaoqian Liu, Xiangya Hospital, Central South University, China

#### \*Correspondence:

Minjie Wei<EMAIL_ADDRESS>Ying Jin<EMAIL_ADDRESS>
#### Specialty section:

This article was submitted to Cancer Molecular Targets and Therapeutics, a section of the journal Frontiers in Pharmacology

Received: 17 January 2019 Accepted: 26 March 2019 Published: 24 April 2019

#### Citation:

Tang H, Liu Y, Yu Z, Sun M, Lin L, Liu W, Han Q, Wei M and Jin Y (2019) The Analysis of Key Factors Related to ADCs Structural Design. Front. Pharmacol. 10:373. doi: 10.3389/fphar.2019.00373

**Abbreviations:** ADCs, antibody–drug conjugates; ALCL, anaplastic large cell lymphoma; AML, acute myeloid leukemia; DAR, drug-to-antibody ratio; MDR, multiple drug resistance; MMAE, monomethyl auristatin E; PDBs, pyrrolobenzodiazepine dimers; PEG, polyethylene glycol; PHF, hydroxymethyl-formal; SMCC, succinimidyl-4-(Nmaleimidomethyl)-cyclohexane-1-carboxylate; SMCC-DM1, succinimidyl 4-(N-maleimidomethyl) cyclohexane-1 carboxylate-maytansinoid; sulfo-SPDB-DM4, N-succinimidyl-4-(2-pyridyldithio)-2-sulfo butanoate-maytansinoid; val-cit-PABC-MMAF, valine-citrulline-p-aminobenzyl-carbamate-monomethyl auristatin F.

of tumor cells to transform cancer treatment from previous chemotherapy drugs to tumor-targeted therapies. Monoclonal antibodies and polypeptides which bind to specific markers on the tumor cell's surface provide targeted therapeutic approaches and are both less toxic. However, whether they are monoclonal antibodies or peptides, they both lack potency in killing tumor cells.

The treatment strategy of antibodies armed with toxins to selectively kill target cells was first proposed in 1970 (Moolten and Cooperband, 1970). The tumor-targeting drug conjugates integrate targeted biomolecules with therapeutic small molecule toxins to specifically recognize the tumor tissues and kill the tumor cells, thereby improving the therapeutic index of the toxins and the insufficient efficacy of antibodies or peptides. The tumor-targeting drug conjugates mainly compose of ADCs that generally couple antibodies which specifically recognize the surface antigens of tumor cells with chemical toxins which effectively kill tumor tissues through linkers, and ADCs exert killing activity by bringing the chemical toxins into the tumor cells. In general, the antibody specifically binds to the tumor cell surface antigen, and the antigen mediates the endocytosis of the ADC and then releases free toxins (**Figure 1**), but the downsides are that immunogenicity, poor internalization and the instability of the linker give rise to insecurity and ineffectiveness (Chari, 2008). More than 60 ADCs have been in the process of clinical development until 2016 (Carter and Lazar, 2017), there are almost 204 ADCs (**Supplementary Table S1**) that aim for cancer in clinical development by 2018, including at least nine of which have entered phases III and IV clinical trials1 . It indicates that ADCs are coming to the center-stage of research field in recent years especially in North America (**Figure 2**). However, until today, only ado-trastuzumab emtansine (T-DM1, Kadcyla@) and brentuximab vedotin (Adcetris@) are approved by FDA and on the market (Mullard, 2013; Thomas et al., 2016). There are many reasons for the dilemma, including the complexity of the composition of the ADC itself, and the fact that the tumor microenvironment or physiological conditions in animals are different from the human so that the evaluation of ADC efficacy by animal models is not applicable to humans. Beck et al. (2017) published a review paper about the strategies and challenges for the next generation of ADCs in 2017. However, ADCs are developing rapidly and some novel technologies may bring new considerations. Thus, this review mainly focuses on imperative factors that are associated with ADC efficacy (**Figure 3**).

### THE SELECTION OF ANTIBODY AND ANTIGEN

Normally, the antigen specific to the cancer cell should be a priority after determining the indications for ADC. Ideally, the antigen should express highly and homogeneously on the surface of the cancer cells (Sievers and Senter, 2013; Damelin et al., 2015). When the antibody combines with the antigen specifically, the antibody-antigen complex should be internalized

1https://clinicaltrials.gov

by antigen-mediated endocytosis, and then the free payloads are released through lysosomal trafficking. As a result, the payloads are concentrated in cancer cells and exert the cytotoxic effect (Erickson et al., 2006). Currently, the predominant therapeutic limitations are the ineffectiveness and the off-target toxicities of ADCs, which are caused by the finite internalization and the low expression of antigens to some extent. Therefore, some researchers came up with some approaches to counteract these problems such as utilizing the anti-tumor angiogenesis antibody, non-internalizing ADC, or bispecific antibody.

#### The Utilization of Anti-tumor Angiogenesis Antibody

Some researchers proposed a strategy that using an anti-tumor angiogenesis ADC to selectively kill cancer cells due to the process without the involvement of internalization, which could improve the deficient efficacy caused by finite internalization. For example, Palumbo et al. (2011) reported that the ADC composes of an anti-angiogenesis LC19 antibody to selectively target to the tumor blood vessels, the strategy showed a long-term anti-tumor effect. However, ADCs of the anti-tumor vessel may elicit offtarget toxicities to normal tissues due to non-specificity of antigen expression and resistance of vessel co-option in some particular tumor tissues (Kuczynski et al., 2016). This requires choosing antibodies based on proper antigens. Seaman et al. (2017) applied the anti-CD276 antibody to the ADC to improve the nonspecificity. The CD276 expresses in angiogenic tumor vessel, existed vasculature and tumor cells. Moreover, the anti-CD276 antibody is capable of identifying the normal and pathological angiogenesis. The anti-CD276 ADC evaded the vessel co-option and displayed a dual-targeting ability thus displaying effective anti-tumor activity (Seaman et al., 2017).

#### Preparing Non-internalizing ADCs

An approach to prepare non-internalizing ADCs to target corresponding antigens needed to be developed. For instance, the ADC took advantage of a diabody without an Fc region to target the matching antigen and an additional chemical activator to cut the linker, and then release the free payload to penetrate into tumor cells (Rossin et al., 2018). This strategy is able to increase the anti-tumor activity and avoid some factors can sacrifice the efficacy of ADCs such as interstitial pressure and epithelial barriers from the tumor cells.

#### The Selection of the Bispecific Antibody

On one hand, in terms of the deficiency of internalization, Li et al. (2016) used a bispecific antibody to target two non-overlapping epitopes of one antigen, which increased the affinity between antibody and antigen. For example, an anti-HER2 biparatopic antibody displayed better internalization, lysosomal trafficking, and degradation of the antibody-antigen complex relative to the traditional T-DM1 (Li et al., 2016). However, the superior affinity also may trigger a controversy about whether the biparatopic ADC would induce on-target toxicities to healthy tissues. Though this study also further indicated that the biparatopic ADC has an acceptable safety profile due to the threshold of antigen.

FIGURE 1 | The process of ADCs exerting activity. (a) ADCs specifically recognize cancer-associated antigens in the blood system. (b) ADCs are internalized into tumor cells during the formation of antibody-antigen complex. (c) ADCs are normally transported to lysosome from endosome. (d) The linker or antibody are broken in the lysosome conditions to release free toxins. ADCs, antibody drug conjugates.

It is unable to form the antibody-antigen complex if the expression level of antigen below the threshold. However, it seems arduous to avoid the problem due to the uncertain threshold. Theoretically, a higher affinity antigen–antibody could make more ADC molecules combine with tumor cells thus having more accumulation, but a lower affinity may allow ADCs to penetrate into tumor cells more effectively. Scientists are still looking for antigen–antibody with proper affinity (Rudnick et al., 2011). It needs further research (Tsumura et al., 2018).

Also, some researchers proposed to use the probody of antibody to solve the on-target toxicities, which may also be applied to ADCs. This strategy used masking peptide to cover up the active sites of the antibody then hydrolysis of the shelter to expose the antibody to target cancer tissues to exert activity (Desnoyers et al., 2013), which allays the indistinct recognition of ADCs in the blood circulation.

On the other hand, the bispecific antibody is able to selectively bind two distinct antigens on a cancer cell to avoid the off-target toxicities. For example, the bispecific antibody simultaneously targets the HER2 and PRLR double positive (HER2+/PRLR+) breast cancer cells to enhance the internalization and activity of the ADC, and to decrease the off-target toxicities to the healthy cells (Andreev et al., 2017). Nevertheless, targeting double-antigens is ineligible for most heterogeneous tumor cells, since it may trigger their escape mechanism. Furthermore, the bispecific antibody could be used to target the immunosuppressive molecule and tumor-specific antigen on the tumor cells simultaneously to improve the efficacy of ADC. The ADC targeting CD47 that an immunosuppressive receptor and TAA double positive (CD47+/TAA+) tumor cells could block the immunosuppression to augment the killing activity of the ADC (Dheilly et al., 2017). Currently, there are more than 70 bispecific antibodies applied in clinical trials (see text footnote 1), two of them have used on the market. These specific antibodies seem to change some imperfect phenomena of ADCs (Piccione et al., 2015). Moreover, the trifunctional antibody also could be used to ADC (Krishnamurthy and Jimeno, 2018), which possess an arm to target the tumor cells, the second is used to target T cells, the remaining Fc region to recruit some immune cells. Using the trifunctional antibody to link a small molecule toxin seems to improve the deficient specificity and the killing potency of ADCs. Though the bispecific or trifunctional prospect is promising to improve potency and specificity to increase market competitiveness of ADCs, the challenge of determining the target combination still remains.

### The Bystander Effect to Heterogeneous Tumors

Some reports also demonstrated that some ADCs may take advantage of the physical and chemical properties of linkers and the microenvironment of the tumor to release free payloads to kill those adjacent negative-antigen cancer cells. The process is the bystander effect (Kovtun et al., 2006; Okeley et al., 2010). ADC was metabolized to release uncharged and membranepermeable toxic metabolites after being internalized in positiveantigen cancer, which is able to kill adjacent antigen-negative cancer cells by membrane-penetration (Kellogg et al., 2011). This has a great significance for some heterogeneous tumor cells. Admittedly this was that the bystander effect may also cause non-specific killing of normal cells. Therefore, it requires to have rational selection and design of payloads and linkers based on the target to avoid the adverse effects from bystander effect.

#### The Selection of Antibody Isotype

Within IgG isotypes, IgG1, IgG2, IgG4 have been used to develop therapeutics, but IgG3 isotypes are not used as therapeutics owing to a significantly faster clearance rate (Jefferis, 2007). Further, most ADCs use IgG1 isotype currently (Beck et al., 2017). IgG1 isotype may exert ADCC (antibody-dependent cell-mediated cytotoxicity) and CDC (complement-dependent cytotoxicity) to improve ADCs activity further, whereas IgG2 and IgG4 are typically deficient in their effector functions (Salfeld, 2007). However, the PD-1 antibodies (Nivolumab and Pembrolizumab) used IgG4 isotype, which may be due to the PD-1 antibody only needing to block the interaction between PD-1 and PD-L1

to increase immune system function to produce anti-tumor activity, which is needed to avoid the toxicity to T cells from ADCC and CDC. Therefore, the choice of isotype also needs careful consideration.

#### The Consideration of Antigen

Glycosylation of antigen also could affect the design of ADCs. On the one hand, if glycosylated antigen specifically exists on the tumor cell surface, it will have an important implication to be a target of an ADC. For example, a monoclonal antibody targeting glycosylated PD-L1 (gPD-L1) to disrupt PD-L1/PD-1 interaction. The gPD-L1 is mainly expressed on tumor tissues, which improves non-specific expression of PD-L1 in some immune cells to limit toxicity (Li et al., 2018). On the other hand, the steric structure of a glycosylated antigen plays a certain protective role, which will block the interaction between the antibody and specific sites on the antigen surface. Therefore, we need to have a more comprehensible understanding on designing ADCs.

Most ADCs utilize the antigens on the tumor cells surface, which are limited in their specificity relative to intracellular antigens. Taking advantage of the antigen presentation feature of MHC that caused tumor-specific endogenous antigen expression on the cell surface overcomes the inaccessibility of intracellular antigens. Further, the MHC-I/peptide complex is recognized by the ADCs that mimic the characterization of TCR, which will produce superior specificity and potency (Lai et al., 2018). **Table 1** and **Figure 4** showed the antigens used in phase III/IV trial currently (see text footnote 1).

### THE SELECTION OF PAYLOADS

Once the target is determined, the proper choice of payload becomes a critical part of ADC. The final potency of ADCs mainly depends on the concentration of payloads in tumor cells; thus some researchers are dedicated on improving the DAR of ADCs to increase the accumulation of drugs in tumor cells. Zhang et al. (2018b) indicated that activity of the ADC still remains constant though augmenting the payload concentration, and this also could magnify the toxicity to normal tissues at the same time. This suggests that the threshold of payload concentration also needs consideration. In summary, choosing the applicable payloads and designing the appropriate DAR is important for the final concentration of the payloads in tumor cells.

#### Improving the Efficacy of ADCs

Early ADCs used drugs that have been approved for clinical use such as vinblastine and doxorubicin, but the low clinical activity of these drugs resulted in suboptimal ADCs efficiency. Some cytotoxins were too toxic to be non-target agents in clinical application, but they seemed to be more promising as payloads for ADCs. At present, the dose of the payloads in tumor cells is required to be the picomolar range to kill these cancer cells (Chari et al., 2014). Also, payloads must possess acceptable solubility and decent sites to react with linkers. These all limited the selection of payloads. Currently, most payloads are derivatives of the microtubule inhibitor family, such as the auristatin and maytansine (Beck and Reichert, 2014). Brentuximab vedotin (Adcetris@), approved by FDA in 2011, composes of MMAE and cAC10 mAb (chimeric IgG1 antibody) via a protease-cleavable dipeptide linker to target tumor antigen CD30 (also known as TNFRSF8) for the treatment of Hodgkin's lymphoma and ALCL (ki-1 lymphoma) (Senter and Sievers, 2012; Younes et al., 2012). Ado-trastuzumab emtansine (Kadcyla@), approved in 2013, consists of a stable thioether linker (SMCC) attached to trastuzumab (anti-human epidermal growth factor receptor-2 antibody, anti-HER2 antibody) and DM1 (maytansine derivative) for the treatment of advanced breast cancer (Lambert and Chari, 2014). Both adopted the microtubule inhibitor family as payloads, yet auristatins and maytansines are only able to exert activity in cell proliferation and they are hydrophobic, which will disturb their activity. Thus, some novel payloads or the original payload structural modifications such as the improvement of hydrophilicity will become the hotspots of the future payload research (Burke et al., 2017). At present, some novel ADCs have better activity and have been through clinical phase III/IV (**Table 1**).

The first commercially available ADC was gemtuzumab ozogamicin (GO) that consists of calicheamicins which damage DNA (Walker et al., 1992) for the treatment of AML. However, GO showed no significant improvement in overall survival (OS) compared with the calicheamicin agent alone, and had a higher mortality rate and was recalled in 2010 (Petersdorf et al., 2013; Kharfan-Dabaja, 2014). This is because calicheamicin is hydrophobic in that almost only 50% could be conjugated, and only approximately 50% of free drugs are eventually released in the conjugated drugs (Beck et al., 2010; Senter and Sievers, 2012), resulting in a significant decrease in potency. To overcome these limitations, some novel targeted DNA agents have been broadly developed. Pyrrolobenzodiazepine dimers (PBDs) have already become a new choice, it may attach to the linker that conjugated to the antibody, and has the ability to overcome MDR relative to the commonly used calicheamicin as a substrate of P-glycoprotein (Kung Sutherland et al., 2013; Stein et al., 2018). The IMGN779 (NCT02674763) utilized DGN462 that a novel drug with DNA-alkylating activity also demonstrated better anti-tumor activity and tolerability (Kovtun et al., 2018).

TABLE 1 | Current clinical phase III/IV trials of ADCs.

fphar-10-00373 April 17, 2019 Time: 16:24 # 6

DUBA, duocarmycin-hydroxybenzamide-azaindole; MMAE, monomethyl auristatin E; MMAF, monomethyl auristatin F; DM4, maytansine 4; PBD, pyrrolobenzodiazepine dimers.

#### Avoiding MDR

The MDR has always been a barrier and one of the important factors affecting the therapeutic effect in the cancer treatment. The MDR is still an impeditive factor of using ADCs. This is because the essence of ADC's activity is that the payloads in tumor cells exert cytotoxicity, and these payloads may be affected by MDR. Many studies concentrate on the modification of drug-linker that, by increasing hydrophilicity, circumvents MDR caused by the overexpression of efflux pumps because the substrates of MDR1 were hydrophobic in general. Moreover, some novel payloads such as PBD, DGN462, and tubulysins cooperate with ADCs to display better anti-tumor activity in MDR+ tumor cells (Burke et al., 2018; Kovtun et al., 2018; Stein et al., 2018). ADCs are susceptible to hydrophobicity to be insensible to MDR+ cells, thereby it is essential to improve the hydrophilicity to escape from MDR to increase the activity of ADCs (Kovtun et al., 2010).

### THE MODIFICATION OF LINKER

Although the linker may be not directly correlated with the final potency of ADC (Lee et al., 2018b), the potency of ADC is dictated by the concentration of payload accumulated in tumor cells, and the payload release is determined by the stability of the linker. Thus, the linker is crucial for a perfect ADC, and it determines the stability, efficacy, and even the ability to overcome MDR. The basic requirement of the linker is to make the payload attach to the antibody, stabilize the payload in the circulation system, and is labile to release the free payload into cancer cells when the antigen–antibody complex is formed (Doronina et al., 2006). Currently, linkers are mainly divided into the cleavable linkers and the non-cleavable linkers.

### The Comparison of Cleavable Linkers With Non-cleavable Linkers

The cleavable linkers normally take advantage of the difference of tumor microenvironment and normal physiological environment to release the payloads that may be membranepermeable and can produce the bystander effect. The non-cleavable linkers need to meet the requirement that the antibody and linker must be disconnected after the formation of the antigen–antibody complex enter the lysosomal trafficking. This may cause the bystander effect that is a passive transport process to weaken, caused by the membrane-impermeability

of linker-payloads connected with polar amino acids. Both types of linkers have their advantages and disadvantages, which are applied to the clinical trials (Chari et al., 2014; Bialucha et al., 2017). However, about 2/3 ADCs used cleavable linkers in the current clinical trials (Lambert and Berkenblit, 2018), in which mainly are dipeptide linkers and disulfide linkers. The non-cleavable linkers are not only more stable to escape from the off-target toxicities than cleavable linkers (Lu et al., 2016), but also may overcome the barrier of multiple-drug resistance (MDR) (Shefet-Carasso and Benhar, 2015; Beck et al., 2017; Nasiri et al., 2018) for the reason that the payload connected with polar amino cannot be a substrate of MDR1, which will improve the MDR phenomenon. However, the non-cleavable linkers need a more elaborate process to produce activity such as the internalization and metabolism of the antibody in the lysosome, which is a prerequisite to release active payloads to exert killing activity (Rosenberg, 2006; Lambert and Berkenblit, 2018), and the polar amino-linker-payload also needs a distinct transporter to carry it from the lysosome to cytoplasm to work (Hamblett et al., 2015; Kinneer et al., 2018; Lee et al., 2018b), which makes the design of ADC more complex to limit the utilization of non-cleavable linkers. The cleavable linkers are more vulnerable to lead to off-target toxicities, but the process of exerting effects is more comprehensible thus researchers are dedicated to modifying the cleavable linkers to overcome their weakness and to increase their stability in the circulation (Sanderson et al., 2005; Kellogg et al., 2011).

#### The Analysis of Cleavable Linkers

The cleavable linker can metabolize some cell-permeable metabolites to exert the bystander killing effect. The cleavable sulfo-SPDB-DM4 linker produced cell-permeable catabolites to display a better activity than non-cleavable SMCC-DM1 linker (Bialucha et al., 2017). Also, the application of the sulfonate group improved hydrophilicity to increase the exposure of ADC to the antigen to promote killing activity. The brentuximab vedotin (SGN-35) took advantage of a cleavable dipeptide linker to release free MMAE, and the MMAE may permeate adjacent cells to exert killing activity which is important to some heterogeneous tumor cells. Moreover, the dipeptide linker offers ADC better stability in the circulation, and is more specific to tumor cells (Katz et al., 2011). The protease cleavage pathway is not restricted to cathepsin B, various cysteine cathepsins can cleavage the dipeptide linker, such as cysteine cathepsins B, K, L, and S. It seems to explain why the dipeptide linkers cannot be insensitive to tumors, caused by the insufficient expression of protease (Caculitan et al., 2017), which is one of the reasons why some protease-sensitive linkers are widely used by ADCs.

In particular, the design of the valine-citrulline (val-cit) linker, the most frequent in dipeptide linkers, needs to consider the connection to the phenol-containing payloads; diverse electron groups affect the degrees of immolation of the linker to influence the different potency of an ADC (Zhang et al., 2018a). However, the val-cit dipeptide linker is not conducive to preclinical research to appraise the efficacy of ADCs due to instability in mice (Dokter et al., 2014). Anami et al. (2018) reported a glutamic acid-val-cit linker replaced val-cit dipeptide linker, which could alleviate the flaw of instability in the mice plasma and retain the cathepsin-mediated cleavage mechanisms, thus boosting preclinical application of some ADCs. The acidic tripeptide linker could increase the polarity of ADCs to improve solubility to increase the therapeutic potency (Anami et al., 2018). However, one of the studies suggests that activity of the ADC with cleavable valine-citrulline-p-aminobenzyl-carbamatemonomethyl auristatin F (val-cit-PABC-MMAF) is much less than the ADC with non-cleavable maleimidocaproyl-MMAF (Doronina et al., 2006), which may be due to the character of payloads rather than the linker. The metabolites of some payloads are more effective than the prototypes. The noncleavable linkers are not widely applied to ADCs since many payload derivatives attached to an amino cannot satisfy the killing potency of ADCs.

The disulfide linker utilized the difference of glutathione (GSH) levels between the tumor microenvironment and the physiological environment of normal tissues to produce activity (Meister and Anderson, 1983; Dubikovskaya et al., 2008), which is more labile in tumoral hypoxia conditions (de Groot et al., 2001). At present, the main obstacle of the disulfide linker is the instability, which is mainly improved by increasing steric hindrance to relieve the vulnerability. ADCs using the disulfide linkers have inferior potency in vivo due to the more rapid clearance of payloads compared with the non-cleavable thioether linkers that displayed more potent activity (Lewis Phillips et al., 2008). The trastuzumab emtansine (T-DM1) consists of noncleavable thioether linker and a maytansine derivate, which has better anti-breast cancer activity. The linker contained a cyclohexane carboxylate and a maleimidomethyl group. The ionized metabolite cannot kill surrounding normal cells due to its impermeability after ADC metabolized, thus the ADC has a better safety (LoRusso et al., 2011). The non-cleavable linkers are stricter in the choice of antigens compared with cleavable linkers, yet fewer toxicities (Polson et al., 2009). Zhang et al. (2016) reported that using methy- and cyclobutyl-substituted disulfide with efficient immolation demonstrated more potent killing activity than cyclopropyl-substituted disulfide with nonimmolation. Also, this reflects that the immolation of the linker is imperative to the potency of ADC (Zhang et al., 2016). However, the anti-tumor activity is more determined by the cleavage of the linkers only when payloads require complete cleaving to exert activity (Caculitan et al., 2017). Thus, new research could focus on developing payloads that do not require the production of pharmacological effects with prototype drugs. Also, future studies could focus on developing some novel technologies of payload-linker to improve the activity of ADCs such as SYD985 based on a cleavable linker-duocarmycin payload (NCT03262935) (Dokter et al., 2014).

#### THE PAYLOAD-LINKER LINKAGE

With the development of ADCs, the drug-linker linkage that goes hand in hand with the efficacy of ADCs is more critical (Nasiri et al., 2018). In order to give full play to ADCs' activity in tumor cells, it is necessary to effectively design the payload-linker

according to the physicochemical properties of the payloads and the characteristics of the linkers.

## The Consideration of the Sites of Payload-Linker

The sites of the payload-linker are essential conditions to consider due to the attaching-sites being correlated with homogeneity that is related to the therapeutic index. In the early stages of ADCs development, the lysine on the antibody was used as the site to attach the linker, which caused great heterogeneity. Later, Adcetris@ used the cysteine that only eight free cysteines per antibody to link through disulfide bonds, which reduced the ADCs heterogeneity. In recent years, to ensure ADC homogeneity, researchers have developed some site-specific methods, such as THIOMAB (Junutula et al., 2008; Chudasama et al., 2016).

#### The Modification of Payload-Linker

The drug-linker linkage determines the DAR that are related to the efficacy of ADC. Generally, the therapeutic potency of ADC gradually increases in vitro with the increase of DAR whereas the therapeutic index in vivo decreases (Hamblett et al., 2004), which may due to with the enhancement of DAR accelerates the clearance of ADC which is closely related to the hydrophobicity of ADC (Lyon et al., 2015). The hydrophobicity is determined by the amounts of payloads per antibody and the design of druglinker (Doronina et al., 2014). It is the main reason for the failure of ADC in the clinical application that the concentration of payload is deficient to treat tumors on account of the DAR of ADC in clinical stage generally control to 3.5–4 (Beck et al., 2017). Thus, augmenting the hydrophilicity of ADC with high DAR by the design of drug-linker exquisitely will improve the efficacy in vivo (Pabst et al., 2017). Some hydrophilic groups such as PEG or PHF may improve this dilemma. Accurately connecting these hydrophilic groups to a linker will effectively improve the efficacy of the ADCs. For example, Trastuzumab–PHF–Vinca ADC with DAR of 20 demonstrated a potent anti-tumor activity and decent pharmacokinetic profile due to the high hydrophilicity of PHF (Yurkovetskiy et al., 2015).

At the same time, MDR+ tumor cells are insensible to some ADCs due to the fact that many payloads applied to ADCs are hydrophobic, which are the substrates of the MDR1 transporter. By improving the hydrophobicity of the drug-linker, it seems to be able to bypass MDR (Kovtun et al., 2010; Shefet-Carasso and Benhar, 2015).

#### OTHER PARAMETERS CORRELATED WITH THE EFFICACY OF ADCs

#### The Relationship Between the Internal Environment and Activity of ADCs

Normally, we consider the internalization that influences the efficacy of ADCs to be regulated by antigen. Recently, Lee et al. (2018a) demonstrated that the internalization may be mainly determined by the cellular environment rather than the antigen, which brought another hint that the development of the ADCs has to consider a variety of parameters besides the choice of target and the design of the linker. The characteristics of tumor cells also affect the activity of ADCs, including the endothelium, interstitial, and epithelial barriers which could limit ADCs uptake in the tumor, resulting in a small fraction of the injected dose reaching the desired tumor target (Perez et al., 2014). Intratumor distribution of ADCs also affects the anti-tumor efficacy (Tsumura et al., 2018).

Sometimes the efficacy of the ADCs does not have a positive correlation with the dose of the injection of ADCs. In addition to being interfered by the payload concentration threshold, the activity of ADCs could be affected by the saturation of the antigen–antibody combination, which causes the concentration of the ADCs in the circulation to be higher than the concentration of the corresponding receptors (Mager, 2006). Some antigens may shed from the tumor cells and circulate in the blood system to alleviate invalid combination with antibodies, which is also able to enhance the efficacy of ADCs (Pak et al., 2012). These internal factors seem to be imperative considerations when designing ADCs in the future.

### The External Conditions Related to Activity of ADCs

Another point worth attention is the choice of assessment method of safety and efficacy of ADCs. Owing to the ADC subjects to some physical and chemical conditions such as storage conditions, which is able to cause degradation or aggregation of ADCs to influence the assessment of ADCs' activity (Mohamed et al., 2018). Therefore, the assessment method must be considered to some extent.

#### CONCLUSION AND PERSPECTIVE

With in-depth understandings of antibodies, linkers, and payloads, ADCs have also achieved great development. The linkage strategy and target diversity have already improved the delivery of the payloads to tumor tissues and reduced exposure to normal tissues. With the development of payloads, some novel potent payloads are used by ADCs, which allows researchers to exploit novel linkers to attach the antibody and payloads without disturbing their potency (Dragovich et al., 2018). Furthermore, some irrelevant antigen-target ADCs also may exert toxicity to tumor cells due to the vascular gap of tumors relative to the normal tissues, which is big enough to make ADCs penetrate into tumor cells (Cardillo et al., 2011), indicating the specific recognition of ADCs by tumor tissues on another aspect.

Some prodrug strategies also are used in ADCs design, which modified the toxic payloads to inactive prodrugs, then utilized self-immolation groups and took advantage of the intratumoral environment to reduce the prodrugs to prototype drugs to exert intrinsic activity (Pei et al., 2018). Moreover, nanoparticles combining with the strategy of ACD prodrugs could also increase the activity and circumvent MDR (Qi et al., 2017). The key issues of ADCs are optimization of the appropriate antibody,

the choice of proper antigen, the selection of high-activity cytotoxic payloads, stable linkage technology and optimization of DAR in future development. These strategies will improve the efficacy of ADCs that give them a larger market share to replace chemotherapy drugs in medical therapy in the future. At present, ADCs in clinical trials mainly focus on hematological tumors especially Hodgkin lymphoma, because the CD30 is an ideal target, overexpressed in Hodgin lymphoma consistently. With the deep investigation of the target, more ADCs to cure other types of cancer will expand to clinical applications. However, the development of ADCs is costly to make, marked by Adcetris@ and Kadcyla@ imposing more family burdens on patients.

In recent years, peptide-drug conjugates (PDCs) are also on the stage of targeted-drug conjugation therapy and are considered as part of ADCs. PDCs replace antibodies with peptides, which minimize the molecular weight to alleviate the reduction of tumor cell absorption caused by the larger molecular weight of the ADCs. Also, PDCs could possess better homogeneity due to the few of the attached sites of the peptides. The cost-effectiveness of PDCs is critical to alleviate the pressure on patients during treatment. However, PDCs also have some weaknesses that need to be improved. The vulnerability of PDCs in the blood system is a nonnegligible obstacle, but it is difficult to improve half-life and reduce off-target toxicity by modifying structure of PDCs without destroying activity. Therefore, we must master more comprehensive knowledge to improve ADCs or PDCs. Whether used alone or in combination with other therapies, the toxicity of ADCs and PDCs must be better understood to adjust the therapeutic index based on the minimum effective dose of

#### REFERENCES

the drug in tumor cells and the maximum tolerated dose for normal tissues.

#### AUTHOR CONTRIBUTIONS

HT wrote the review manuscript. YL, ZY, MS, and WL modified the review. LL and QH edited the review including grammar. The correspondent authors MW and YJ provided the thought.

#### FUNDING

This work was supported by the National Natural Science Foundation of China and Liaoning Joint Fund Key Program (Grant No. U1608281), the National Natural Science Foundation of China (Grant Nos. 81601370 and 81673475), the Key Laboratory Foundation from Shenyang S&T Projects (Grant No. Z18-4-020), the National Key Research and Development Plan, Digital Diagnosis and Treatment Equipment Research and Development Key Special Project (Grant No. 2017YFC0114203), and the Liaoning Provincial Science and Technology Public Welfare Research Fund Project (Grant No. 20170030).

### SUPPLEMENTARY MATERIAL

The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fphar. 2019.00373/full#supplementary-material

TABLE S1 | The ADCs of cancer therapy in clinical development.

**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Copyright © 2019 Tang, Liu, Yu, Sun, Lin, Liu, Han, Wei and Jin. This is an openaccess article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

# Glesatinib, a c-MET/SMO Dual Inhibitor, Antagonizes P-glycoprotein Mediated Multidrug Resistance in Cancer Cells

Qingbin Cui 1,2, Chao-Yun Cai 2 , Hai-Ling Gao2,3, Liang Ren1 , Ning Ji 2,4, Pranav Gupta2 , Yuqi Yang2 , Suneet Shukla5 , Suresh V. Ambudkar 5 , Dong-Hua Yang2 and Zhe-Sheng Chen2 \*

1 School of Public Health, Guangzhou Medical University, Guangdong, China, 2 Department of Pharmaceutical Sciences, College of Pharmacy and Health Sciences, St. John's University, Queens, NY, United States, 3 Department of Histology and Embryology, Clinical Medical College, Weifang Medical University, Weifang, China, 4 Tianjin Key Laboratory on Technologies Enabling Development of Clinical Therapeutics and Diagnostics, School of Pharmacy, Tianjin Medical University, Tianjin, China, 5 Laboratory of Cell Biology, Center for Cancer Research, National Cancer Institute, NIH, Bethesda, MD, United States

#### Edited by:

Massimo Broggini, Istituto Di Ricerche Farmacologiche Mario Negri, Italy

#### Reviewed by:

Fabrizio Martelli, Istituto Superiore di Sanità (ISS), Italy Kamini Singh, Memorial Sloan Kettering Cancer Center, United States Sandro Cosconati, Università degli Studi della Campania Luigi Vanvitelli Caserta, Italy

> \*Correspondence: Zhe-Sheng Chen<EMAIL_ADDRESS>
#### Specialty section:

This article was submitted to Cancer Molecular Targets and Therapeutics, a section of the journal Frontiers in Oncology

Received: 25 January 2019 Accepted: 08 April 2019 Published: 25 April 2019

#### Citation:

Cui Q, Cai C-Y, Gao H-L, Ren L, Ji N, Gupta P, Yang Y, Shukla S, Ambudkar SV, Yang D-H and Chen Z-S (2019) Glesatinib, a c-MET/SMO Dual Inhibitor, Antagonizes P-glycoprotein Mediated Multidrug Resistance in Cancer Cells. Front. Oncol. 9:313. doi: 10.3389/fonc.2019.00313 Multidrug resistance (MDR) is one of the leading causes of treatment failure in cancer chemotherapy. One major mechanism of MDR is the overexpressing of ABC transporters, whose inhibitors hold promising potential in antagonizing MDR. Glesatinib is a dual inhibitor of c-Met and SMO that is under phase II clinical trial for non-small cell lung cancer. In this work, we report the reversal effects of glesatinib to P-glycoprotein (P-gp) mediated MDR. Glesatinib can sensitize paclitaxel, doxorubicin, colchicine resistance to P-gp overexpressing KB-C2, SW620/Ad300, and P-gp transfected Hek293/ABCB1 cells, while has no effect to their corresponding parental cells and negative control drug cisplatin. Glesatinib suppressed the efflux function of P-gp to [3H]-paclitaxel and it didn't impact both the expression and cellular localization of P-gp based on Western blot and immunofluorescent analysis. Furthermore, glesatinib can stimulate ATPase in a dose-dependent manner. The docking study indicated that glesatinib interacted with human P-gp through several hydrogen bonds. Taken together, c-Met/SMO inhibitor glesatinib can antagonize P-gp mediated MDR by inhibiting its cell membrane transporting functions, suggesting new application in clinical trials.

Keywords: multidrug resistance, P-gp, glesatinib, reversal effects, mechanism

## INTRODUCTION

Multidrug resistance (MDR) is the one of the major challenges in cancer treatment (1). MDR refers to a phenomenon that cancer cell once becomes resistant to one chemotherapeutic, accompanied by cross resistant to other chemotherapeutics that are structurally and mechanistically different (2). MDR is one of the major causes of failure in cancer treatment. The mechanisms of MDR involve dynamic ATP-binding cassette (ABC) transporters (3, 4), oncogenes mutations (5), microenvironment changes (6), reprogramed cancer cell metabolism (7, 8), efficient DNA repairing (9, 10), survived cancer stem cells (11, 12), and activated detoxifying systems (13, 14). Novel effective remedies are urgently needed to circumvent MDR.