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Immunotherapy is considered to be the fourth pillar of cancer treatment (in parallel with surgery, radiotherapy, and chemotherapy), by enhancing the ability of the immune system to target and kill tumor cells, including:

1) Blocking antibodies to the inhibitory immune checkpoint pathway
2) Cell therapy based on dendritic cells and engineered T cells
3) Vaccines that trigger antigen-specific immune responses in tumors, etc.

In recent years, antibodies that block the two immunological checkpoint proteins, CTLA-4 and PD-1, have become the regulators of the rules of cancer therapy. These antibodies liberate T cells from CTLA-4 and PD-1 pathway-mediated immunosuppression and have been shown to promote effective, long-lasting T cell responses, thereby eliminating tumors and leading to cancer remission.
However, unfortunately, only 10%-30% of cancer patients can benefit from such immune checkpoint blocking therapy, which has led to a strong interest in improving patient response. One strategy to achieve this goal is to combine immunological checkpoint blocking therapy with cell therapy or therapeutic vaccines.

 

 

The engineering approach to personalized immunotherapy:
1) Collect tumor samples from cancer patients
2) Aligning the tumor genomic sequence with the somatic genomic sequence to determine the mutation, and then predicting the new antigen by means of an algorithm
3-4) Production of new antigen-specific DNA, RNA and peptide vaccines, and preparation of personalized nanomedicine for use in combination with cancer immunotherapy
5) New antigens can also be used to produce dendritic cell vaccines or new antigen-specific T cells
6) Genes encoding new antigen-specific scFv or TCRs can be transduced into peripheral blood lymphocytes to produce tumor-reactive T cells for adoptive transfer into a patient.

 

I. Adoptive T cell therapy

T cells isolated from the patient’s blood can be purified to contain specific T cell populations, which can then be genetically engineered to enhance antitumor effects. In fact, in addition to blocking by immunological checkpoints, adoptive T-cell therapy (ACT) has become the mainstream of cancer immunotherapy, including in vitro purification and manipulation of patient-derived T cells, and subsequent injection of engineered T cells. Back to the patient. Tumor-infiltrating lymphocytes (TILs, isolated from patient biopsy and expanded in vitro by IL-2 stimulation) represent the first class of adoptive T-cell therapies, however, despite their superiority in tumor specificity, limited efficacy . TCR-T and CAR-T are two other adoptive T cell therapies, both based on peripheral blood T cells.
Among them, CAR-T therapy has been proven to be effective in the treatment of hematological malignancies, and the FDA approved two of these products in 2017 (disagenlecleucel and axicabtagene ciloleucel) for the treatment of acute lymphoblastic leukemia and large B-cell lymphoid tumor.

A major factor in the success of T-cell engineering therapies such as CAR-T is antigen-specificity. However, if the target antigen is expressed in both cancer cells and normal cells, such T-cell therapies may cause severe toxicity, so how to produce It is a research focus in this field to minimize the toxic side effects and increase the efficacy of CAR-T cells targeting cancer cells as much as possible. Studies have shown that one way to improve the specificity of T cell engineering tumor cells is to develop T cells that target new antigens (neoantigen, mutant proteins expressed by tumor cells, which are not found in healthy cells).

Another limitation of adoptive T cell therapy is the need to produce a sufficient number of cells in vitro, while in vitro T cell expansion is a labor intensive process requiring specialized skills, which limits the availability of such therapies. One possible solution is to transfect the CAR gene in situ in peripheral blood T lymphocytes, which can be achieved by CD3 targeting nanoparticles carrying the CD19 CAR gene. Another solution strategy is to develop artificial antigen presenting cells to more efficiently amplify functional T cells during in vitro proliferation.

 

In situ targeted delivery of CAR gene to peripheral blood T lymphocytes

When the T-cells such as CAR-T are reintroduced into the patient, maintaining the viability and tumor infiltration characteristics of these cells is also a challenge, especially when using adoptive T-cell therapy against solid tumors. It is important to ensure that T cells that are transferred to the patient proliferate and homing to the tumor. To this end, studies have added lipid nanoparticles (implementing local delivery of cytokines) carrying IL-15 super agonists and IL-21 cytokines on the surface of CD8+ T cells. These biomaterials enable the drug to be specifically targeted to cells that are transferred to the patient, avoiding off-target immune regulation, thereby safely improving the efficacy of T cell therapy. In addition, other studies have shown that biopolymers (such as alginate hydrogels) can also support the survival and proliferation of T cells after adoptive transfer.

 

II. Dendritic cell vaccine

Cellular therapies based on patient-derived, tumor-associated antigens (TAAs) expressing dendritic cells (obtained by in vitro differentiation of peripheral blood mononuclear cells) can also be returned to the patient to enhance T cell activation and tumor cells. Killing.

As early as 2001, there were studies showing that 10 children with metastatic solid tumors (including neuroblastoma, osteosarcoma, and renal cell carcinoma) received five patients after receiving biennial dendritic cell vaccination. Stable, one patient’s tumor significantly subsided; afterwards, studies have confirmed the potential of dendritic cell vaccine against other types of cancer (such as pleomorphic glioblastoma, ovarian cancer). However, the dendritic cell vaccine used in these previous studies, although well tolerated and improved in patient survival, did not completely relieve or resolve the tumor. Based on this situation, scientists began to develop DC vaccines that present new antigens in anticipation of improving the efficacy of such vaccines.

In 2015, a Phase I clinical trial involving 3 patients with melanoma who had previously been treated with anti-CTLA-4 therapy used a systematic approach to generating a DC vaccine carrying a new antigen. First, the researchers performed exome sequencing of the patient’s tumor to identify neoepitopes with missense mutations and to use predictive human leukocyte antigen (HLA) binding affinity tools and gene expression analysis pairs. These new epitopes are screened; then, dendritic cells presenting the new antigen are infused into the patient. In each patient’s treatment regimen, half (3) of the new antigen induced significant new antigen-specific CD8+ T cell expansion.

Although some studies have shown promising results, it is challenging to convert DC vaccines into reliable therapies for patients, in part because this requires well-trained personnel and specialized equipment to treat peripheral blood. Nuclear cells differentiate into dendritic cells in vitro, and dendritic cells are incubated with tumor antigens or tumor lysates. Furthermore, the production of dendritic cells presenting MHC-peptide complexes is not stable, depending on the condition of the peripheral blood mononuclear cells of the patient. On the other hand, after administration, only <4% of dendritic cell vaccines can homing to lymphoid tissues. Fortunately, these limitations are expected to be solved by new engineering methods, such as in situ vaccination with injectable alginate hydrogels (this method does not require manipulation of dendritic cells in vitro) to initiate localization. Dendritic cells trigger an anti-tumor immune response.

Another engineering solution to improve dendritic cell vaccines is to use exosomes. Studies have shown that the addition of exosomes in the culture of dendritic cells promotes antigen presentation of dendritic cells by “naturally occurring membrane exchange between exosomes containing tumor antigens and dendritic cells”.

 

III. Vaccine based on new antigen

In addition to patient-specific cell therapies, some scientists have confirmed that vaccines based on new tumor antigens are also expected to improve cancer immunotherapy.

 

 

Personalized vaccine preparation process

Peptide vaccine

Since 2012, a large amount of evidence has shown that the new antigen peptide vaccine can release the killing potential of new antigen-specific T cells (especially when combined with immune checkpoint blockade) and play a role in killing tumors with limited side effects. Related trials have also shown that new antigen vaccines can be used as a therapeutic strategy to prevent tumor recurrence and metastasis. However, the development of safe and effective anti-tumor new antigen therapy is very challenging, because the amino acid composition of the new antigen has an important influence on its isoelectric properties, which will make the new antigen peptide vaccine accumulate in non-target tissues, or not limited. Lymphatic tissue is transmitted through the system circulation. This delivery disorder results in only a small fraction of the vaccine injected into the body reaching the target tissue, reducing the efficacy of the vaccine and, therefore, an efficient delivery strategy is needed to enhance the transport of the new antigen and its adjuvant molecules to the lymph nodes. Some studies have confirmed that technologies based on synthetic high-density lipoprotein (sHDL)-based nanodiscs, DNA-RNA nanocapsules, and PC7A nanoparticles can help improve the targeting of new antigenic peptide vaccines. These nano-vaccines provide exciting proof-of-concept results for vaccination based on new antigens.

Individualized new antigen vaccination with nanodisks based on synthetic high-density lipoprotein (sHDL)

mRNA vaccine

In addition to peptide vaccines, some early clinical trials and preclinical studies have tested the safety and immunogenicity of mRNA vaccines based on new antigens. In addition, there is evidence that synergy between mRNA new antigen vaccines and immunological checkpoint blocking provides a clinically appropriate approach to promote T cell survival and enhance the strength and efficacy of anti-tumor immune responses.

 

IV. Imaging Technology / Photothermotherapy / Photodynamic Therapy

Imaging techniques, photothermal therapy (PTTs), and photodynamic therapy (PDT) are other joint pathways that are expected to improve cancer immunotherapy. Image-guided methods, including magnetic resonance imaging (MRI) and PET-CT, are expected to limit the off-target toxicity of combination immunotherapy by precisely controlling the timing and location of drug release.
Photothermal therapy uses photothermal agents or gold nanoparticles (GNPs) that generate heat under near-infrared light.
When the photothermal agent or gold nanoparticles are delivered to the tumor, the tumor can be ablated by infrared light irradiation.
A major challenge in cancer immunotherapy is that when applied to large tumors, efficacy is often limited.
Although in most cases the preferred treatment procedure is surgical removal of the primary tumor, direct surgical resection is not always feasible.
In this case, photothermal therapy may offer another option.
Photodynamic therapy is a similar technique that uses photosensitizers to produce reactive oxygen species to induce apoptosis in cancer cells.
Several studies have supported the use of photothermia and photodynamic therapy to eliminate primary tumors and trigger the release of tumor antigens (including new antigens), endogenous risk signals, and pro-inflammatory cytokines.
There is also evidence that photo-therapy and photodynamic therapy-induced immune responses can be combined with chemotherapy and immunotherapy.
A study published in 2018 showed that photothermotherapy combined with chemotherapy can induce powerful systemic anti-tumor immunity against diffuse and untreated tumors.

Combined chemotherapy and photothermal therapy to remove distal secondary tumors

 

Summary

Each immunotherapy has its own unique advantages:

1) Delivery of a new antigenic peptide vaccine via nanoparticles provides a true patient-specific treatment that can be tailored to individual tumors
2) Gene therapy using nanoparticles or polymers to deliver RNA encoding new antigens has shown good efficacy
3) Personalized cell therapy including adoptive transfer of genetically engineered T cells can effectively promote anti-tumor immunity and enhance effector cell function
4) Dendritic cell vaccines can present new antigens to T cells of patients in vivo.

For now, the most important thing is how to combine these strategies to promote synergistic anti-cancer effects, thus achieving an unprecedented therapeutic effect.
In the future, the most ideal goal is that personalized immunotherapy for cancer patients not only eradicates tumors, but also maintains anti-tumor immunity for the rest of the patient’s life.

Clinical advantages and barriers to different technologies for personalized cancer immunotherapy

 

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