With the rapid development of tumor immunotherapy, drugs have been approved at PD-1, CTLA-4, PD-L1, LAG-3 and other immune checkpoints, bringing a lot of enthusiasm for research and development to the industry. The principle of using mRNA in tumor immunotherapy is to use mRNA to encode mutant tumor suppressor protein and modify the tumor microenvironment so as to achieve the purpose of treatment. The application of this method is limited by mRNA delivery technology. With current delivery capabilities, mRNA cannot reach every tumor cell in the patient’s body. Therefore, researchers are paying more and more attention to using mRNA as a therapeutic vaccine to induce the immune system to recognize and kill tumor cells.

In the research and development of COVID-19 mRNA vaccines, the experience gained by the industry in the rapid development and production of mRNA drugs and encoding complete disease antigens in vivo makes mRNA vaccine technology very promising in tumor therapy. In addition, some patients’ resistance to current immune-targeting drugs also creates opportunities for mRNA vaccines to be used for tumor therapy.

​There are several common challenges in the development of therapeutic vaccines. First of all, infectious disease vaccines achieve preventive function by inducing humoral immunity, while therapeutic tumor vaccines must also be able to induce a strong CD8+T cell response to completely eradicate all tumor cells. Second, the development of a tumor therapeutic mRNA vaccine needs to in vivo encoded antigens that can induce a highly specific immune response to the tumor. Because of the high variability of antigens among individuals, researchers have developed a series of new patient-specific antigens to indeal with this challenge. Lastly, even if antigens can induce the cellular immune response, the inhibitory tumor microenvironment can prevent T cells from infiltrating tumor tissue and may even lead to T cell failure.  Therefore, therapeutic vaccines need to be used in combination with drugs to overcome the inhibitory tumor microenvironment, such as immune checkpoint inhibitors.

Tumor-associated Antigen

Tumor-associated antigen (TAA) is mainly distributed on the surface of tumor cells and is the recognition site for the immune system to attack tumors. The targets of tumor vaccines include a variety of known TTA.

BNT111 consists of mRNA that encodes four melanoma-associated antigens: New York esophageal squamous cell carcinoma 1 (NY-ESO-1), tyrosinase, melanoma antigen A3 (MAGE A3) and transmembrane homologous phosphatase-tensin (TPTE). The mRNA sequences of the four TAA were optimized and the corresponding proteins could be translated into immature DC cells. Each sequence also contains a signal peptide, tetanus toxoid CD4+P2 and P6 epitopes, and a major histocompatibility complex (MHC) Ⅰ transport domain used to enhance the antigen presentation and immunogenicity of human leukocyte antigen (HLA).

The results of 18-fluoro-2-deoxy-D-glucose positron emission tomography (PET) of the spleen showed that the metabolic function of the spleen was enhanced, indicating the activation of lymphoid tissue. The results of enzyme-linked immunosorbent assays showed that about 75% of the 50 subjects had positive results for at least one of the four kinds of TAA, that is, an immune response. Antigen-specific T cells are OD1+CCR7-DD27+/-D45RA-effector memory T cell phenotypes, which can secrete IFN- γ and tumor necrosis factor after activation. In patients with continuous vaccination, the number of TTA-specific cells remained stable or even increased, while in patients who no longer received the vaccine, T cells survived for several months, followed by a gradual decline. By transfecting TAA-specific T cell receptors cloned from vaccinated patients into healthy donor CD8+T cells, the transfected cells can cleave melanoma cell lines.

After each administration, the levels of IFN- α, IFN- γ, IL-6 and other cytokines increased, which usually reached the peak a few hours after inoculation and returned to the normal level 24 hours later, and this was consistent with the observed characteristics of adverse reactions. The adverse reactions are mainly mild to moderate influenza-like symptoms, which are usually short-lived and self-limited (self-limited diseases are diseases that can stop automatically after the disease develops to a certain extent and gradually recover. Such as chicken pox, pityriasis rosea, alopecia areata and the common cold). The results of the first imaging evaluation of 42 patients were encouraging. Of the 25 subjects who received monotherapy, 3 had partial remission and 7 had stable disease, while of the 17 patients who received vaccine combined with PD-1, 6 had partial remission. Interestingly, two patients who had progressed after anti-PD-1 therapy responded to PD-1 therapy again after vaccination, which supported the conclusion that induced T cells belonged to the PD1+ effect memory T cell phenotype. Currently, BNT111 is conducting a phase II clinical trial of melanoma.

Personalized New Antigen

In the process of tumorigenesis and development, malignant tumor cells continue to mutate to produce protein sequences that are not expressed by normal cells. These proteins are processed into peptides by the proteasome and recognized by T cells. These new antigens are usually unique to each patient and bring opportunities and challenges to tumor-specific and patient-tailored immunotherapy.

When designing an mRNA vaccine that encodes a new patient-specific antigen, it is necessary to collect tumor samples from patients and identify the patient-specific new antigen by next-generation sequencing technology. The mRNA encoding the new antigen was then injected into the patient to induce the immune system to attack the tumor. However, this process must be accelerated and patients must be treated effectively before the tumor progresses further. It is reported that the above steps must be controlled within 30-40 days. Since the production of mRNA vaccine must be carried out under GMP conditions, the products must meet certain quality standards, which brings great challenges to pharmaceutical companies.

So far, most of the work on personalized new antigen vaccines is still focused on new antigen polypeptide vaccines, but the above work has not been substantially successful. In theory, the malignant tumor with the highest tumor mutation burden (TMB) is the best application scenario for the new antigen vaccine, but it is also most likely to develop drug resistance. Compared with the polypeptide vaccine, the new antigen encoded by mRNA has moderate immune irritation, can provide stronger immunogenicity, and patients can get more clinical benefits. Unlike polypeptide vaccines, mRNA can encode the whole antigen and present multiple epitopes. In addition, mRNA vaccines can simultaneously express multiple new antigens (multiple mRNAs express different new antigens, or fuse different new antigens into the same mRNA sequence). Some tumors can produce dozens of new antigens. Based on the requirement of inducing a wider immune response, the expression of multiple epitopes is more likely to induce the T cell response.