Innovations in cancer treatment as novel potential and the myth of personalized vaccines, immunotherapy, CRISPR/Cas9, and bacteriotherapy as promising therapeutic strategies

Abstract

Recent advancements in cancer treatment, including targeted therapy, tailored vaccines, immunotherapy, and bacteriotherapy, have demonstrated remarkable potential in addressing various types of cancer. However, the pursuit of novel therapeutic strategies remains challenged by significant obstacles, including toxicity to healthy tissues, limited penetration into malignant tissues, and the potential for tumor cell resistance to pharmacological agents. This study explored the potential of tailored vaccinations, immunotherapy, CRISPR/Cas9, and bacteriotherapy for cancer treatment. Randomized and non-randomized studies were reviewed using the Cochrane Central Register of Controlled Trials (CENTRAL), PubMed (NCBI), Scopus (ELSEVIER), and Web of Science (CLAVIRATE) databases. This research highlights the promising use of personalized cancer vaccines, which induce tumor-specific immune responses targeting neoantigens unique to each patient. Immunotherapy, which enhances the ability of the immune system to identify and destroy cancer cells, has revitalized tumor immunology. While the effectiveness of immunotherapies, such as immune checkpoint inhibitors (ICIs) and adoptive cell transfer (ACT), is variable, certain cancer patients show substantial benefits. Bacteriotherapy has also proven effective in promoting tumor remission and inhibiting metastasis, either alone or in combination with conventional treatments, by reducing tumor proliferation and metastasis. Additionally, bacteriotherapy serves as a potential platform for delivering therapeutics, genes, or medications directly to tumors. The CRISPR-Cas9 genome-editing technique shows promise for advancing cancer treatment, with the potential to target and treat genetic mutations at the tumor level, although its application in human cancer therapy is still under development. This study highlights the potential of these innovative approaches in the fight against cancer.

Introduction

The 2023 cancer statistics report released by the American Cancer Society revealed a substantial 33% reduction in the death rate attributed to cancer since 1991 and a modest 1.5% decline in the period from 2019 to 2020 [1]. This decline in mortality rates has been observed across various types of tumors, including leukemia, melanoma, and renal cancer, despite an increase in the incidence of these cancers. Anticipated new cancer cases for the year 2023 stand at approximately 2 million, with prostate, lung, and colorectal cancer (CRC) being the predominant diagnoses in men, comprising a total of 48% of cases [2]. In women, breast, lung, and (CRC) (CRC) account for 52% of diagnoses. Among these, prostate cancer is projected to be the most prevalent among men at 29%, while breast cancer is expected to have the highest incidence rate among women at 31%. Improved survival rates have been steadily rising, with a 68% 5-year survival rate for patients diagnosed between 2012 and 2018, in contrast to the 49% survival rate for those diagnosed in the 1970s [3]. One contributing factor to these improvements is the enhanced categorization of patients for treatments that target specific driver mutations [4] Common treatments for cancer are surgery, chemotherapy, or radiation therapy but there are complications associated such as the destruction of normal body cells, Anemia, bleeding, and infection. These treatment options are not working in a good manner due to adverse side effects. Novel therapeutic options such as personalized cancer vaccines, immunotherapy, bacteriotherapy, and CRISPR-CAS9 are the modern options due to its accuracy, long-term memorization, and complete elimination of the cancer microenvironment with minimum side effects. This review aims to combine all novel therapeutic options and current progress on these treatments [5]. Personalized cancer vaccines bacteriotherapy, immunotherapy, and CRISPR-CAS9 are the most suitable and highly advanced treatment options for eliminating cancer. the goal of developing individualized cancer vaccines is to reduce side effects while increasing the likelihood of a strong immune response targeting tumor-specific neoantigens and establishing long-term protection against cancer [6, 7]. Bacteria have the ability to multiply in tissues, their distinct virulence factors that can be used to fight tumors, and their antibiotic-controlled population, bacteria are being considered as potential micro-medication for cancer treatments [8]. Cancer treatment includes immunotherapy. It boosts the immune system and helps find and kill cancer cells using body-produced or lab-made chemicals. Immunotherapy treats many cancers. It can be used alone or with chemotherapy. Because of its precision and accuracy, CRISPR/Cas9 is being considered as a cancer treatment. It treats brain, colorectal, hepatocellular, renal cell, and urinary bladder cancers worldwide. The trails vaccines of cancer are represented in Table 1 [9].

Table 1.

Represent the class of different vaccines under trial for various cancers (https://clinicaltrials.gov) the table represent different trials of vaccines, common side effects and number of doses

Personalized vaccine (study, trial, name)	Cancer types	Treatment	Study evaluation
Personalized Peptide Vaccine for Pancreatic Cancer or Colorectal Cancer NCT02600949	colorectal d pancreatic ductal adenocarcinoma	Subcutaneously pembrolizumab injection	Toxicity, PFS, RR, tumour biomarker CA19-9,
Personalized and Cell-based Antitumor Immunization MVX-ONCO-1 in Advanced HNSCC NCT02999646	Squamous Cell Carcinoma (HNSCC)	MVXONCO-1 subcutaneous vaccination	Assess OS at 26 weeks, time to next treatment, response duration, AEs, PFS, and OS.
Personalized Tumor Vaccine Follicular Lymphoma NCT03121677	Follicular Lymphoma	subcutaneously injection	Review response time and select OS and PFS.
mRNA Vaccine Encoding Neoantigen in Patients with Advanced Digestive System Neoplasms NCT03468244	squamous carcinoma, gastric adenocarcinoma, pancreatic colorectal	Subcutaneous mRNA injection	Assess tumour vaccine’s safety, tolerability, and effectiveness.
Neoantigen Vaccine for Pancreatic Tumor NCT03645148	Pancreatic cancer	Subcutaneous Personalized neoantigen vaccine	Use ELISPOT to assess neoantigen immune response.
Vaccine to Nab-Paclitaxel, Durvalumab and Tremelimumab NCT03606967	breast carcinoma,	Subcutaneous injection	Evaluate PFS
Smoldering Multiple Myeloma NCT06574126	Multiple myeloma	Subcutaneous injection	Assess the feasibility, intensity and longevity
Neo-antigen Vaccine (NeoPepVac) NCT03715985	lung cancer (NSCLC),	Subcutaneous with 5 doses successively	Incidence of treatment related Ages
NeoAntigen Cancer Vaccine Combined with Anti-PD-1 in Melanoma NCT04072900	Melanoma	Administration of a personalized neoantigen cancer vaccine.	Assess AEs, complete remission rate.
Neoantigen Vaccination With Dendritic Cells NCT04912765	Triple-negative breast cancer (TNBC)	Subcutaneous	Assess AEs and safety
PCV13 NCT04613375	Diffuse large-cell lymphoma,	3 times injection of intramuscular injection	Determine humoral RR to PCV13 vaccine.

Personalized vaccine for cancer treatment

A tailored neoantigen-based vaccination called NEO-PV-01 was coupled with PD-1 therapy in the first open-label phase IB clinical study (NCT02897765) of 122 advanced solid tumor patients. Out of 82 patients, the median progression-free survival (PFS) for vaccinated melanoma was 23.5 months, NSCLC was 8.5 months, and bladder cancer was 5.8 months. Melanoma and NSCLC patients did not attain the median overall survival (OS) of 20.7 months for vaccinated people. However, bladder cancer patients had a 20.7-month OS. The research focused on NEO-PV-01 and nivolumab safety and tolerability. The vaccine’s most prevalent side effects were injection site responses (52% of patients) and flu-like symptoms (35%). No significant medication adverse effects were detected. This treatment was safe and effective for advanced solid tumours [10].

Individualized neoantigen-specific immunotherapy (iNeST) RO7198457 was tested in 144 locally or metastatic solid tumor patients in a phase IB research (NCT03289962) utilizing atezolizumab. RO7198457, an mRNA vaccine, has 20 neoantigens. Of the 108 patients evaluated, one had a complete response (CR) among the nine who responded to medication (ORR 8%), and 54 (49%) had stable illness. Vaccination produced neoantigen-specific T cells in 77% of people. Infusion-related reactions, tiredness, cytokine release syndrome, nausea, and pyrexia were the most common combined side effects, affecting over 10% of patients. Many people tolerated the drug. Compared to anti-PD-1 monotherapy, immune-mediated adverse effects were unchanged. RO7198457 with atezolizumab may induce a strong neoantigen-specific immune response [11].

In a second phase I multicenter research (NCT03313778), mRNA-4157 and pembrolizumab will be tested for safety, immunogenicity, and tolerability in 13 solid tumor patients who have undergone surgery and 20 untreated patients. Indications include melanoma, bladder cancer, non-small cell small cell carcinoma, (CRC) with metastases, and non-small cell lung cancer—neoantigen-based lipid-encapsulated RNA vaccination mRNA-4157. After eight months of follow-up, twelve of thirteen patients are disease-free. One complete response (CR), two partial responses (PR), five progressive diseases (PD), two newly detected immune-unconfirmed progressive illnesses, and one patient who could not be tested were found among the other twenty patients. No drug-related severe adverse events (SAEs) or AAEs above grade 3 were recorded. Treatment-related AEs are usually mild and short-lived. These findings move mRNA-4157 to phase 2 by confirming that pembrolizumab and neoantigen-specific T cells are safe and effective against tumours [12]. The ADXS-NEO-02 Phase 1 study (NCT03265080) is testing ADXSNEO’s effectiveness and safety in solid tumors alone or conjunction with anti-PD-1 antibody radiation treatment. Listeriolysin O (tLLO) and 20-21mer peptides make up the ADXS-NEO-tailored tumour antigen system. The best and safest ADXS-NEO dosage is shown by this early finding. The only mild to moderate, controllable, and transient side effects at this dosage were chills, fever, and tachycardia. Chemokines that promote T-cell trafficking into the tumour microenvironment were released at a greater rate, and many CD8 + T cells were primed against most neoantigens soon. The synergy between ADXS-NEO and anti-PD-1 antibody treatment is currently being studied [10]. GEN-009 and anti-PD-1/L1 are being tested in another phase I/IIA research (NCT03633110) to treat advanced cancers. GEN-009, a customized neoantigen vaccine, uses poly-ICLC to synthesize four to twenty synthetic long peptides. The first solid tumor research using GEN-009 alone showed persistent peripheral neoantigen-specific CD4 + and CD8 + responses in all eight patients. Multiple doses have caused mild local pain, but no dosing-related adverse effects (DLT). We eagerly await vaccine-PD-1 inhibitor research outcomes for advanced tumour patients [13].

Whole tumor cell vaccines admixed with adjuvant

An essential first step toward the development of tailored vaccinations for use in therapeutic settings is the isolation of tumor cells from a patient and their subsequent use in the production of an autologous whole tumor cell vaccine [14]. By introducing a wide variety of tumor antigens, including tumor-associated antigens (TAAs) and neoantigens unique to the individual patient, this strategy aims to trigger a potent and precise immune response against the tumor [15]. Additionally, this strategy aims to prevent immune evasion by the tumor. In several clinical studies spanning phases I and II, the efficacy of autologous cell vaccines against advanced metastatic cancers such as prostate, lung, colorectal, melanoma, and renal cell carcinoma (RCC) has been investigated [16]. These are just a few of the advanced malignancies that have been investigated. In this research, therapeutic immunological responses and excellent safety profiles have been observed [17]. The tumor cell lysate vaccination, known as Reniale, has shown promising results when used in cases of locally limited renal cell carcinoma [18]. Reniale substantially improved the 5-year (PFS) in patients with non-metastatic (RCC) across all tumor stages when administered post-nephrectomy [19]. The improvement increased the (PFS) rate to 77.4% from 67.8% in the control group. One other method for boosting the immunogenicity of tumor cell vaccines is to use immunomodulatory adjuvants in combination with the vaccinations [20].

Genetically modified tumor cells as vaccines

Several methods have been found to be effective in developing vaccines that utilize genetically modified tumor cells. Combining chemotherapy with Autologous tumor cell–based vaccines (ATVs) improves objective responses in ovarian cancer patients [21]. Hydrogels and microneedle patches have been employed in both preventative and curative mouse models to encode foreign dendritic cells (DCs), cytosine-phosphodiester-guanine oligodeoxynucleotide, or even whole tumor cells or lysates. However, postoperative malignancies still do not respond to ATVs-based therapy when T cell induction is the only strategy used [22]. It is advised that modified ATVs be made in an easy way to accommodate the limited period between getting autologous cell sources and giving postoperative immunization [23]. It is possible that enhancing the immunogenicity of autologous tumor cells through genetic modification to include immunostimulatory components could be advantageous [20]. A Phase I clinical trial utilized autologous tumor cells engineered to produce the co-stimulatory B7-1 (CD80) molecule to vaccinate patients with metastatic (mRCC) using systemic interleukin (IL)-12 [24]. In the treated group, there were a total of 15 people. Out of these 15 individuals, two cases of disease stability and two instances of partial response were observed [25]. Numerous preclinical and clinical studies have utilized autologous tumor cells transformed to express GM-CSF (GVAX) [26]. These genetically modified cells attract and grow dendritic cells (DCs), which transmit tumor antigens and stimulate cytotoxic CD8 + T lymphocytes [27]. Table 1 represents various vaccine trials conducted between 2010 and 2021. The autologous vaccine against (OC) known as Vigil, which is a product of Gradalis^®, utilizes modified cells that express and block GM-CSF. There is some evidence, which is cause for optimism, that this immunization may enhance (PFS) in individuals with advanced r (OC) [28].

Cell-derived exosomes as vaccines

Exosomes are released into the extracellular environment by a variety of cell types, including normal cells, malignant cells, and (DCs) [29]. They play a crucial role in the processes that occur within the cell, such as the transportation and communication of molecules. Tumor cells produce exosomes containing numerous tumor antigens, MHC, HSPs, and inducible costimulatory molecules [30]. Exosomes produced from tumors, in conjunction with the appropriate immunostimulatory drugs, have been shown in several studies to be capable of inducing potent anti-tumor responses in CD8 + T cells [31]. During the Phase I clinical investigation, immunotherapy was administered subcutaneously once per week for a total of four weeks to forty patients with advanced (CRC). Both GM-CSF (granulocyte-macrophage colony-stimulating factor) and Aex (autologous ascites-derived exosomes) were used in the administration of these immunizations [32]. Following vaccination, there was a noticeable increase in the number of cytotoxic T lymphocytes (CTL) that responded favorably to tumors, and patients had a positive response to the therapy itself [33]. Similarly, in a Phase I study, autologous dendritic cell-derived exosomes (DEX) expressing MAGE-3 peptides were used to immunize individuals with stage III/IV melanoma. These individuals received four doses of the exosome immunization; however, only mild grade 1 adverse effects were reported after the treatment [34]. Two out of the fifteen patients exhibited signs of disease stability (SD), while one patient showed signs of a partial response (PR) [35]. Patients with advanced cases of non-small cell lung cancer (NSCLC) received a vaccination containing DEX, which included peptides from MAGEA3, MAGEA4, MAGEA10, and MAGE-3DPO4 as part of a phase II clinical trial [36].

DNA and RNA-based vaccines derived from tumor cells

There is reason to be optimistic about the potential of using nucleic acid vaccines as a means of protecting multiple tumor antigens simultaneously. They are both safe and effective in clinical trials for eliciting antitumor immune responses at both the humoral and cellular levels [37]. The target genes for DNA vaccines are inserted into bacterial plasmids or viruses, which subsequently function as vectors for delivering the genes and enabling vaccine production [38]. They can be produced in large quantities at a low cost and do not require a refrigerated supply network [39]. It has been shown that DNA vaccines may elicit immune responses; however, the magnitude of these responses is often lower when compared to those generated by cell-based cancer vaccines [40]. At least a portion of this issue might be attributed, at least in part, to the difficulty DNA vaccines face in effectively stimulating the body’s immune system, or to the inefficiency with which DNA plasmids are transported into the nucleus of target cells. Figure 1 represent different bases of vaccines development [41].

Fig. 1.

Represent the different ways vaccine development for cancer. There were six different approaches utilized in the development of personalized cancer vaccines such as (a) autologous molecules (RNA, DNA) (b) autologous (neo) antigen peptides (c), autologous subunits (d) autologous whole tumor lysates (e), genetically modified autologous tumor cells (f), autologous tumor cells and adjuvants to treat the cancer

Since RNA cannot be integrated into the host genome, RNA vaccines are rapidly translated into the host cytoplasm without activating oncogenes. PCR can amplify RNA from even small amounts of tumor or stroma [42]. Because RNA is less stable than DNA, additional measures must be taken during the first biopsy. To evaluate the success of RNA vaccines in treating melanoma, prostate cancer, and (RCC), several clinical studies have been conducted [43]. The major issue with RNA based vaccine is that they are rapidly degraded by RNases and crossing cell membrane barrier. One of the most promising ways to deliver messenger RNA (mRNA) is using liposome complexes or liposome nanoparticles (LNPs), which can transport both hydrophobic and hydrophilic molecules. These molecules can include small molecules, proteins, and nucleic acids. The first liposome delivery materials were cationic liposomes, which protected messenger RNA (mRNA) against RNase breakdown [44]. Production of pH-responsive cationic lipids is underway with the aim of improving the efficacy of mRNA delivery. This is because, in addition to lipids, immune cells may take in additional substances with negative charges that interact with them [45]. A vaccine against neoantigen mutations in melanoma, based on RNA, has successfully completed Phase I of its clinical trials [46]. Two synthetic RNAs generated in the lab were used to encode a total of five large synthetic peptides, which were then combined to create the vaccine. The vaccine was then administered to patients. Patients exhibited T cell responses against numerous vaccine-encoded neoepitopes, and they displayed selective tumor cell death after receiving the vaccination [47]. One patient who experienced a cancer relapse achieved tumor remission after receiving combination therapy with an anti-PD-1 antibody, and eleven out of thirteen patients remained disease-free for up to 26 months [48]. At the moment, ModernaTX Inc. is conducting a Phase II trial in melanoma patients to investigate the efficacy of an RNA vaccine named mRNA-4157. This vaccine encodes 20 neoantigens. The mechanism of DNA/RNA based are shown in Fig. 2 [48].

Fig. 2.

Immunotherapy of cancer: DNA and RNA-based vaccines for tumor antigen targeting

Tumor neoantigen peptides

As discussed previously, the genomic instability of tumor cells has been shown to potentially trigger the generation of neoantigens. Tumor antigens, on the other hand, are exclusively expressed by malignant neoplasms [49]. This contrasts with self-tumor-associated antigens, or TAAs. Since they are not regulated by thymic selection or central tolerance mechanisms, they have the potential to activate highly reactive T cells [50]. Using prediction algorithms and cutting-edge technologies such as mass spectrometry, it may be possible to determine which neoantigen candidates offer the greatest potential for personalized vaccination [51]. Chen and colleagues’ novel research has demonstrated that synthesized long peptides are safe and effective in human clinical trials [52]. Four out of six cancer patients in the phase I clinical trial showed no recurrence at 25 months after receiving NeoVax (comprising up to 20 long synthetic tailored neoantigen peptides mixed with Poly IC: LC as an adjuvant) [53]. Neon Therapeutics has initiated a Phase I clinical research study to combat melanoma, (NSCLC), and bladder cancer by combining nivolumab with synthetic personalized peptides (at least 20 peptides per vaccination) [54]. A Phase I clinical research study (NCT03673020) of the AutoSynVax (ASV^®) AGEN2017 vaccine has been initiated by Agenus Inc. in patients with advanced cancers that have shown resistance to traditional therapies [54]. The AutoSynVax (ASV^®) was developed by coupling synthetic peptides isolated from a patient’s cancer neoantigens with HSP70 to enhance the immune system’s capacity to process and recognize antigens (NCT02992977) [55].

Tumor neoantigen peptides have garnered interest for their capacity to enhance the specificity and personalization of cancer therapy; yet, they are accompanied by many limits and drawbacks. A significant issue is tumor heterogeneity, cancer cells within the same tumor may possess distinct mutations, indicating that a neoantigen vaccination tailored for one subset of cells may not efficiently target others, resulting in partial tumor eradication. A further problem involves the identification of appropriate neoantigens, necessitating intricate and time-intensive bioinformatics analyses, sequencing, and predictive algorithms that are not consistently reliable. Furthermore, not all anticipated neoantigens elicit a robust immune response, and some ones may not be effectively presented by the patient’s HLA molecules [56].

Moreover, tumor immune evasion might diminish efficacy; cancer cells may downregulate antigen presentation mechanisms or establish an immunosuppressive tumor microenvironment that impedes T cell functionality. The production of tailored neoantigen vaccines is both expensive and time-consuming, hence restricting accessibility and prompt therapy, particularly for patients with aggressive malignancies. Safety issues exist, including the potential for causing autoimmunity if a neoantigen closely mimics a normal human peptide. Currently, clinical data on neoantigen peptide vaccines are scarce; several studies demonstrate encouraging immune activation but provide modest clinical results, suggesting that while the idea is potent, its conversion into a reliable and successful treatment poses a considerable obstacle [25].

Virus-like particle vaccines

A groundbreaking therapeutic cancer vaccine has been created, harnessing the power of P22 VLPs to showcase the crucial B and T epitopes of ovalbumin (OVAB peptide and OVAT peptide). These OVAB and OVAT peptides are intriguing tumor-specific neoantigens that result from somatic mutations within tumor cells. They possess an impressive skill in stimulating cytotoxic lymphocytes (CTLs), which initiates a potent immune response against tumors. When the adjuvant poly (I: C) was used in combination with the OVAB-P22 VLPs, a robust immune response against the OVAB antigen was observed. In addition, the OVAT-P22 VLPs successfully inhibited the growth of tumors by promoting immune responses that specifically target the tumor cells. Testing was conducted on mice with a melanoma model using PLPs, which have a connection to polyomaviruses. By combining the primary coat protein VP1 with two antigens, namely ovalabumin (OVA257-264) and tyrosinase-related protein (TRP2180-188), both of which are H2-Kb-restricted T-cell epitopes, CD8 T cells can be generated. We conducted experiments using VP1-OVA252-270 and VP1-TRP2180-188 on C57BL/6 mice with MO5 (B16-OVA) melanoma to evaluate their efficacy as therapeutic agents. The VP1-OVA252-270 treatment significantly improved survival rates by 80–100%, while the VP1-TRP2180-188 treatment showed a 60% increase in survival [57]. The HBV surface antigen is their basis. Three vaccines that protect against human papillomaviruses—Cervarix, Gardasil-4, and Gardasil-9—have been approved globally based on virus-like particle (VLP) technology. The majority of cervical cancer cases are caused by two to seven different types of HPV, notably HPV16, 18, 31, 33, 45, 52, and 58 [58]. These VLPs derive from the primary capsid protein L1 and provide protection against these types of HPV. They also protect against the two types of human papillomavirus (HPV) that are associated with over 90% of genital warts: HPV6 and HPV11. Cervarix and Gardasil-4 need an immune response that lasts 13 years, but Gardasil-9 just needs a 6-year duration. There is a 21–64% chance that the Cervarix vaccine will continue to protect against HPV35, 31, 33, 45, and 58 after 11 years. Nevertheless, 52% of Gardasil-4 vaccinees had antibodies against HPV18, and 90% had antibodies against heterologous HPV types (HPV6, 11, and 16) after 14 years [59, 60] .

Immunotherapy

CAR-T cell therapy

CAR-T cell therapy is a groundbreaking treatment that uses a patient’s own immune cells to fight cancer. Doctors start by collecting T cells, a type of white blood cell, from the patient’s blood. These cells are then genetically modified in the laboratory to carry a special receptor called a Chimeric Antigen Receptor (CAR) [61]. The CAR works like a tracking device, allowing the T cell to recognize and attach to proteins on cancer cells. Once re-infused into the patient, these engineered T cells can find cancer cells more effectively, activate themselves, multiply, and release toxic substances that directly destroy the tumor [62]. CAR-T cells do not require cancer cells to display antigens by a mechanism that utilises MHC molecules as the tumour does (as normal T cells do), but instead home in on the tumour itself. The remarkable success of this unique mechanism has already been achieved in the treatment of certain blood cancers such as leukaemia and lymphoma [63]. Several CAR-T therapies are approved and in clinical practice. In 2017, the first to be approved was called Tisagenlecleucel (Kymriah), which targets CD19 in B-cell cancers. It has been applied in the treatment of children and young adults with acute lymphoblastic leukemia (ALL) and in the treatment of adults with some forms of lymphoma [64]. This was followed by Axicabtagene ciloleucel (Yescarta), which is also based on CD19, and is commonly used in large B-cell lymphoma. Brexucabtagene autoleucel (Tecartus) is licensed for mantle cell lymphoma and B-cell precursor ALL, whereas Idecabtagene vicleucel (Abecma) and Lisocabtagene maraleucel (Breyanzi) were licensed for large B-cell lymphoma and multiple myeloma, respectively [65]. More recently, Ciltacabtagene autoleucel (Carvykti) was given the green light in relapsing or refractory multiple myeloma. These treatment methods have demonstrated good response rates, and many patients were brought to complete remission, even after other treatments had failed [66].

There are numerous benefits of CAR-T cell therapy, but it is not safe. CRS occurs when the CAR-T cells become hyperactive and produce huge doses of chemical messengers called cytokines. These cytokines play a crucial role in combating cancer, but when produced out of control, they cause an unregulated inflammatory reaction commonly known as the cytokine storm [67]. CRS causes the patient to experience the symptoms of a flu, such as fever, fatigue, and muscle aches, as the first signs [68]. The reaction can get bad to such an extent that it can lead to very low blood pressure, difficulty breathing, and even organ failure. CRS is very common (the majority of patients have mild symptoms and about 10–20% develop severe cases requiring urgent care) [69]. Immune Effector Cell-Associated Neurotoxicity Syndrome (ICANS), which is a brain and nervous system side effect, is another serious side effect [70]. ICANS has been believed to be a result of injury to the blood-brain barrier and diffusion of inflammatory cytokines into the brain. The initial symptoms in patients may include confusion, inability to communicate (either verbally or in writing), tremors, or headache. In even more severe cases, there may be seizures, consciousness loss or even swelling of the brain. ICANS typically occurs a few days following CRS, but may occur independently. Doctors use standardized grading systems developed by the American Society for Transplantation and Cellular Therapy (ASTCT) to manage these complications. CRS is rated as mild (gets a fever but does not need any other medications); to life-threatening (needs more than one medication to stabilise blood pressure or a ventilator to breathe) [71]. ICANS can be evaluated by a test known as the ICE score, which evaluates the ability of a patient to respond to orientation questions, write a sentence, name things, and count in reverse. The normal score is 4 and below corresponds to a higher degree of severity [72]. Treatment depends on the severity of symptoms. For mild CRS, supportive care such as fluids, fever reducers, and oxygen is usually enough. If symptoms worsen, the first-line treatment is tocilizumab, a drug that blocks IL-6, one of the main cytokines responsible for CRS. Steroids may also be used if symptoms do not improve quickly. ICANS is treated differently because tocilizumab does not cross into the brain. Instead, steroids like dexamethasone are the main therapy, along with seizure prevention and, in severe cases, intensive neurological monitoring [73].

Immune checkpoint inhibitors (CPIs)in cancer treatment

Immunological checkpoints are receptors found on the surface of immune cells that play a crucial role in regulating the activation or suppression of the immune response. Immunotherapy, specifically immune checkpoint inhibitors (CPIs), enhances the immune response against tumors by obstructing the receptors on T cells’ surface. This class of immunotherapy is extremely important in the treatment of numerous types of cancers, and it has been extensively researched compared to any other class so far [74–76]. In the past decade, blocking programmed cell death protein-1 and its ligand-1 (PD-1/PD-L1) and cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) molecules has emerged as highly effective and widely employed strategies for checkpoint suppression. There are several other potential targets that can be considered, such as inhibitory receptors like Tim-3, VISTA, Lag-3, and GITR, as well as activating molecules such as OX40 (CD134) and glucocorticoid-induced TNFR-related protein. Through its interaction with ligands B7-1 (CD80) and B7-2 (CD86), CTLA-4, a member of the immunoglobulin superfamily and a molecule that inhibits immune response, regulates T cell activation in a negative manner. The genes for CTLA-4 and CD28 are found in proximity on chromosome 2q33, and their protein sequences exhibit significant similarities. They both bind the same ligands in homodimers, but their affinities vary. Negative signaling of T cells happens due to the higher affinity binding and ligand scavenging of CTLA-4 compared to CD28. CTLA-4 plays a crucial role in the early stage of the T-cell response in the lymph nodes, specifically in the context of tumor immunoregulation. This suggests additional evidence that the absence of CTLA-4 could result in unregulated multiplication of T cells, providing a fresh perspective on the potential of CTLA-4 inhibition to bolster the immune response against tumors. A member of the immunoglobulin superfamily, PD-1, reacts to a process controlled by T cells by inducing programmed cell death. It is prevalent in several immune cell types inside the tumor microenvironment (TME) and expresses itself more broadly than CTLA-4. The death of T cells is caused by an inhibitory signal sent when PD-1 and its ligand PD-L1 (B7-H1) bind. While CTLA-4 regulates T-cell activity during the priming phase, PD-1 mostly suppresses T-cell activity in peripheral tissues as tumors progress.

Researchers have shown that inhibiting PD-1 successfully counteracts tumor cells in several types of cancer, including colon, melanoma, and pancreatic ductal adenocarcinoma. Pembrolizumab, nivolumab, and cemiplimab are the three PD-1 inhibitors that have been approved by the FDA after extensive clinical studies [77–79] .

When PD-1 interacts with PD-L1 and PD-L2, it can potentially hinder the function of activated immune cells. Various types of tumors and immune cells express PD-L1, while normal dendritic cells typically express PD-L2. When cancer cells utilize the PD-1/PD-L1 pathway to dampen T-cell-mediated immunity, it can result in abnormal cell growth. Through extensive research, it has been discovered that PD-L1 can interact with certain entities, thus presenting itself as a promising candidate for immunotherapy. There are three PD-L1 inhibitors that have been approved by the Food and Drug Administration: Atezolizumab, durvalumab, and avelumab. In 2016, the treatment for urothelial cancer called atezolizumab, a humanized IgG1 anti-PD-L1 mAb, received approval. Due to the improved response rate, the indication was later expanded to include small cell lung cancer, melanoma, hepatocellular carcinoma, non-small cell lung cancer, and melanoma. It is worth mentioning that the therapy was originally developed for TNBC. However, due to the unsuccessful outcome of the IMpassion130 clinical trial, it is no longer considered a viable treatment option [77, 80].

Immune checkpoint inhibitors (ICIs) such as anti–CTLA-4, anti–PD-1, and anti–PD-L1 antibodies have transformed cancer treatment by unlocking the body’s immune system to attack tumors [81]. By removing the “brakes” on T cells, they allow a stronger and longer-lasting immune response. However, this same mechanism can also cause the immune system to attack healthy tissues, leading to a wide range of side effects known as immune-related adverse events (irAEs) [82]. These toxicities are different from those seen with chemotherapy or targeted drugs, and they require a specific approach to recognition and management [83]. irAEs can affect nearly every organ system. The most widespread of them are skin reactions (including rash and itching), gastrointestinal (including diarrhoea and colitis), and endocrine (including thyroid disorders, hypophysitis, and adrenal insufficiency) conditions. Inflammation of the liver and the lungs (pneumonitis) also occurs, with rarer but life-threatening complications such as heart inflammation (myocarditis), neurological (encephalitis or myasthenia gravis-like syndromes), and kidney inflammation [84]. Most irAEs are mild to moderate but others may become severe or even life threatening when they are not detected early [85].

The Common Terminology Criteria for Adverse Events (CTCAE) often characterises the severity of irAEs on a scale of 1 (mild) to 4 (life-threatening). The timing also differs according to the organ system [86]. Skin toxicities are usually evident in the first few weeks, and gastrointestinal and liver side effects are seen in the first three months. Later, the endocrine problems can occur and become permanent [87]. Neurological and cardiac complications are particularly dangerous and prone to manifest themselves at an early stage and develop rapidly. Both severity and recovery determine the decision to resume the ICIs on an irAE. Mild cases can often be managed without stopping therapy, but severe or life-threatening toxicities generally require permanent discontinuation, especially if they involve the brain, heart, or lungs [88]. Managing irAEs requires a thoughtful, organ-specific approach. Corticosteroids remain the backbone of treatment for most moderate to severe cases, but the details matter. For skin reactions, topical creams and antihistamines may be enough for mild cases, while severe blistering diseases may require high-dose steroids and discontinuation of therapy [89]. Diarrhea and colitis are usually treated with systemic steroids, and if symptoms do not improve, additional drugs such as infliximab or vedolizumab can be used [90]. Liver inflammation is treated with high-dose steroids, and in resistant cases, mycophenolate may be considered, but infliximab is avoided due to its risk of worsening liver injury. Endocrine toxicities are often irreversible, meaning hormone replacement (such as thyroid hormone or insulin) becomes the mainstay rather than steroids [91]. Pneumonitis is managed with steroids, but severe cases frequently require permanent cessation of ICIs. Neurologic and cardiac toxicities are medical emergencies, treated immediately with high-dose intravenous steroids, sometimes alongside intravenous immunoglobulin or plasmapheresis. In these cases, rechallenge with ICIs is generally not safe [92]. While most immune-related side effects improve with timely treatment, some are fatal or irreversible. Myocarditis and severe neurological toxicities carry some of the highest mortality rates, sometimes exceeding 30–50% despite aggressive care [92]. Endocrine disorders like hypothyroidism, adrenal insufficiency, and diabetes usually do not resolve and require lifelong hormone replacement. On the other hand, many patients recover fully from skin, gastrointestinal, or hepatic events, and there is evidence that patients who experience irAEs may actually have better cancer outcomes, suggesting a link between immune activation and treatment success [93]. Nevertheless, the long-term effects of ICIs are still being studied, and careful follow-up is critical for survivors who may face chronic fatigue, hormonal issues, or other lasting effects [94].

Precise cancer treatment using clustered regularly interspaced short palindromic repeats (CRISPR-CAS9)

Three components that constitute the CRISPR/Cas9 system. These include a DNA-specific guide RNA (sgRNA), the Cas9 protein, which can endonuclease DNA, and an interaction between Cas9 and tracrRNA. Just like a biologist, the 20-base-pair gRNA forms a binding with the precise genomic location, providing instructions to the Cas9 protein [95]. Having a deep understanding of the gRNA sequence is crucial to maximize the effectiveness, precision, and reliability of CRISPR/Cas9-mediated genome editing. The seed sequence, found at the 3′ end of gRNA and adjacent to a protospacer adjacent motif (PAM), plays a vital role in determining the binding specificity to the target sequence. By utilizing truncated gRNAs with fewer than 20 complementary nucleotides, the potential for off-target effects can be reduced by a remarkable 5000-fold, all while ensuring a strong on-target efficiency. Furthermore, extending the length of the gRNA duplex by 5 base pairs has been demonstrated to significantly improve the efficiency of the knockout procedure [96]. Proto-oncogenes, which regulate cell formation, also have the potential to control cell proliferation and differentiation. If these proto-oncogenes undergo mutations and transform into oncogenes, the conversion of typically dividing cells into cancerous cells is theoretically possible [97]. There is a possibility that this change led to the development of cancer. On the other hand, genes that function as tumor suppressors can be utilized to inhibit abnormal cell divisions, thus averting the onset of cancer [98]. Proteins known as tumor suppressors may prevent oncogenes from promoting abnormal tissue growth [99]. Tumor suppressor genes provide a defense mechanism against cancer by inhibiting the overexpression of the cell cycle in normal human cells [100]. They keep track of the rates at which different types of cells proliferate, die off, and repair damage to their DNA. The BRCA1 and BRCA2 genes, along with the TP53 gene, are the three most well-known tumor suppressor genes. These genes play a role in regulating various stages throughout the cell cycle [100, 101]. The process by which tumors develop as a consequence of mutations in the DNA of the affected cells is known as carcinogenesis. Incubation, growth, transition to a malignant state, and spread are the four stages that constitute cancer progression [102]. Even if a single mutation in a cell is not harmful, the accumulation of mutations that naturally occurs with aging might increase carcinogenesis and the development of malignancies [96]. This is because age naturally causes mutations to arise. Cancer cells can exhibit a characteristic known as genetic instability. The most common type of genetic instability is chromosomal instability, which occurs when the structure of chromosomes frequently changes [103]. Chromosomal instability may be defined as the frequent shifting of chromosome structures. It is still not completely understood when exactly during the progression of cancer this genetic instability will manifest itself [33, 104]. Because cells with unstable genes divide at a quicker pace, they may bypass the usual checkpoints that occur during the cell cycle. It is possible that this will transform cancer cells into more aggressive forms of the disease [105]. If the factors contributing to the development of cancer are better understood, novel strategies to block or delay the expansion of malignant cells may be uncovered [106]. The purpose of CRISPR technology and other gene editing approaches used to regulate or prevent carcinogenesis is to modify DNA nucleotides by deleting defective genes and replacing them with functional equivalents. This is accomplished by replacing the faulty genes with functional counterparts [107]. CRISPR/Cas9 technology may be used in oncology to create cancer models, identify targetable genes, and more [108] The clarification of resistance mechanisms and the improvement of immunotherapies. As checkpoint inhibitors and T-cell therapies become first-line treatments, enhancing their efficacy is crucial. CRISPR/Cas9 can address challenging tumor microenvironment issues, such as T-cell depletion and immunosuppression [108]. The technology’s ability to create very effective gene knockouts allows this. The ability of CRISPR/Cas9 to modify genes holds promise for personalized cancer therapies. Previous and ongoing preclinical and clinical investigations have established that CRISPR/Cas9 systems work. Different genes of cancer are knock down via CRISPR-CAS9 as shown in Table 2 [109].

Table 2.

Represent different targets genes of cancer in a CRISPR-CAS9. The various genes such as TERT, TR53, PKC, CTLA-4, EGFR, and non-metastatic cancer cell in various cancer tissue were knocked-down through CRISPR-CAS9

Target genes	Cancer types	CRISPR-CAS9	References
TERT	Glioblastoma	knockdown	[110]
TP53	Prostate cancer	knockdown	[111]
PKC	Colon cancer	knockdown	[112]
Genes on non-metastatic cancer cell line	Lung metastases	knockdown	[113]
Colorectal cancer driver genes	Intestinal tumors	knockdown	[114]
CTLA-4	Bladder cancer	knockdown	[115]
EGFR	NSCLC	knockdown	[116]
KRAS, BRAF	Colorectal	. knockdown	[117]

T-cell depletion is particularly problematic in immunosuppressive microenvironments because it significantly impairs the effectiveness of CAR-T cells and the body’s endogenous anticancer responses [118]. People with persistent or chronic lymphocytic leukemia (CLL) who received CD19-targeted chimeric-antigen receptor (CAR) T-cell therapy showed clinical improvement in T-cell depletion following apheresis [119]. It is conceivable to achieve improved clinical outcomes by modifying CAR T cells using the gene-editing tool CRISPR/Cas9 to eliminate regulators of T cell activity and persistence [120]. Table 3 represents different genes and CRISPR-Cas9 trials for the treatment of cancer. EGFR is mainly expressed in lung tissue and neurogenic tissue. Eliminating EGFR mutations using CRISPR may limit lung cancer cell growth in vitro and in mice [121]. Genome-wide CRISPR screening, using extensive libraries of guide RNAs targeting numerous genes of interest, has unveiled promising therapeutic targets in preclinical (CRC) research [122]. These laboratory achievements establish the framework for studying how CRISPR/Cas9 may help in creating cancer treatments [123].

Table 3.

Represent The clinical trial of CRISPR-CAS9. (https://clinicaltrials.gov). The table represent different clinical trials of phase 1 in various cancer using CRISPR-CAS9 system

Clinical Trial	CANCER TYPE	Related CRISPR Method
Phase 1	Refractory cancers	Delete two genes that encode TCR and a gene encoding PD-1
	Advanced NSCLC	Edite PD-1 on T cells
	Mesothelin-positive solid tumors	PD-L1 possible target for knockout
	Refractory solid cancers	Knockout two (TCR) gene

Since 2018, researchers have started utilizing CRISPR/Cas9 technology to develop personalized therapy options for different types of cancer. Several potential therapies currently in development are based on proteins suspected of having oncogenic properties [124]. Overexpression of the large ribosomal subunit component RPL15 has been associated with breast cancer metastasis. These genes can be identified through CRISPR/Cas9 genome screening. CRISPR/Cas9-mediated deletion of the estrogen receptor signaling gene FASN may reduce breast cancer cell proliferation and metastasis [125]. It is known that BRCA1 mutations are responsible for 80% of triple-negative breast cancer cases. However, there are currently no CRISPR/Cas9 applications for treating TNBC [126]. The poly (ADP-ribose) polymerase 1 (PARP1) gene may be targeted using CRISPR/Cas9 to treat various types of breast cancer. This gene causes synthetic lethality in BRCA1-deficient cells and mechanism of cancer is shown in Fig. 3 [127].

Fig. 3.

Virus-like particle vaccines and the CRISPR-Cas9 mechanism for gene editing in cancer therapeutics

Additionally, the estrogen receptor (ER) gene was found in prostate cancer and was effectively excised from the patient’s genome using CRISPR. The signaling of androgen receptors is largely responsible for controlling the development and function of the prostate [128]. However, estrogen receptors (ER) help in the formation of prostatic epithelial cells and inhibit the growth of prostate cancer cells. ER signaling is still poorly understood, but this suggests another potential cancer therapy utilizing CRISPR [129]. Stadtmauer et al. generated T cells using CRISPR-Cas9 in phase I. CRISPR/Cas9 was used to delete two natural. (TCR) genes in three refractory cancer patients, reducing TCR mispairing and increasing cancer-specific TCR transgenic expression. The PD-1 gene was removed to improve antitumor immunity. The CRISPR/Cas9 technology may edit genes for immunotherapies, since all three T cell transplants were successful and lasted nine months. This innovative experiment enabled focused treatment research and immunotherapy efficacy improvements [130]. Both phase I lung cancer trials indicated CRISPR/Cas9 T cell editing is safe and effective. Lu et al. treated 12 advanced NSCLC patients with CRISPR/Cas9-edited T cells for PD-1. Edited T cells injected into peripheral blood had no significant side effects. Median progression-free and overall survival were 7.7 and 42.6 weeks. Next-generation sequencing detected off-target events, and median mutation frequency was 0.05%, indicating CRISPR/Cas9 modified T cells are safe and practical. Most recently, Wang et al. recruited 15 mesothelin-positive solid tumor patients and used CRISPR/Cas9 to produce PD-1 and TCR-deficient mesothelin-specific CAR-T cells and evaluated dose escalation [131] Two patients had stable disease; circulating changed T cells peaked at days 7–14 and were undetectable after one month without any side effects or toxicity, indicating CRISPR/Cas9 altered T cells are safe and practical. In osteosarcoma patients, Liao et al. found that CRISPR/Cas9 could knock down PD-L1. The PD-1/PD-L1 axis is crucial to cancer immune evasion and therapy, therefore our results are the first steps toward proving CRISPR/Cas9’s safety and efficacy in treating NSCLC, sarcoma, and other cancers [132].

CRISPR in cancer therapy: real-world translational and clinical hurdles

CRISPR has changed how we think about treating cancer, but moving from elegant lab experiments to safe, effective patient care is not straightforward [133]. The first practical barrier is delivery. Ex vivo editing (for example, editing a patient’s T cells outside the body and reinfusing them) is relatively controllable and already in trials, but it is expensive and logistically complex [134]. Getting CRISPR components to solid tumours in vivo is far more difficult. AAV vectors have poor cargo capacity and can integrate at low frequency; lipid nanoparticles can be concentrated in the liver and spleen; and local injections do not yet cover multi-site or metastatic disease [135]. Tumours are also located under physical and biological obstacles of dense stroma, high interstitial pressure, and irregularity of blood flow that dulls delivery to the very cells we must edit [136]. The second is the issue of precision and safety. Even high-fidelity nucleases and better guide design do not eliminate off-target edits. More importantly, on-target, off-tumour editing has the potential to damage normal cells that contain the target gene [137]. Large deletions, translocations, or p53-mediated stress could be induced by a double-strand break and, ironically, favour the survival of p53-deficient (more aggressive) clones. Base and prime editors do not require DNA cutting; however, they do have their own editing windows as well as bystander effects, which need to be well-mapped and monitored over the long term [138].

The real-life issues are immunogenicity and re-dose. Most human beings have prior immunity against Cas proteins or viral vectors. Edited cells may be cleared by immune recognition, or repeat dosing may be unsafe and ineffective. LNPs can be re-dosed more easily than viral vectors and can induce complement. In the case of ex vivo, patients might require lymphodepletion to allow proliferation of the edited cells-increasing both toxicity and cost [139].

Even tumour biology rebels. Cancer is a heterogeneous and rapidly evolving disease. You can edit works in one subclone and still have another escape [140]. Since a single edit can be eroded by redundant pathways, copy-number variation, and the immunosuppressive tumour microenvironment, it can be easily eroded. Combination (e.g., editing T cells and using them with checkpoint blockade) is a promising approach, but it creates new questions regarding timing, dosing, and compounded toxicity [141]. CRISPR therapies put pressure on existing systems on the clinical operations side. Production should be GMP-level, repeatable, and fast with a closed chain-of-identity on customised products [142]. Without delaying treatment too long, release testing must establish potency, specificity, residual nuclease/guide levels, and absence of contamination. It is expensive and uneven in access, particularly where specialised facilities are limited [143].

Lastly, regulatory and ethical authorities consider CRISPR a form of gene therapy, and there is greater concern about long-term risks, including genotoxicity, unintentional germline introduction (but highly unlikely in the oncology setting), and delayed adverse events. Trials should be developed with long-period monitoring, excellent off-target analytics, and explicit stop rules. Patient selection, biomarker strategies (to show editing actually happened and mattered), and clinically meaningful endpoints (beyond molecular editing rates) are all essential to convince regulators, clinicians, and payers [144].

Cancer treatment through bacteria

Because cancer is a complex illness with many different manifestations, effective treatment of the disease necessitates the use of several therapeutic modalities [145]. Bacteria have been the subject of intensive research for more than a century, to determine their potential for use as therapeutic agents in the treatment of cancer. In the beginning, cancer patients were treated with live bacteria such as Streptococci and Clostridia by medical professionals [146]. There are certain challenges in bacteriotherapy such immune response against anti-tumours bacteria, cytotoxicity of bacteria. To overcome with challenges bacteria are modified so that they are unable to detect by our immune system by masking the pathogen associated molecular patterns (PAMP) receptors. The second approach is using attenuated bacteria of the treatment such bacille Calmette-Guerin (BCG) vaccine. The third approach to use probiotics and gut microflora for bacteriotherapy for cancer because they not recognized by immune as foreign antigens [147]. However, in today’s modern times, this task is often accomplished through the use of genetically engineered microorganisms [148]. The use of bacteria in the treatment of cancer encompasses several different strategies, including the expression of antigens specific to tumors, facilitating gene transfer, utilizing RNA interference, and enabling the activation of pro-drugs, as shown in Table 4 [149]. Some of these strategies include harnessing the bacteria’s intrinsic toxicity, integrating them with complementary therapeutic methods, using bacteria that have the ability to control the production of anticancer substances, and integrating them with complementary therapeutic methods [150]. The study of cancer has utilized a wide variety of experimental models, including the examination of whole live bacteria, attenuated strains, and genetically modified versions, either individually or in conjunction with conventional therapies [151]. Salmonella, Clostridium, Bifidobacterium, Lactobacillus, Escherichia coli, Pseudomonas aeruginosa, Caulobacter, Listeria, Proteus, and Streptococcus are among the most often described bacterial genera in this setting. The potential of Clostridia, Bifidobacteria, and Salmonellae as vectors for delivering or expressing tumor suppressor genes, anti-angiogenic genes, suicide genes, and (TAAs) has been extensively studied in animal models with a wide range of malignancies [152]. These bacteria might potentially carry or express these genes. Due to the limited results shown in the ongoing clinical studies, additional research involving human subjects is necessary [153]. Imaging techniques such as MRI and PET can identify genetically modified bacteria. This indicates that besides their therapeutic benefits, these microorganisms also have diagnostic applications [154].

Table 4.

Different bacteria used for cancer as a therapeutic purpose. The tables represent different types of bacteria used in various types of cancers

Treatment Options	Types of bacteria	Results	References
Immunotherapeutic agents	Streptococcus pyogenes, Clostridium spp, Salmonella typhimurium Salmonella typhimurium	Rapid tumor regression Reduced frequency of cancer Tumor regression An antitumor effect	[162]
Vectors/spores to carry tumoricidal agent	Clostridium novyi-NT, C novyi‐NT and C. sporogenes, C novyi‐NT,	Elimination of tumors Hemorrhagic necrosis of tumors	[163]
Bacterial toxins/enzymes	Salmonella enterica Serovar Typhimurium, Streptococci and Serratia marcescens, Serratia marcescens (Coley’s Toxin) E coli BM2-1 strain)	Vaccine as an adjuvant against different types of cancer.	[164] [165]
Clostridium novyi-NT	Injection 3 × 108 spores of C novyi-NT	C novyi-NT plus external beam radiation led to tumor shrinkage in mice bearing HCT116 tumors	[166] [167]

Genetically modified bacteria in cancer therapy

Gene therapy is a cutting-edge method that holds enormous therapeutic promise for the treatment of cancer. The high degree of specificity with which this method eradicates cancer cells is the key benefit of using it [155]. Cancer therapy using genetically altered bacteria has the potential to be more effective and less harmful to the host [156]. In recent years, a unique approach to treating cancer has emerged: the use of genetically engineered bacteria to produce reporter genes, cytotoxic proteins, anticancer drugs, and tumor-specific antigens [157].

According to the findings of several studies, genetically engineered bacteria may reproduce more quickly inside tumor tissue than they do within normal cells [158]. Tumor-colonizing bacteria have been utilized as delivery vehicles in animal tumor models, however this strategy has been met with poor results and unwanted side effects [56]. This has been achieved without the use of conventional chemotherapy. Salmonella typhimurium serovar VNP20009 and Clostridium butyricum M55 are two of the microorganisms that make up this group. Nevertheless, the results of not all of the research have been favorable [159].

The anticancer properties shown by genetically modified Clostridium strains (C. acetobutylicum and C. beijerinckii), which express genes encoding bacterial enzymes (cytosine deaminase and nitroreductase, respectively), have exhibited promising outcomes [160]. Moreover, scientific investigations have shown that bacteria possess the capability to generate antibodies that may effectively attach themselves to hypoxia-inducible factor 1, a pivotal transcription factor that is associated with the formation of malignancies [161]. Experiments conducted in clinical settings have shown that modified S. typhimurium and Clostridium novyi-NT have the potential to promote tumor regression and necrosis by stimulating the immune system of the host. The production of cytokines, including (IL-2), IL-4, IL-18, and CC chemokine-21, facilitates this process. These results imply that bacteriotherapy, when combined with other cancer treatments such as radiation therapy, immunotherapy, or chemotherapy, may offer an innovative and effective strategy for combating the illness when utilized in combination with these other treatments as shown in Table 4.

Bacteria as immunotherapeutic agents in cancer therapy

The efficacy of immunotherapy in cancer treatment stems from its ability to stimulate the immune system to specifically target malignant cells. The proposed methodology primarily focuses on the activation and stimulation of CD8 + and CD4 + T lymphocytes, which possess the ability to recognize tumor antigens. Upon activation, these immune cells will actively participate in the process of identifying tumor cells and eradicating them [168]. Following infection by bacteria such as C. novyi, necrotic cells possess the capacity to produce pathogen-associated molecular patterns (PAMPs) and heat shock proteins, including Hsp70. The Hsp70 protein is of significant importance in the dendritic cell maturation process, which is vital for the effective initiation of immune responses targeted towards antigens [169]. The synthesis of pro-inflammatory cytokines and costimulatory molecules is triggered by the interaction between pathogen-associated molecular patterns (PAMPs) and toll-like receptors. The mediators in question induce the production of interferon gamma (IFN- α), which subsequently initiates a cellular immune response that is primarily driven by CD8 + effector cells and is reliant on Th1 cells. The treatment of Clostridium novyi NT to mice has shown the capacity to induce tumor-specific acquired immunity via the mediation of CD8 + cells [170]. The capacity of intracellular bacteria, namely S. typhimurium, to invade host cells plays a pivotal role in the efficacy of the melanoma immunotherapy strategy presented by Avogadri et al. The use of a type-3 secretion system (T3SS) by Salmonella typhimurium in order to facilitate the invasion of neoplastic cells has been shown. Both in vivo and in vitro studies have provided empirical data indicating that mutant strains missing the type III secretion system (T3SS) exhibit an inability to infiltrate tumor cells [171]. The elimination of infected tumor cells by Salmonella does not occur by direct means. Rather, the presence of bacterial antigens facilitates the activation of T lymphocytes that specifically recognize Salmonella, so acting as targets for immune response. Nevertheless, more investigation is required to fully understand the intricate mechanism behind this process [172].

Clostridia strains may generate cell suicide genes like cytosine deaminase or eukaryotic host molecules like TNF-α [61]. C. novyi-NT has RecA, a DNA repair and maintenance protein, which boosts host immunity and pro-inflammatory cytokines and chemokines, which shrink tumors.

Engineering less virulent Salmonella and Listeria may lower dose toxicity and infection. Three commonly used attenuated Salmonella strains for therapy are VNP20009, AR-1, and ΔppGpp. By eliminating purI and msbB genes, VNP20009 lowers septic shock and boosts antibiotic sensitivity A phase I clinical trial found better tumor colonization without severe toxicity or side effects and quick bloodstream clearance throughout experimental testing [173].

A study by Yun et al. observed that mice treated with ΔppGpp showed reduced cytoskeletal components and increased iron and heme metabolism, suggesting that bacteria proliferation might disturb tumor tissues and give free heme and iron for growth [174] without maintenance treatment. The live-attenuated double-deleted (LADD) strain of Listeria exhibits lower liver toxicity and fast liver and spleen clearance, making it the main vaccine candidate in clinical trials. The XFL-7 serovar indicates that prfA gene deletions greatly reduce Lm strains. A tumor-associated antigen-cloned PrfA plasmid recovers immunogenic partial virulence. A plasmid alone may partially restore virulence in the Δdal/Δdat (Lmdd) strain, which lacks peptidoglycan genes and needs alanine co-administration. Medical trials use Lmdd and XFL-7 strains [175].

The regression of B16F10 melanoma tumors in mice is seen in its entirety when (DCs) are activated by bacterial compounds, namely CpG oligonucleotides, which are recognized for their capacity to initiate the immune response [176]. Nearly thirty years have elapsed since the first use of recombinant Escherichia coli strains in the realm of molecular biology and the synthesis of recombinant proteins [177]. These strains have the potential to serve as vehicles for the delivery of tumor antigens to dendritic cells. Upon internalization by macrophages, the OVA Kb-restricted epitope, SIINFEKL, is presented by MHC class I molecules after the concurrent synthesis of listeriolysin O (LLO) and ovalbumin (OVA) inside a single Escherichia coli strain. After the process of phagocytosis and subsequent breakdown inside phagocytic vesicles, LLO, along with the proteins that are co-expressed, is released into the cytosol [158]. Following that, it proceeds through processing and presentation via the major histocompatibility complex (MHC) class I route. Bacterial species are involved in the synthesis of LLO, a protein that is mostly located inside the cytoplasmic compartment. Based on current study findings, the use of a distinct assemblage of pathogen-associated molecular patterns (PAMPs) has shown promise in achieving comprehensive elimination of solid tumors in mice suffering from cancer [178].

Bacterial toxins or enzymes in cancer therapy

Several types of pathogenic bacteria are capable of producing and secreting protein toxins in order to weaken the immune system of the host. Researchers have investigated the idea of using a few of these carcinogens as potential cancer treatments [179]. They do this by accelerating covalent modifications in the target proteins, which in turn prevents the production of antibodies and cytokines [180]. These toxins not only prevent macrophages from migrating, but they also prevent epithelial cells from performing their barrier role. Many of these toxins are potent enzymes with incredible selectivity, and the biological substrates that they target are often signaling molecules. These enzymatic poisons are created by bacteria after they have successfully infiltrated a host cell [154]. They have the potential to modify the cytosolic substrates of the host cell, which may result in a change in the function of the cell or even cell death [181]. These toxins have been used to investigate or alter a variety of mammalian cell signaling pathways because of the familiarity of their structures, cellular receptors, molecular processes, and absorption routes [182].

Cytotoxins produced by bacteria are among the most lethal substances on the earth. Cytolysin A, also known as ClyA or HlyE, is an enzyme toxin that is generated by bacteria. It causes the membranes of eukaryotic cells to become perforated, which then leads to the caspase cascade causing the cells to die [183]. According to the findings of a number of research, mice who were given Escherichia coli or Salmonella typhimurium strains that released the ClyA toxin had much less development of tumors than mice that were not given the treatment. The tumor necrosis factor (TNF), also known as TRAIL, and the FAS ligand, also known as FAS-L, are all examples of cytokines that belong to the TNF superfamily. Caspases 3 and 8 are examples of apoptotic mediators that are activated when these proteins preferentially induce programmed cell death via pathways associated with death receptors. Various types of bacteria use for use cancer which are shown in Fig. 4 [184].

Fig. 4.

Represent different bacteria used for cancer treatment. The figure represent various of bacterial strains specially modified to remove the cancer cell from the tissue

Carbon nitride (C3N4) and an E. coli strain with the ability to produce nitric oxide (NO) were used in the creation of a “photo-controlled bacterial metabolite therapy” that was recently created by Zheng and colleagues. When C3N4-containing bacteria were introduced into a mouse model tumor, this resulted to a substantial anticancer impact as well as an approximately 80% reduction in the growth of the tumor. Toxins produced by bacteria may either inhibit or stimulate the cell cycle progression of eukaryotic organisms. Cell-cycle stimulators, on the other hand, cause cells to divide more quickly than they would if the stimulant were not there, while cell-cycle inhibitors, often referred to as “cyclomodulins,” are toxic substances that prevent cells from dividing [185]. The secretion of CNF by some bacteria, such as E. coli, has been seen to enhance the progression from the G1 to the S phase of the cell cycle and promote DNA replication, leading to the formation of multinucleated cells. There is a potential for toxins such as Cif and CDTs, produced by many Gram-negative bacterial species like Salmonella typhi and Campylobacter jejuni, to inhibit the clonal proliferation of lymphocytes. Cif is found in both enterohemorrhagic E. coli and enteropathogenic E. coli. CDT toxins are synthesized by a wide range of Gram-negative bacterial species [186]. It’s possible that treating cancer using bacterial toxins has fewer adverse effects than conventional therapy does. It is possible that the treatment of cancer might be improved by combining the use of these poisons with conventional chemotherapy or radiation therapy [187].

Bacteriotherapy in cancer: real-world translational and clinical hurdles

Therapeutic bacteria promise to do things drugs cannot—find hypoxic tumor cores, remodel the microenvironment, and deliver payloads locally. But turning this promise into routine care faces unique obstacles [188]. The former is strain selection and control. Attenuated Salmonella, Clostridium, Listeria, or modified E. coli may colonise tumours, but may also disseminate, mutate, or cause sepsis, risks that are aggravated in immunocompromised cancer patients. Biocontainment design (auxotrophy, kill switches, quorum-sensing lysis circuits) is beneficial, but genetic circuits may fail in a selective environment, and horizontal genetic transfer to commensal or pathogen hosts is a practical consideration. The issue of safety and dosing is not a trifle. In contrast to conventional medicines, live bacteria replicate unpredictably, and their pharmacokinetics are influenced by the immune state of the patient, antibiotics, tumour perfusion, and oxygen levels in the region. Inflammation mediated by endotoxins may lead to high fevers or hemodynamic instability; aggressive antibiotic rescue may, on the other hand, eliminate the treatment effect. Tumor targeting and efficacy remain variable. While hypoxic or necrotic regions attract some strains, many solid tumors are patchy; incomplete colonization leads to uneven payload delivery and mixed responses [189]. The tumor microenvironment (TME) is a double-edged sword: it provides niches for bacterial growth but also contains dense stroma, abnormal vasculature, and immune suppressive cells that limit penetration and durable control [190]. When bacteria are used as delivery vehicles for cytokines, checkpoint inhibitors, or toxins, the payload control (how much, where, and when) must be precise to avoid systemic toxicity. Bacteriotherapy is a living product about manufacturing and logistics: it has to be sterilised to a high level (GMP), it has to maintain a consistent genotype, it has to be predictable in CFU, it has to be free of phage contamination, and it has to be closely regulated in terms of temperature during shipping and storage [191]. This is because it is difficult to test the batch-to-batch reproducibility and real-time release without slowing down care. Hospitals must also have infection-control measures such as patient isolation, environmental shedding, and waste management information, which is critical to regulators and institutional review boards [192]. There must be a change in clinical trial design. Conventional oncology dose-escalation (in search of a maximum tolerated dose) might not identify the biologically effective dose at which colonisation and payload expression capabilities occur. Trials require an imaging or molecular readout of colonization of the tumor, shedding measurements to control environmental risk, and an objective definition of when the antibiotics are to be removed [193]. A combination of bacteriotherapy, radiotherapy, chemotherapy, or immunotherapy is desirable but creates scheduling issues, drug-microbe interactions (e.g., antibiotics blunting effect), and overlapping toxicities [194]. Lastly, regulatory and public acceptance are substantial hurdles. Engineered microbes are regulated as live biotherapeutic products and genetically modified organisms (GMOs), with additional biosafety requirements and environmental risk assessments [195]. Long-term follow-up is needed to monitor persistence, resistance, and microbiome disruption. On the access and equity side, specialized manufacturing, containment infrastructure, and monitoring increase cost and limit availability, particularly outside major centers [196].

Combined therapies for cancer

Using a combination of different agents is a key strategy for effectively treating the disease. Various types of combination therapy are currently being used in both clinical settings and ongoing clinical trials. Many creative combinations have been developed. Given the surge in popularity of this treatment, it is reasonable to anticipate that technological advancements will lead to the development of even more innovative approaches. Furthermore, information from fields like nanotechnology, DNA sequencing technologies, and computational analysis can be employed to improve different combination approaches. Combination therapies have significant advantages over monotherapies. It’s important to highlight that there are still challenges to address, including preventing drug resistance, managing toxicity and drug interactions, and conducting more comprehensive survival analysis [197]. Furthermore, it is essential to carefully adjust combination therapies based on the unique reactions of patients. Having a deep understanding of the different combination approaches used in clinical settings or trials is essential for assessing the most effective treatment options for patients. Utilizing a variety of therapeutic agents is crucial in the battle against cancer, as it allows for a more effective approach known as combination therapy. When different anti-cancer drugs are combined, they synergistically enhance their effectiveness, surpassing the efficacy of using a single drug alone. These pathways are targeted in a manner that either works together or adds up. This approach holds great promise in reducing drug resistance and providing therapeutic advantages in the fight against cancer. These advantages encompass the deceleration of tumor growth and the inhibition of cancer cell proliferation, as well as the decrease in the population of cancer stem cells and the initiation of cellular demise [198]. Survival rates for most metastatic cancers are unfortunately still quite low, and the process of developing new anti-cancer drugs is both expensive and time-consuming. Thus, scientists are investigating alternative approaches that prioritize the pathways for survival to attain the best outcomes at a reasonable expense. Another approach involves using therapeutic agents that were initially created to treat diseases other than cancer. This method is most efficient when the FDA-approved substance focuses on pathways that are also found in cancer. By incorporating an FDA-approved drug into combination therapy, the expenses associated with research are greatly diminished. By optimizing the cost efficiency of therapy, it ensures that individuals with limited access to medical services can also reap the benefits. In addition, a technique that combines repurposed pharmaceutical agents with other therapeutics has shown promising results in reducing tumor burden [199].

Comparative study of four therapeutics options for cancer

Cancer vaccines. Therapeutic cancer vaccines (not preventive vaccines like HPV) have generally produced modest clinical outcomes to date [200]. The clearest real-world example is sipuleucel-T for metastatic prostate cancer, which in the pivotal trial prolonged median overall survival by roughly 4 months versus control [201]. This statistically significant but clinically modest benefit established proof of principle rather than a broad cure [202]. Several tumor-antigen and neoantigen vaccine platforms are in development (peptide, dendritic cell, viral vectors, mRNA), and some early-phase trials show immunologic responses and occasional durable remissions when combined with other therapies. However, single-agent vaccine response rates remain low in most solid tumors, and success has been limited to niche indications or combination strategies [203]. Key limitations are the generally weak magnitude of induced T-cell responses in the hostile tumor microenvironment (TME), antigen heterogeneity and loss, and the difficulty of generating neoantigen vaccines quickly and cost-effectively for each patient. Immune checkpoint inhibitors (ICIs) and cell therapies. ICIs (anti-PD-1/PD-L1 and anti-CTLA-4) are the most broadly impactful immunotherapies so far [204]. In immunologically “hot” tumors such as melanoma and some lung cancers, single-agent anti-PD-1 therapies produce objective response rates (ORRs) often in the 20–45% range and durable long-term survival for a subset of patients; combinations (e.g., nivolumab + ipilimumab) push response and long-term survival rates even higher in melanoma, with multi-year survivors now well documented [205]. In non-small cell lung cancer (NSCLC), trials show ORRs around 20% overall but much higher in PD-L1-high tumors, and 5-year survival benchmarks are now in the low-to-mid-20% range for some populations. CAR-T cell therapy for hematologic cancers has shown very high complete-remission rates in relapsed/refractory B-cell malignancies (often >50–80% in certain trials), but its success in solid tumors is far more limited [206, 207]. Limitations across ICIs and CAR-T include primary and acquired resistance (driven by the TME, antigen loss, and immune exclusion), immune-related toxicities that can be severe or fatal, and variable biomarkers to reliably predict responders [208]. The clinical payoff is real and sometimes dramatic, but benefits are heterogeneous and patient selection remains an unmet need.

CRISPR/Cas9-based approaches.

Clinical use of CRISPR in oncology is still nascent and mainly experimental. Most early clinical work involves ex vivo editing of patient immune cells (for example, knocking out PD-1 in T cells or inserting engineered receptors) and reinfusing them; a few bespoke trials have shown safety signals and hints of anti-tumor activity in small cohorts, but objective response rates are low and follow-up is short [209]. In vivo CRISPR editing of tumor cells remains largely preclinical because of delivery challenges. Key translational hurdles are safe, tumor-specific delivery (avoiding liver/spleen accumulation), limiting off-target edits and large genomic rearrangements, managing immune responses to CRISPR components, and building GMP manufacturing and long-term monitoring for genotoxicity [210]. Regulatory and ethical scrutiny is high, and clinical evidence of meaningful, reproducible cancer cures from CRISPR is still pending. In short, strong biological potential, but early, small, and cautious clinical signals so far.

Bacteriotherapy (engineered/attenuated bacteria). Using bacteria to treat cancer is attractive because some microbes naturally home to hypoxic tumor niches and can be engineered to deliver drugs, immune modulators, or provoke local inflammation [211]. Clinical experience is mixed: several Listeria- or Salmonella-based vaccines and Clostridium or C. novyi-NT intratumoral approaches have shown safety in phase I–II studies and immunologic activity, and a handful of trials reported encouraging survival signals in specific settings [212]. But robust, reproducible objective response rates across large trials are still rare. Major limitations include controlling bacterial replication and preventing systemic infection (especially in immunocompromised patients), ensuring predictable tumor colonization, biocontainment and horizontal gene transfer risks, and complex manufacturing and infection-control logistics [213]. Like CRISPR, bacteriotherapy is promising in principle, but translation to routine, widely used therapy requires solving safety, dosing, and regulatory challenges. Comparative takeaways. ICIs (and, in a few cases, CAR-T) are at the forefront with respect to current clinical maturity and established population value, as they have already achieved long-term remissions and survival advantages in large patient groups [214]. There have been isolated, small successes with cancer vaccines, and they are best combined with other agents rather than acting alone. CRISPR and bacteriotherapy are promising experimental platforms. CRISPR has exciting potential in terms of precise editing, but must address delivery, safety, and regulatory challenges; bacteriotherapy has the capacity to target unique tumour niches and deliver payload locally, but must overcome safety, reproducibility, and manufacturing challenges.

In all modalities, overarching themes that hinder success are the tumour microenvironment, tumour heterogeneity, technology of delivery, patient selection/biomarkers, and scalable manufacturing [215, 216]. Immune checkpoint blockade and CAR-T therapy have produced the clearest and most reproducible clinical benefits to date, with durable remissions in subsets of patients (notably melanoma, some lung cancers, and B-cell malignancies) [217]. Therapeutic cancer vaccines have delivered modest survival gains in selected settings and are likely to be most useful in combination with other immunotherapies. CRISPR-based cancer therapies remain early-stage: ex vivo edited immune cells have shown safety and preliminary activity in small trials, but in vivo editing faces major delivery and safety challenges. Bacteriotherapy shows promise for targeting difficult tumor niches, but it requires reliable control of live organisms, tight biocontainment, and clear strategies to prevent systemic infection [218].

Conclusion

Personalized cancer vaccines are a promising strategy for enhancing the precision and efficacy of tumor targeting in individual cancer patients. The progress and enhancement of cancer vaccines have made significant advancements, while not achieving the same remarkable efficacy and results as immune checkpoint inhibitors and T-cell therapy. The CRISPR/Cas9 system has garnered significant attention because of its potential to revolutionize the treatment of cancer and other diseases in the preclinical domain. This is now recognized as an advanced technique for genome editing. The potential medicinal applications of phases 1 and 2 are currently under investigation. The CRISPR/Cas9 system represents a promising and transformative advancement in the realm of immunological oncology and its associated disciplines. However, further research and development are required to ensure its widespread use. Tumor-targeting bacteria have favorable characteristics for the transportation of cancer therapeutic payloads, mostly attributed to their selectivity towards tumors and their ability to efficiently encapsulate genes. The remarkable capacity of unlimited gene packaging not only facilitates the activation of many or sizable target genes but also establishes the foundation for the manipulation of signaling networks in bacteria, thereby empowering them to perform intricate functions in the realm of cancer therapy. Cancer immunotherapy has recently seen significant advancements with the introduction of cancer vaccines, adoptive cell transfer (ACT), and immune checkpoint inhibitors (ICIs). These breakthroughs have brought about the long-anticipated promise of cancer immunotherapy. The cellular composition of the tumor microenvironment (TME) encompasses various cell types, including cancer, stromal, and immune cells. Various cell types have the potential to engage in interactions that may modify the tumor microenvironment (TME) and exert control over the proliferation of cancerous cells. Emphasizing this significance lies in acknowledging the collaborative efforts of immune cells residing inside tumors in regulating the progression of cancer. Synchronized reactions from both innate and adaptive immune cells play a pivotal role in determining the efficacy of immunotherapeutic interventions. Integration of different omics techniques for cancer treatment and biomarker identification is a promising and expanding field. It provides significant information regarding the biological processes and therapies of diseases. Integrating data from DNA, RNA, proteins, metabolites, and epigenetic markings is necessary to understand the complex interactions in living systems. This multi-omics technique holistically shows complex systems. Multi-omics has been used in biomedical research to identify new diseases, explore new medications, customize treatments, and optimize cancer therapies.

Author contributions

A.Z. wrote the manuscript. Data curation was done by S.W.

Disclosure

This paper has been uploaded to [https://www.authorea.com/] as a preprint: [https://www.authorea.com/users/854474/articles/1239719-unlocking-the-potential-and-myth-of-personalized-vaccines-crispr-cas9-and-bacteriotherapy-as-therapeuticoptions-for-cancer]

Funding statement

This study received no government or private financial support.

Data availability

The data used to support the findings of this study are included in this article.

Declarations

Ethics approval and consent to participate

Not applicable.

Informed consent

Not applicable.

Competing interests

The authors declare no competing interests.