Immunological checkpoint inhibitors currently have been dramatically investigated and the most reported inhibitors are cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) and programmed death receptors 1/programmed death receptor-ligand 1 blockade (PD-1/PD-L1). The body’s immune system uses the immune checkpoints to control the corporeal immune balance and maintain self-tolerance (63). In normal conditions, the activated T cells express PD-1 to recognize abnormal or cancerous cells and then eliminate them to protect the body from their development (64, 65). However, the tumor cells might up-regulate the expression of PD-L1 or PD-L2 that bind to PD-1 to evade recognition and attack of immune cells (66, 67). Therefore, anticancer immunotherapy can be achieved by using blocking inhibitors of PD-1 or its ligand. Another immune checkpoint is CTLA-4, which diminishes the T-cell activity and assists the maintenance of self-tolerance (68). The anti-CTLA-4 antibody has been used to block the CTLA-4 to induce T cell activation, which inhibits tumor growth.

Nonetheless, many challenges remain with these checkpoint blockade strategies that limit their application for complete cancer treatment. As mentioned above, the administration of immunological checkpoint inhibitors can cause severe harm to different normal organs (69, 70). Moreover, only a small portion of patients show positive effectiveness of checkpoint inhibitor treatments, this phenomenon is still under study (71, 72). In addition, the extracellular matrix and microenvironment of different tumors suppress the immune recognition and activation (73). With the new development of polymers and biomaterials science, these challenges can be overcome by utilizing various polymers to obtain targeting tumor delivery ( Table 1 ).

One of the typical strategies in ICI is applying siRNA gene transfection to express the protein knockout of the PD-1/PD-L1 immunosuppressive pathway. This strategy has performed promising results for cancer treatment (84). Specifically, even though adoptive T cell immunotherapy performs promising results in epithelial ovarian cancer (EOC) treatment, the EOC cell-expressed PD-L1 can interact with PD-1 from T cells and induce undesired immunosuppression resulting in low therapeutic effect (85). To overcome this problem, Teo et al. have designed a folic acid (FA) functionalized PEI polymer complexed with PD-L1 siRNA. The research showed the polyplex, which consisted of FA/polymer/siRNA, has successfully blocked the PD-1 and PD-L1 pathways and repelled the immunosuppression of T cells, leading to promote the recognition of T cells toward EOC cells (74). It is important that the FA not only lower the cytotoxicity of the PEI but also effectively enhance specificity of uptake into EOC tumor cells.

On the other hand, transforming growth factor-β (TGF-β) mediated tumor microenvironment also plays an important role in immune suppression besides the PD-1/PD-L1 pathway (86, 87). Unfortunately, the TGF-β signaling is necessary for many cellular processes so utilizing TGF-βR1 inhibitors can cause severe side effects such as hemorrhagic, degenerative, and inflammatory lesions in heart valves (88). To overcome this problem, diverse polymeric nonviral vectors have been generated to encapsulate TGF-βR1 inhibitors and target them to the tumor site. In 2017, Schmid et al. introduced a CD8+ T cell-specific nanoparticle system based on PLGA-PEG polymer conjugated with anti-CD8a F(ab’)2 fragments to encapsulate TGF-βR1 inhibitor SD-208 (50). The nanoparticles system successfully reduced the cytotoxicity of TGF-βR1 inhibitor SD-208 to normal cells and recover the immune function of T cells. Moreover, the PD-1-PLGA-PEG nanoparticle co-delivery of Toll-like receptor (TLR7/8) agonist (R848) showed recruited a significantly high number of T-lymphocytes at the tumors. In addition, a nanoparticle system containing poly(ethylene glycol)-block-poly(D,L-lactide) (PEG5k–PLA11k) and the cationic lipidN,N-bis(2-hydroxyethyl)-N-methyl-N-(2-cholesteryoxycarbonyl-aminoethyl) ammonium bromide (BHEM-Chol) has been used to deliver immunosuppressive factor siRNA to a tumor (75). The siRNA encapsulated in the copolymer-based nanoparticles not only was protected from enzymatic degradation but also enhanced the cell internalization compared with negatively charged bare siRNAs. The system exhibited a favorable modulation implement in tumor invasive CTL. The loaded CTL-associated molecule-4-siRNA nanoparticles (NPsiCTLA-4) effectively stimulate T cells’ activation and hinder tumor growth in melanoma mice.

To overcome the limitations of checkpoint blockers, the polymeric micelles have also been a promising system. According to their special structure which consists of polymeric amphiphiles, micelles can carry and deliver manifold hydrophobic drugs to the target tumor. Recently, Peng et al. introduced a polymeric micelle system for tumor immunity post photothermal therapy (PTT) based on amphipathic polymer MPEG-PCL to co-deliver photosensitizer IR780 and NLG919 (an indoleamine 2,3-dioxygenase (IDO) inhibitor) (51). This nano system has shown sufficient accumulation at the tumor site and shifts to lymph nodes to promote the activation of T lymphocytes. In another research, an immunostimulatory dual-functional nanocarrier based on a prodrug conjugate of PEG with NLG919 was studied by Chen et al. The system was also equipped with a Fmoc group, a drug-interactive motif for enhancing drug loading capacity and formulation stability. The PEG2k-Fmoc-NLG alone showed greatly stimulated T-cell immune responses and excellent tumor inhibition in vivo. It is noteworthy to mention that the systemic administration of paclitaxel (PTX) loaded PEG2k-Fmoc-NLG nano system exhibited significant tumor inhibition in both melanoma and breast cancer mouse models (76). A redox-responsive immunostimulatory polymeric prodrug carrier which can controllably co-deliver chemotherapeutic DOX and immune checkpoint inhibitor NLG was introduced by Sun et al. (77). The system, which is called POEG-b-PSSNLG prodrug (PSSN10), was a synthesized poly(oligo(ethylene glycol) methacrylate)-poly(N,N′-(tbutyoxycarbonyl)cystamine) copolymer conjugated NLG919 prodrug which can self-assemble into nano-sized micelles. The PSSN10 carrier can improve cell immune responses in the lymphocyte-Panc02 co-culture experiments and exhibited significant anti-tumor activity in vivo. Moreover, the DOX/PSSN10 micelles displayed higher efficacy in the tumor growth inhibition and longer survival rate of 4T1.2 tumor-bearing mouse model compared with free DOX or a clinical formulation of liposomal DOX (DOXIL). Another IDO inhibitor, indoximod, was also co-delivered with DOX in a synthesized copolymer POEG-b-PVBIND micelles by Wan et al. in 2019 (78). The indoximod conjugating copolymer micelles effectively promoted the anti-tumor immunity resulting in an extreme tumor inhibition effect in a preclinical breast cancer model. In a different concept, Lan et al. developed a dual functional indoximod-based carrier PEG2K-Fmoc-1-MT also for breast cancer chemo and immunochemotherapy (79). The polymeric micelle itself successfully inhibited IDO effect with decreased kynurenine (KYN) production leading to the proliferation of CD4+ and CD8+ T cells. The DOX/PEG2kFmoc-1-MT micelles can generate an immunogenic cell death process, subsequently secreting many cytokines [such as interferon (IFN)-γ, IL-2, and tumor necrosis factor-alpha (TNF-α)] inducing later T cell-mediated immunity. The 4T1 murine breast cancer model tumor inhibition profile of DOX/PEG2kFmoc-1-MT micelles was dramatically high with long survival time compared with other groups. Another amphiphilic PEGpoly-1-Methyl-l-Tryptophan (MLT) block of copolymer self-assembled polymeric micelles was investigated by Huang et al. which showed effectively reduced levels of KYN in activated macrophages (80).

As mentioned above, CTL-4 can suppress the activation of T cells and promote self-tolerance. The anti-CTLA-4 antibody can be used for blocking the CTLA-4 and stimulating the activity of T cells toward the tumor. CTLA-4 antibodies have been loaded in poly(lactic-co-hydroxymethyl-glycolic acid) (PLHMGA) by Sima et al. for cancer immunotherapy. The nanoparticle system has been proven to block inhibitory receptors on T cells and obtained promising therapeutic efficacy than the IFA formulation in colon carcinoma tumor model (MC-38) (81). Lei Zhang et al. reported a study on PLGA microparticles to co-deliver IL-2 and CTLA-4 antibodies. The surface of the PLGA microparticles was conjugated with a H-2Kb/TRP2-Ig dimer and anti-CD28. The polymeric macroparticles successfully co-released IL-2 and anti-CTLA-4 inducing dual effects in activating and promoting tumor antigen-specific T cells. The systems exhibited enhancement in anti-tumor efficacy in a mouse melanoma model (82).

Similar to CTL-4, diverse PD-1/PDL-1 inhibitors have been used for combinatorial therapy in recent clinical trials (89). Different kinds of polymers have been exploited with PD-1/PD-L1 blockade for cancer immunotherapy presently. Especially, utilizing the synergistic effect of diverse types of delivery systems can exploit the maximized potential of immune therapeutics. For example, Chao et al. reported a hyaluronic acid (a biocompatible natural polymer) microneedle integrated with pH-sensitive dextran nanoparticles (NPs) that can encapsulate and release PD-1 antibodies in a controlled manner to melanoma tissue. The report showed that this self-degradable microneedle encapsulated PD-1 antibody system generated a higher robust immune response compared to free anti-PD-1 antibody at the same dose in a B16F10 melanoma model (83). Another study conducted by Ye et al. has also produced immunotherapeutic nanoparticles from hyaluronic acid but modified with 1-methyl-DL-tryptophan (1MT) to deliver anti-PD-1 antibody. The particles combined with microneedle successfully sustain release and increased the accumulation of anti-PD-1 antibodies in the TME. The system indicated the improvement in tumor growth inhibition and lowered the immunosuppression in a B16F10 melanoma model (52). In addition, the specific tumor targeting nanoparticles can possibly enhance the performance of antitumor immunity. For example, a pH and matrix metalloproteinase dual-sensitive micellar nanocarrier which can spatiotemporally control the release of anti-PD-1 and PTX in solid tumors has been developed by Su et al. (53). The report interestingly indicated that the PTX-induced immunogenic cell death (ICD) can activate the antitumor immunity along with the blocking of the PD-1/PD-L1 axis from anti-PD-1. Together, they hinder the immune escape of tumor cells due to PTX-induced PD-L1 up-regulation. Of note, the pH-sensitive polyethylene glycol (PEG) shell could be sheddable at the tumor acidity site resulting in release of anti-PD-1 and PTX. In general, polymer-based material can be utilized as a superlative and effective delivery system to sustain biocompatible antibodies and other immunological checkpoint inhibitors in cancer immunotherapy ( Figure 2 ).

The term “adoptive cell transfer” (ACT) involves a group of cell-based anticancer immunotherapies and is very attractive for its smart and patient-tailored strategies (90, 91). ACT contains different steps to induce immune-mediated clearance of cancer. First, the circulating or tumor-infiltrating lymphocytes are collected from the patient. Then the cells are elected, activated ex vivo, genetically modified to express a cancer-targeting receptor, and multiplied to a therapeutic quantity. Finally, the cells are reinjected into the treated patient to recognize and eliminate cancer cells.

CAR-T cell is the most representative of ACT recently and has obtained optimistic clinical success. This therapy utilizes CAR to engineer autologous T cells for achieving immune activation without major histocompatibility complex (MHC)-restriction. The CAR is mainly composed of an extracellular antibody-derived antigen binding domain for cancer targeting and a one or more linked intracellular signaling domain. The extracellular binding domains are commonly constructed from single-chain variable fragments (scFvs) derived from tumor antigen-reactive antibodies (92). The intracellular signaling domain comprises CD3ζ chain domain and co-stimulatory domains, such as CD28 and/or 4-1BB to provide costimulatory signals for promoting CAR-T cell expansion, persistence, and function (93).

However, the manufacturing of CAR-T cell is time-consuming, expensive, and technically complex compared to other small therapeutic drugs. There are multiple aspects that need to be carefully controlled to archive a secure, therapeutically safe and effective CAR-T cell therapy, such as balanced CD4/CD8 ratio, the viability of differentiated CD3+CAR+ cells, and in vitro cytotoxicity and cytokine release against cells expressing the target antigen, which makes it costly and limits its widespread use (94). For instance, the list cost of Kymriah is US$475,000 and of Yescarta is US$373,000 which is higher than the current common cancer therapies’ cost (95). The manufacturing time to archive enough therapeutic cell numbers is from 3 to 4 weeks at least depending on different methods (96–98). It is noteworthy that the consistency of cell products plays a significant role in the tumor clearance effect of the patient. An optimum manufacturing of engineered T-cell process is required for reducing the cost and escalating clinical translation of CAR-T cell therapy.

Notably, utilizing engineered polymers can improve and streamline CAR-T cell manufacturing process. Diverse kinds of polymers have been synthesized and modified to apply in many medical engineering processes including cell culture (99), tissue engineering (100), separation (101), and drug and gene delivery (102). Similarly, many polymeric systems have been used to optimize streamlined CAR-T cell manufacturing process, mainly focusing on activation and genetic modification of the CAR-T cell.

The ex vivo CAR T-cell activation consists of three central signaling steps, which are T cell receptor (TCR) stimulation, CD28 co-stimulation, and cytokine signaling. Usually the T cell receptor (TCR) stimulation and CD28 co-stimulation utilize independent anti-CD3 and anti-CD28 monoclonal antibodies (mAbs) adhered to solid materials for receptor clustering. On the other hand, the cytokine signaling commonly uses soluble cytokines dissolved in the culture medium. Therefore, an activation platform should match the above requirements and these following criteria. The platform first needs to be “friendly” and can promote T cells to multiply to around 1 to 5 × 10⁸ CAR+ T cells for one patient (103), and maintain the stably therapeutic state in vivo. It is favorable that the platform can keep the balance number of CD4+ and CD8+ T cells in expansion (104). One more crucial point is the activation materials should be convenient to process and easily separate from T cells.

In this part, we discuss different polymeric T cells activation systems and their properties for adequately activating and expanding T cells.

Compared to viral transduction and nucleofection with expensive cost and safety issues, polymer-based nonviral gene delivery systems have recently become a potential candidate for CAR T-cell genetic modification. Cationic polymers which can electrostatically interact with negatively charged DNA and RNA to form polyplexes are the most favorable polymers for this purpose. Moreover, the positive charge of polyplex facilitates cellular uptake by the ionic interaction with negatively charged proteoglycans on the cell surface (133). Especially, if the cationic polymers contain amine groups, the amine groups will be protonated in the pH range of 5.0 to 6.8 and induce the proton sponge effect resulting in endosomal escape (134). Numerous polymers have been exploited such as poly(β-amino ester) (PBAE) (135), poly(2-dimethyl)aminoethyl methacrylate) (pDMAEMA) (136), polyamidoamine (PAMAM) (137), and branched polyethylenimine (bPEI) (138).

In this part, we report different polymer architectures for nonviral gene delivery to T cells, arranging them by ex vivo and in situ application. We also review main key barriers that require further improvement to achieve more efficient transfection with these systems.

Oncolytic virotherapy is the most auspicious access for tumor immunotherapy. The vital advantage of oncolytic virotherapy is based on the ability of replication-competent viruses that can proliferate selectively at tumor cells (146). Among many different viruses, adenoviruses can be represented as an example for illustrating oncolytic viruses as a whole. In 2005, the State Food and Drug Administration of China approved Oncorine, a replicative, oncolytic recombinant ad5 (rAd5-H101) for treating refractory nasopharyngeal cancer. This was considered the first approved oncolytic virotherapy for clinical use in the world (147, 148). Many types of cancer cells lose the p53 gene which causes drug resistance and lower survival rates in cancer patients (149). The p53 gene inactivation cell can halt the activation of apoptotic pathway. The Oncorine is a human serotype 5 adenovirus which is deleted by the E1B 55K gene. The elimination of the E1B 55K gene prohibits viral proliferation in normal cells, tolerating only multiples in the p53-lacking host cells. Therefore, the rAd5-H101 selectively proliferates in tumor cells and causes cancer cell lysis. The newly generated viruses release and infect surrounding cancer cells which leads to a chain-reaction of ultimate cancer cell destruction (150). The Oncolytic Adenovirus (oAd) recently become one of the most interesting generic immunotherapies for cancer in numerous phases of clinical trials (151, 152). The adenovirus (Ad) possesses several advantages that are beneficial for generic immunotherapy, such as the transduction ability of dividing and non-dividing cells with high efficacy, high loading capacity, easy modification of Ad genome, high production of viral progenies, and subsequent spreading of progenies to adjacent cancer cells (153–156).

It is noteworthy that the virus propagation inducing cell lysis is an extremely immunogenic process (157). This aspect is crucially relevant considering that the cancer cells usually disguise themselves from the host immune systems. The cell lysis releases multiple immunogenic molecules, such as abundant tumor-associated antigens for presenting to dendritic cells. The released virus genomes induce immunological danger signals through pathogen-associated molecular pattern (PAMP) and damage-associated molecular pattern (DAMP) receptors. These simultaneous actions stimulate the adaptive immune system, including helper CD4+ T cells and cytotoxic CD8+ T cells, toward the tumor resulting in disabling the tumor immunosuppression (158). Moreover, the T cell immunity toward the replicated adenovirus can enhance the overall antitumor process (159). The adenovirus infection can also indirectly activate the nature killing cells response for further immunogenic tumor inhibition (160). Indeed, many reports have investigated and confirmed the ability to inhibit the growth of different tumors of locally administered oAds, both in preclinical and clinical cases (161–166). The Oncorine is representative of oAds that have been used for clinical cancer treatment. However, the oAds still face certain challenges that narrow the therapeutic effectiveness for clinical trials.

One of the major hurdles for oAds’ efficacy is the pre-existing humoral immunity of the host body. Nowadays the human adenovirus serotype 5 (Ad5) is the most popularly used for adenoviral virotherapy and many reports showed that high percentages of the general population possess anti-Ad5 neutralizing antibodies (Nabs) which can easily terminate the bioactivity of Ad5. On the other hand, another utmost limitation of utilizing oAds for cancer therapy is the internalization of oAds dramatically depends on appropriate receptors, such as the Coxsackievirus and adenovirus receptor (CAR) on the surface of targeted cells. The CAR is a protein that belongs to a type I membrane receptor for subgroup C adenoviruses. CAR protein is expressed in many human tissues, including brain, heart, and some endothelial and epithelial cells (167–169). The efficiency of adenoviral transgene expression and CAR expression have been corresponded in numerous studies, suggesting that adenoviral binding and entry into target cells play an important role in achieving successful adenoviral gene expression (170, 171). Unfortunately, several cancer cell lines and clinical cancer tumors have been frequently observed in the loss of CAR expression, preventing attempts to achieve adequate oncolytic adenovirus virotherapy for cancer patients (172–175). It is impossible for oAds to obtain satisfactory remedial efficacy without overwhelming this CAR-dependent internalization. To overcome this, most currently ongoing clinical trials of oAds have been genetically modified to equip the beneficial fiber region. This adjusted fiber region can improve cellular internalization of the adenovirus and is independent of CAR expression level in heterogenic clinical tumor or tumor-specific internalization (176–178). However, processing genetic engineering for optimization of fiber-modified virus is risky and contains various disadvantages such as excessive cost, time, and labor-consuming. Inappropriate genetic editing can cause viral replicability loss, viral inactivity, and inadequate gene sequence expression (163).

To overcome these severe limitations of both local and systemic administration of oAd, many advanced polymer-based delivery techniques have been developed in recent years. The polymer-based delivery techniques can enhance the bioavailability while at the same time provide the necessary ( Figure 4 ) protection of oAd. The delivery and tumoral targeting profile of encapsulated oAd can be adapted to the specific medical goals by choosing a proper encapsulation polymer (179, 180). By covering the outer surface of oAd, the polymer-based delivery systems can avoid adverse problems caused by the viral capsid. Furthermore, the applied materials can sufficiently equip the oAd with beneficial properties and override the natively disadvantageous attributes of viral vectors, which efficiently contribute to improving the tumor-specific accumulation of oAd (58, 181–183) ( Table 2 ).