In recent years, antibody therapies (including traditional antibodies and nano-antibodies) have become an important means of treating a wide range of human malignant diseases, and more than 100 antibody therapies have been approved for clinical use. Since 2014, the U.S. Food and Drug Administration (FDA) and the European Medicines Agency (EMA) have approved 6-13 antibody therapeutics per year. The main form of antibody approved for marketing is IgG, in addition to bivalent and trivalent antibodies and antibody fragments. Currently, antibody therapeutics are most commonly used in the clinic to treat diseases such as cancer, autoimmune disorders and chronic inflammation. As novel antibodies such as nano-antibody therapies continue to emerge, treatments for infectious diseases, hematologic disorders, neurological disorders, ophthalmic disorders, metabolic disorders, musculoskeletal disorders, and organ transplantation have been widely explored and applied. This article will learn about the 8 most popular targets of nano-antibodies and traditional antibodies: Her2, EGFR, CD19, CD20, CTLA-4, BCMA, VEGF, and TNFα.
| Target name | Uniport number | molecular weight (kDa) | Length (a.a.) | Subcellular localization |
| Her2 | P04626 | 137.9 | 1255 | Class I unit sub-transmembrane proteins |
| EGFR | P00533 | 134.3 | 1210 | Class I unit sub-transmembrane proteins |
| CD19 | P15391 | 61.13 | 556 | Class I unit sub-transmembrane proteins |
| CD20 | P11836 | 33.08 | 297 | Quadruple transmembrane proteins |
| CTLA-4 | P16410 | 24.66 | 223 | Class I unit sub-transmembrane proteins |
| BCMA | Q02226 | 20.17 | 184 | Class Ⅲ unit sub-transmembrane proteins |
| VEGF | P15692 | 27.04 | 232 | Scretory protein |
| TNFα | P01375 | 25.64 | 233 | Class Ⅱ unit sub-transmembrane |
Her2
HER2 (uniport: P04626) is short for Human Epidermal growth factor Receptor 2, a member of the receptor tyrosine kinase family in humans. HER2 is normally involved mainly in the regulation of cell growth and differentiation processes, and is usually expressed on the cell membranes of a wide range of organs (e.g., breast and skin) as well as epithelial cells of the gastrointestinal tract, respiratory tract, reproductive system, and urinary tract. However, when it is mutated or overexpressed, it leads to abnormal cell proliferation and tumor formation.
The HER2 protein, also known as ErbB2, is located in the region of human chromosome 17q12. HER2 overexpression occurs in approximately 15-30% of breast cancers and 10-30% of gastric/gastroesophageal cancers, and is a marker that has been implicated in a variety of malignancies, including breast cancer and gastric/gastroesophageal cancer. HER2 overexpression is also seen in other cancers such as ovarian, endometrial, bladder, lung, colon and head and neck cancers. Therefore, the detection of HER2 is important in the diagnosis, treatment and prognosis of these tumors. HER2 positivity means that the HER2 protein is overexpressed or genetically amplified on the surface of the tumor cells, suggesting that the cancer cells may be sensitive to HER2-targeted therapy. There are 3 main mechanisms of action of anti-HER2 therapeutic antibodies (Figure 1). 1. Blocking HER2 receives growth signals from HER2-positive breast cancers. By blocking growth signals, anti-HER2 drugs can slow or stop the growth of HER2-positive and HER2-low-expressing breast cancers. 2. Initiates an ADCC (antibody-dependent cell-mediated cytotoxicity) effect that mediates direct killing of target cells by killer cells. 3. Coupling with small molecule drugs to achieve target cell killing.HER2 antibodies can also be used in immunomolecular imaging of HER2, such as the nanoantibody imaging agent 68GaNOTA-Anti-HER2 VHH1 (NCT03331601), which has already entered phase 2 in the clinic.
EGFR
EGFR (epidermal growth factor receptor) is a transmembrane tyrosine kinase receptor that belongs to the ErbB receptor family along with HER2. EGFR is normally expressed in low amounts in tissue cells such as skin and liver. GFR abnormalities or mutations have been associated with a variety of diseases such as non-small cell lung cancer (NSCLC), colorectal cancer, breast cancer, and head and neck cancers, as well as with kidney disease and certain skin diseases such as psoriasis and atopic dermatitis. High expression of EGFR promotes the proliferation, survival and invasion of cancer cells, leading to tumorigenesis and progression.
A variety of therapeutic agents have been developed to target EGFR, including monoclonal antibodies (e.g., cetuximab, nimotuzumab, panitumumab, etc.) that inhibit the activity of EGFR mainly by blocking the binding of growth factors to EGFR, thereby achieving the effect of tumor treatment (Figure 2).
CD19
CD-19 is a single transmembrane glycoprotein widely expressed on the surface of B cells. It plays an important role in B cell development, differentiation and functional regulation. CD19 is expressed on the surface of leukemic cells in more than 90% of patients with acute lymphoblastic leukemia (ALL). Thus, CD-19 has become an important target in the therapeutic direction of hematologic malignancies. Especially for B lymphoma and acute lymphoblastic leukemia (ALL). Therapeutic agents targeting CD19 mainly include CAR-T cell therapies (e.g., CD19/20 bispecific nanoantibody-derived CAR-T cell therapy, NCT03881761) and bispecific antibodies (e.g., blinatumomab), which can induce apoptosis in B cells or activate T cells to attack tumor cells through different mechanisms.
CD20
CD20 is a B cell-specific membrane glycoprotein that is widely expressed on the surface of B lymphocyte precursors, mature cells, and memory B cells, and is involved in biological processes such as B cell differentiation, proliferation, and antibody production. Because CD-20 is highly expressed on B cells in patients with B lymphoma and some autoimmune diseases, it has become one of the targets for the treatment of these diseases. A variety of CD-20 antibody products have been developed, such as the nanobody CAR-T therapy mentioned above (NCT03881761), Rituxan, Ofatumumab, Ublituximab for the treatment of non-Hodgkin’s lymphoma or chronic lymphocytic leukemia, and chimeric proteins (e.g., Obinutuzumab), iopathic lesion-removing drugs (TOTEM) for the treatment of rheumatoid arthritis, and Orelizumab for the treatment of relapsing multiple sclerosis (MS) in adults. These therapies or drugs can cause B cells to undergo apoptosis through different mechanisms, leading to a therapeutic effect.
CTLA-4
The CTLA-4 (cytotoxic T lymphocyte antigen 4) gene was first discovered by French scientists in 1987, and subsequently, its function has been successively revealed (Figure 1). CTLA-4, a member of the immunoglobulin superfamily, is a checkpoint molecule that performs signaling and immunoregulation mainly between immune cells (e.g., T cells) and antigen-presenting cells. CTLA-4 binds to the same ligands as CD28 (CD80 and CD86) but with opposite functions, CTLA-4 is a key inhibitory receptor that plays an important role especially in the initiation phase of the immune response. In resting T cells, CTLA-4 is located intracellularly and translocates to the T cell surface when the T cell is activated by CD28 stimulation. CTLA-4 competes with CD28 for binding and blocks T cell activation, thereby inhibiting the T cell activation process. Through this action, CTLA-4 can inhibit the immune system from attacking the body’s own tissues and avoiding autoimmune diseases, as well as potentially preventing T cells from attacking tumor cells. Therefore, drugs targeting CTLA-4 have been widely used in the field of tumor therapy.
CTLA-4 monoclonal antibody (Ipilimumab, approved in the United States in 2011 for the treatment of advanced melanoma) was an early developed immune checkpoint inhibitor that preceded PD-1 monoclonal antibody but was surpassed by PD-1 monoclonal antibody and is no longer recommended as a first-line therapeutic monoclonal antibody for advanced melanoma due to its high toxicity and limited efficacy. Clinical studies of Tremelimumab, another CTLA-4 monoclonal antibody analog, have progressed more tortuously. It was approved by the FDA as an orphan drug for the treatment of malignant mesothelioma (MM) in 2015, failed Phase III clinical trials for the treatment of non-small cell lung cancer (NSCLC) or bladder cancer in 2017, 2019, and 2020, and was granted orphan drug status for the treatment of Hepatocellularcarcinoma HCC in February 2020 by the FDA. Tremelimumab, an hIgG2 subtype, has no ADCC effect and is less effective as a single agent. To solve the dilemma faced by CTLA-4 monoclonal antibody drugs, bispecific antibodies and nanobodies have become breakthroughs, such as Cadonilimab, a tetravalent antibody targeting PD-1/CTLA-4, KN046 targeting PD-L1/CTLA-4, and AstraZeneca’s MEDI5752, a bispecific antibody targeting PD-1/CTLA-4, all of which have strong antitumor activity. In addition, HBM4003, a nano-antibody drug developed by Harbour, was modified to significantly enhance ADCC and increase Treg cell depletion.
BCMA
BCMA (B cell maturation antigen), a member of the tumor necrosis factor (TNF) receptor family, is a transmembrane protein expressed on more mature B cells and plasma cells. BCMA is involved in physiological processes such as regulation of the immune system and inflammatory responses, mainly by mediating signal transduction pathways during B cell to plasma cell differentiation.
In oncology, BCMA has also emerged as one of the therapeutic targets for multiple myeloma (MM). Currently, therapies such as monoclonal antibodies, biospecific drugs, and CAR-T cells have been widely explored for BCMA (Figure 2). Among them, BCMA monoclonal antibody drugs depend on antigen specificity and can promote macrophage-mediated antitumor activity by recognizing BCMA and acting directly on the surface of multiple myeloma (MM) cells. In contrast, CAR-T cells have the ability to specifically kill CD19- as well as CD38+ malignant plasma cells, which makes it possible that CAR-T cell therapy targeting BCMA may have a significant therapeutic effect on some hard-to-treat cancers such as MM. 2022 Legend Biotech’s first nanoantibody CAR-T cells targeting BCMA developed by Legend Biotech were approved by the FDA for marketing, making it the first cell product from China to enter the U.S. market.
VEGF
VEGF (vascular endothelial growth factor) is a vascular endothelial growth factor that is overexpressed in many tumors. VEGF can promote neovascularization and provide nutrients and oxygen to tumors, thus promoting tumor growth and metastasis (Figure 4). Therefore, VEGF has become an important target.
Several antibody drugs have been developed against VEGF, including:
Bevacizumab: Approved for marketing by the U.S. FDA in 2004, it is currently approved for the treatment of a wide range of cancers, including advanced colorectal cancer, lung cancer, breast cancer, kidney cancer, and malignant glioma.
Ablibercept is a VEGF-binding Fc fusion protein developed by Regeneron Pharmaceuticals. It was approved for marketing by the FDA in 2011. Currently, Ablibercept is approved in Europe and the United States for the treatment of four conditions: wet age-related macular degeneration (AMD), central retinal vein occlusion secondary to macular edema (CRVO-ME), diabetic macular edema (DME), and visual impairment due to myopic choroidal neovascularization (mCNV).
Conbercept is a new-generation anti-VEGF fusion protein developed by Chengdu Kanghong Phamaceutical Group Co., Ltd. Conbercept received marketing approval in 2013 for the initial indication of age-related macular degeneration (AMD). In 2017, it gained a new indication – vision loss due to choroidal neovascularization secondary to pathologic myopia (PM) (pmCNV). New indications such as RVO-ME and DME have entered clinical phase III trials in December 2020. These drugs can inhibit VEGF activity through different mechanisms, thereby preventing tumor vascularization and achieving therapeutic disease outcomes.
TNFα
Tumor Necrosis Factor (TNF) is a class of cytokines produced primarily by macrophages. It has a variety of biological functions, including regulating the activation, proliferation, and differentiation of immune cells, promoting inflammatory responses, and inducing apoptosis (Figure 4). TNF-α (tumor necrosis factor-α) is the most common and functionally important member of the TNF family.
Since TNF-α plays a key role in many inflammatory and autoimmune diseases, inhibiting the biological activity of TNF-α could be an effective strategy for treating such diseases. Anti-TNF-α drugs are mainly monoclonal antibodies or soluble receptor proteins against TNF-α that can inhibit its biological activity by interfering with the binding of TNF-α to its receptor. Several anti-TNF-α drugs have been used for clinical treatment:
Infliximab is a murine-human chimeric monoclonal antibody primarily used to treat inflammatory diseases such as rheumatoid arthritis, ankylosing spondylitis, Crohn’s disease (CD) and ulcerative colitis.
Adalimumab is a fully humanized monoclonal antibody indicated for a variety of autoimmune diseases such as rheumatoid arthritis, ankylosing spondylitis, psoriatic arthritis, Crohn’s disease and ulcerative colitis.
Golimumab is a fully humanized monoclonal antibody used to treat rheumatoid arthritis, ankylosing spondylitis, psoriatic arthritis, and ulcerative colitis.
Ozoralizumab (also known as ATN-103) is a backbone-optimized, fully humanized nano-antibody drug against tumor necrosis factor-alpha (TNF-α). It works by specifically binding and neutralizing TNF-α, thereby inhibiting its biological activity for the treatment of inflammation-related diseases. The primary initial development of ozoralizumab was to improve the safety and tolerability of existing anti-TNF-α drugs. Due to its unique “trimer” structure, ozoralizumab has a lower molecular weight and higher selectivity, and is able to accumulate in the joint cavity and remain biologically active for a long time. This allows ozoralizumab to potentially reduce the risk of infections and other systemic side effects while maintaining efficacy compared to traditional anti-TNF-α drugs. Currently, ozoralizumab is being studied primarily in the treatment of autoimmune disease areas such as rheumatoid arthritis and ankylosing spondylitis. Clinical trials have shown that ozoralizumab has demonstrated a favorable safety and tolerability profile in the treatment of rheumatoid arthritis and other diseases.
V565 is an oral TNFα nano-antibody drug that has completed Phase II clinical trials. Antibodies to TNFα are very effective in the treatment of Crohn’s disease, but the primary mode of administration is intravenous or subcutaneous; V565 is innovatively administered orally. The phase II clinical trial of V565 evaluated the efficacy of oral V565 in patients with active CD over a 6-week period. The study included 125 patients with ileal or colonic disease, of whom 2/3 were randomly assigned to receive oral V565 treatment and 1/3 to receive placebo, with a follow-up period of 28 days. The primary assessment metric was clinical response, defined as a reduction in CDAI-70 (Crohn’s Disease Activity Index) at day 42 and a reduction in inflammatory markers. The results of the study showed no significant difference in clinical remission rates between the two groups (35.4% in the V565 group versus 37.2% in the placebo group), but endoscopic improvement rates were higher in the treatment group (56.3% in the V565 group versus 30.0% in the placebo group). The incidence of serious adverse events was similar between the two groups.
References
1 Wynn, C. S. & Tang, S.-C. Anti-HER2 therapy in metastatic breast cancer: many choices and future directions. Cancer and Metastasis Reviews 41, 193-209, doi:10.1007/s10555-022-10021-x (2022).
2 Han, C.-B., Ma, J., Li, F. & Zou, H.-w. Molecular Markers for the Prediction of Anti-EGFR Monoclonal Antibody Treatment Efficacy in Metastatic Colorectal Cancer. Journal of Cancer Therapy 02, 675-682 (2011).
3 Wang, K., Wei, G. & Liu, D. CD19: a biomarker for B cell development, lymphoma diagnosis and therapy. Experimental Hematology & Oncology 1, 36, doi:10.1186/2162-3619-1-36 (2012).
4 Kellner, C., Peipp, M., Gramatzki, M., Schrappe, M. & Schewe, D. M. Perspectives of Fc engineered antibodies in CD19 targeting immunotherapies in pediatric B-cell precursor acute lymphoblastic leukemia. Oncoimmunology 7, e1448331 (2018).
5 de Sèze, J. et al. Anti-CD20 therapies in multiple sclerosis: From pathology to the clinic. Frontiers in Immunology 14, doi:10.3389/fimmu.2023.1004795 (2023).
6.Brunet, J.-F. et al. A new member of the immunoglobulin superfamily—CTLA-4. Nature 328, 267-270 (1987).
7.Van Coillie, S., Wiernicki, B. & Xu, J. Molecular and cellular functions of CTLA-4. Regulation of Cancer Immune Checkpoints: Molecular and Cellular Mechanisms and Therapy, 7-32 (2020).
8.Cho, S.-F., Anderson, K. C. & Tai, Y.-T. Targeting B cell maturation antigen (BCMA) in multiple myeloma: potential uses of BCMA-based immunotherapy. Frontiers in immunology 9, 1821 (2018).
9.Leone, G. M., Mangano, K., Petralia, M. C., Nicoletti, F. & Fagone, P. Past, Present and (Foreseeable) Future of Biological Anti-TNF Alpha Therapy. Journal of Clinical Medicine 12, 1630 (2023).