Cancer is a serious threat to human health and has become a global public health challenge. According to data from the World Health Organization, cancer is one of the leading causes of death worldwide, resulting in millions of deaths every year^[1]. It is estimated that there are tens of millions of new cancer cases diagnosed globally each year, and this number continues to rise.

The global cancer incidence and mortality data are alarming. According to the International Agency for Research on Cancer (IARC) "Global Cancer Statistics 2020," there were 19.3 million new cancer cases and approximately 10 million cancer deaths worldwide in 2020. By 2022, it is estimated that there were 20 million new cancer cases and 9.7 million cancer deaths globally. It is projected that by 2050, the global cancer incidence will exceed 35 million cases, a 77% increase compared to 2022. The top 10 cancers in terms of new cases accounted for about 63% of all cases, with lung cancer, breast cancer, colorectal cancer, prostate cancer, and stomach cancer being the top five. The top 10 cancers in terms of deaths accounted for 72% of all cancer deaths, with lung cancer, colorectal cancer, stomach cancer, liver cancer, and female breast cancer being the major culprits. Cancer incidence and mortality rates vary significantly among different regions, genders, ages, and ethnicities^[2-5]. Asian and African countries generally have higher cancer incidence and mortality rates compared to North America and Europe. Men have higher cancer incidence and mortality rates than women, with higher rates for lung cancer, colorectal cancer, liver cancer, and prostate cancer in men, and breast cancer and cervical cancer in women. Elderly individuals, especially those aged 65 and above, have significantly higher cancer incidence and mortality rates. Additionally, different ethnic groups have varying cancer risks; for example, African Americans have a higher risk of colorectal cancer, while Asians face a higher risk of liver cancer^[6-7].

In China, the situation regarding cancer is equally severe. According to the latest data from the National Cancer Center of China, there were approximately 4.064 million new cancer cases in China in 2016, with 2.552 million cases in men and 1.512 million cases in women. The number of cancer deaths was about 2.414 million, with 1.632 million deaths in men and 0.781 million deaths in women, with higher incidence and mortality rates in men than in women. In 2022, there were approximately 4.8247 million new cancer cases and 2.5742 million new cancer deaths in China, with lung cancer ranking first in both incidence and mortality rates. In terms of incidence, the overall crude cancer incidence rate in 2022 was 341.75 cases per 100,000 people, with the top five new cancer cases being lung cancer, colorectal cancer, thyroid cancer, liver cancer, and stomach cancer, accounting for 57.42% of all new cancer cases. By gender, the top five new cancer cases in men were lung cancer, colorectal cancer, liver cancer, stomach cancer, and esophageal cancer, while in women, they were lung cancer, breast cancer, thyroid cancer, colorectal cancer, and cervical cancer. In terms of mortality, the overall crude cancer mortality rate in 2022 was 182.34 deaths per 100,000 people, with the top five causes of cancer deaths being lung cancer, liver cancer, stomach cancer, colorectal cancer, and esophageal cancer, accounting for 67.50% of all cancer deaths. By gender, the top five causes of cancer deaths in men were lung cancer, liver cancer, stomach cancer, colorectal cancer, and esophageal cancer, while in women, they were lung cancer, colorectal cancer, liver cancer, stomach cancer, and breast cancer^[8-9]. From 2000 to 2018, the overall cancer incidence rate in China showed an upward trend, increasing by approximately 1.4% annually, while the mortality rate decreased slightly, with an annual decline of about 1.3%. The overall age-standardized cancer incidence rate for men remained relatively stable, while for women, it increased significantly by 2.6% annually due to the increased diagnoses of thyroid cancer and cervical cancer. The decrease in the overall age-standardized cancer mortality rate during this period was mainly due to the reduction in mortality rates for esophageal cancer, stomach cancer, and liver cancer.

Among all types of cancer, lung cancer is considered the "number one killer." In 2022, there were 730,000 deaths from lung cancer in China, accounting for over 30% of all cancer deaths, with an estimated 1.8 million deaths from lung cancer globally, representing 18.7% of all cancer deaths.

In recent years, the development of biotechnology has brought new hope for cancer treatment. Monoclonal antibody therapy, as an important treatment modality, has made significant breakthroughs in the field of cancer treatment. Monoclonal antibodies can specifically bind to antigens on the surface of cancer cells, activate the immune system to attack tumor cells, and achieve therapeutic effects. However, the current production methods for monoclonal antibodies are costly, complex, and inefficient, limiting their widespread clinical application^[2-6]. To address the challenges facing monoclonal antibody production, red light-induced nanobody production technology has emerged as a promising new approach. This technology utilizes the unique properties of red light to precisely control the synthesis and yield of nanobodies, providing a new possibility for cancer treatment. By conducting in-depth research and applying red light-induced nanobody production technology, we have the potential to overcome the limitations of traditional monoclonal antibody production, improve the efficiency and quality of monoclonal antibody production, and provide more effective treatment options for cancer patients^[7].

In the field of biomedicine, red light-induced nano-antibody production technology has garnered significant attention as an emerging research direction. Traditional antibody production methods typically involve complex experimental procedures and expensive equipment, limiting their efficiency and cost-effectiveness in large-scale applications. However, the emergence of red light-induced nano-antibody production technology has brought new hope to the field of antibody production.

Red light-induced nano-antibody production technology leverages the unique properties of nanomaterials under red light irradiation to enhance antibody synthesis and yield by controlling the red light wavelength and intensity. Compared to traditional methods, red light-induced nano-antibody production technology offers advantages such as ease of operation, high efficiency, and low cost, bringing revolutionary changes to the field of antibody production^[10].

Red light-induced systems, as an emerging gene expression regulation technology, have been proven to have unique advantages in non-toxic regulation and precise spatiotemporal expression. However, current research efforts have mostly focused on basic research, and the application of red light-induced systems in antibody drug production is still in its early stages^[3]. Currently, there are cases of using blue light-induced systems to induce antibody production, but there has been no utilization of red light-induced systems for producing nano-antibodies.

The reasons or advantages for choosing red light-induced systems are as follows: Reducing side effects and energy consumption in the production process significantly enhances the expression efficiency and quality of nano-antibodies. Additionally, this technology platform exhibits excellent scalability and can be applied to the production of other biopharmaceuticals, bringing about innovation-driven and economic benefits to the biopharmaceutical industry. Compared to blue light systems, red light has a smaller impact on bacterial growth status, whereas blue light can inhibit bacterial growth.

In the rapidly evolving field of biotechnology, the application potential of red light-induced nano-antibody production technology is immense. By conducting in-depth research on the principles and mechanisms of red light-induced nano-antibody production technology, novel solutions can be provided for future biomedical research and clinical therapy, driving innovative development in the field of antibody production.

Lung cancer is one of the most common malignant tumors worldwide. Among these, non-small cell lung cancer accounts for approximately 80% of all lung cancer cases. Current main methods for treating cancer: antibody drugs, among which nanobodies have garnered significant attention in anti-tumor therapy due to their high affinity, low immunogenicity, and good tissue penetration. Nanobody (nanobody, Nb): is an artificially designed antibody molecule, also known as single-domain antibodies (sdAbs), VHH antibodies or camelid antibodies, discovered in alpacas, dromedaries, etc., as a type of heavy-chain antibody (heavy-chain antibodies, HCAbs) naturally lacking light chains (Figure 1).

Figure 1. Schematic structure of a conventional nanobody(cited by：http://omicsspace.com)

To improve lung cancer treatment efficacy, this study selects non-small cell lung cancer as the therapeutic target, focusing on developing nanobodies against PD-L1 and PD-1. Their mechanism of action is based on the principle of tumor immune escape: tumor cells inhibit T-cell immune killing function by overexpressing PD-L1/PD-L2 ligands that bind to PD-1 on T-cell surfaces, thereby evading immune system clearance(Figure 2).

Figure 2. Targeting mechanism of PD-L1 and PD-1[1]

First, construct a red light-induced system, whose photosensory module (PSM) has the bacterial phytochrome (DrBphP) as the key gene. The primary mechanisms are: a. Perception of red and far-red light through the biliverdin chromophore. Biliverdin (BV) can be produced by heme oxygenase (HO) alone. b. Light-induced structural changes in PSM are transmitted to the effector HK domain (YF1), thereby altering its basal kinase and phosphatase activities. FixJ: an inverter cassette encoding the λ phage repressor cI and λ promoter pR.

The research plan is divided into three progressive stages. The first is red light-induced system validation: first construct the red light-induced system, detect the system's response sensitivity under red light irradiation at different time points, while evaluating the impact of light parameters on the host bacteria (E. coli) growth status to ensure compatibility between induction conditions and microbial cultivation(Figure 3).

Figure 3. Components of the red light-induced system

The second is nanobody production and verification: clone Anti-PD-L1 and Anti-PD-1 nanobody genes into expression vectors(Figure 4), transform DH5α and BL21 E. coli strains respectively. After red light-induced expression, employ dual verification with ELISA and functional experiments: confirm the red light system can effectively drive antibody production. The third is production condition optimization: systematically investigate the effects of parameters such as light intensity and duration on antibody yield, establishing relatively ideal production conditions.

Figure 4. Production mechanism of PD-L1 and PD-1 nanobodies.

This study aims to develop an efficient and controllable nanobody production platform by utilizing a red light-induced gene expression system in Escherichia coli. By integrating optogenetic regulation with the robust protein synthesis capacity of E. coli, we achieve precise control over nanobody expression for the preparation of anti-tumor therapeutics, significantly enhancing both the production efficiency and quality of nanobodies.

Product Description:

Our innovation is a modular nanobody production kit, enabling customizable synthesis of diverse nanobodies (e.g., anti-PD-1/PD-L1 et.al). The product components are illustrated in Figure 5.

Figure 5. Schematic diagram of the product (nanobody production kit)

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