Release date:2022/9/15 15:00:46

Monoclonal antibodies (mAbs) are a class of molecules that can specifically bind to target antigens and induce cytotoxicity through neutralizing or proapoptotic effects, as well as promote innate immune responses. The researchers found that mAbs showed remarkable performance in boosting the innate immune system, suppressing cancer cell activity and eliminating cancer cells. The successful development of hybridomas by Georges Kohler and Cesar Milstein in 1975 led to continued mass production of monoclonal antibodies. The introduction of antibody humanization technology by Greg Winter in 1988 further promoted the application and development of mAbs in the field of cancer treatment. To date, the US Food and Drug Administration (FDA) has approved more than 100 mAbs for the treatment of different human diseases, including cancer, autoimmune diseases, and chronic inflammatory diseases. With the development of modern biotechnology, the development and application of new therapeutic antibodies have made exciting progress.

Antibody Conjugates (ADCs And ARCs)

Antibody-drug conjugates (ADCs)

In recent years, the application of ADCs has gradually increased and become a rising star in the field of tumor therapy. ADCs contain three main components: mAb, cytotoxic drug (payload), and linker. After binding to target antigens on tumor cells, ADC can deliver cytotoxic drugs to the cytoplasm of target cells through receptor-mediated endocytosis. During lysosomal degradation, fine cytotoxic drugs are released from ADC to inhibit cell division and proliferation by destroying DNA and eventually kill tumor cells.

Schematic diagram of ADC drug structure

Figure 1 Schematic diagram of ADC drug structure

High degree of tumor targeting is a key factor that determines the druggability of an ADC. Tumor-specific or overexpressed target antigens can improve the targeting of ADCs. More than 50 antigens, including human epidermal growth factor receptor 2 (HER2), trophoblast cell surface antigen 2 (TROP-2), and B-cell maturation antigen (BCMA), are currently being developed as targets for preclinical or clinical applications of ADCs.

Free cytotoxic drugs are not suitable for direct administration as chemotherapeutic drugs due to their high potency, but the damage to normal cells of the body can be minimized by conjugating them with tumor-specific antibodies. Cytotoxic drugs used for ADCs are mainly divided into 3 types: 1) tubulin inhibitors, such as monomethyl auristatin E (MMAE); 2) DNA synthesis inhibitors, such as calicheamicin; 3) topoisomerase inhibitors and RNA Polymerase 2 inhibitors, such as α-amanitin. ADCs loaded with these drugs showed higher toxicity potency against tumor cells compared to the effects of conventional chemotherapy.

Linkers that covalently bind cytotoxic drugs to antibodies are also important components of ADC. The ideal linker is stable until it reaches the target tumor site and is properly designed to rapidly release cytotoxic drugs from the ADC upon entry into the lysosome. Linkers can be classified as cleavable or non-cleavable according to the mechanism of drug release. Cleavable linkers can respond to changes in the tumor microenvironment, such as the acid-unstable hydrazone linker in gemtuzumab ozogamicin (GO). Non-cleavable linkers rely on the degradation of antibodies in lysosomes to release drugs, for example, the thioether linker in ADo-trastuzumab emtansine.

To date, 12 ADC drugs have been approved by the FDA for cancer treatment.

ADC Drugs approved by the FDA for cancer treatment

Table 1 ADC Drugs approved by the FDA for cancer treatment

ADCs at phase III clinical trial

Table 2 Antibody-drug conjugates at phase III clinical trial

Antibody-small interfering RNA conjugates (ARCs)

As of January 2021, four siRNA drugs loaded with lipid nanoparticles (LNPs) or N-acetylgalactosamine (GalNAc) delivery systems have been approved by the FDA or the European Medicines Agency (EMA). However, these two siRNA delivery therapies have the problems of low delivery efficiency and limited target organs (only liver and eye). In late 2015, Genentech achieved site-specific, large-scale siRNA conjugation with antibodies using the THIOMAB platform. Recently, Alnylam Pharmaceuticals reported the generation of structurally defined ARCs (DVD-ARCs) without introducing mutations or the use of enzymes, for which three siRNA drugs (patisiran, givosiran, and lumasiran) have been previously developed. The study showed that ARC significantly down-regulated the expression levels of target mRNA and protein in tumor cells, which further demonstrated the basic principle and feasibility of using antibody as a carrier to specifically deliver siRNA to non-liver tissues.

Schematic diagram of ARC drug structure

Figure 2 Schematic diagram of ARC drug structure

Multispecific Antibodies

Over the past three decades, multispecific antibodies have received considerable attention as therapeutic agents. More than 100 multispecific antibody formats have been developed that work by interfering with different mechanisms in cancer immunotherapy, including: 1) Binding to T cells or other immune cells (such as NK cells) to specifically eliminate tumor cells; 2) Bridge receptors block or activate synergistic signaling pathways; 3) Target multiple tumor antigens or different antigenic epitopes on tumor cells to increase tumor selectivity.

Forms of Multispecific Antibodies

In the 1960s, Nisonoff and colleagues first described the original concept of mixing monovalent antibody fragments to generate multispecific antibodies. Initially, BsAbs were produced by chemical conjugation of two antibody fragments, followed by somatic fusion of two different hybridoma cell lines. The development of recombinant DNA technology and antibody engineering technology has made it possible to assemble different antibody domains into multispecific antibodies with targeting and stability. Formats of multispecific antibodies can be divided into two broad categories: IgG-like antibody formats (with an Fc domain) and non-IgG-like antibody formats (without an Fc domain). Full-length IgG-like multispecific antibodies contain an Fc domain that can bind to FcRn, exhibit better pharmacokinetic properties than antibody fragments, and exhibit multiple antitumor mechanisms. Compared to full-length IgG-like multispecific antibodies, non-IgG-like multispecific antibodies lack the Fc domain and thus have lower molecular weights, allowing better penetration in solid tumors. Due to their rapid metabolic rate, non-Ig-like polyspecific antibodies have a relatively short serum half-life and can be used to prevent the innate immune system as well as FC-mediated non-specific activation of ADCC or CDC.

structure of multispecific antibodies

Figure 3 Schematic diagram of the structure of multispecific antibodies

Mechanism of action of multispecific antibodies in cancer therapy

Targeting CD3 to engage T cells: BiTEs, bispecific single chain antibodies that use T cells as effector cells. BiTEs are able to simultaneously bind CD3ε in TCR complexes and specific tumor-associated antigens (TAAs) on tumor cells, and cytotoxic T cells are then redirected to kill specific tumor cells. Blinatumomab is a BiTEs composed of scFvs targeting CD3ε on T cells and the B lymphocyte antigen CD19. Results of a phase II clinical trial showed a 43% (81/189) CR rate in adult R/R B-ALL patients after two cycles of continuous intravenous infusion of blinatumomab. Currently, the FDA approved the expansion of the clinical indication for Blinatumomab to patients with Philadelphia chromosomal positive R/RB-ALL and ALL with R/R ALL or MRD-positive.

Targeting T cell costimulatory receptors: To reduce hepatotoxicity after systemic T cell costimulation with monoclonal antibodies, anti-TAA antibodies were fused with agonistic antibodies that recognize costimulatory receptors for tumor localization, TAA-dependent aggregation, and costimulatory receptor activation. CD28 and members of the TNF receptor (TNFR) superfamily, such as 4-1BB and OX40, are promising targets for T-cell costimulation-related cancer immunotherapy. For example, the 4-1BB/HER2 bispecific molecule PRS-343 significantly inhibited tumor growth in HER2+ mouse xenograft tumor models by inducing T-cell costimulation through HER2-dependent 4-1BB aggregation and activation, increasing lymphocyte infiltration and activation in tumors.

Targeting CD16 to engage NK cells: NK cells play an integral role in the innate immune system, reducing tumor burden and performing tumor immune surveillance. CD16 (FcγRIII) is a low-affinity receptor mainly expressed on mature NK cells that binds to the Fc domain of IgG antibodies to mediate ADCC. AFM13 employs a tetravalent bispecific CD30/CD16A tandem diabody with two binding sites for the CD16A isotype on NK cells and two binding sites for the CD30 antigen on lymphoma cells. A phase I study in patients with R/R HL showed an overall disease control rate of 61.5% and a partial response rate of 11.5% with AFM13 in 26 patients. Another recent phase Ib study of AFM13 in combination with pembrolizumab in patients with R/R HL showed an overall response rate of 83% and was well tolerated.

Targeting checkpoint receptors on T cells: Multiple studies have shown that the high expression of PD-L1 in advanced tumor cells inhibits T-cell responses and promotes immune escape from cancer cells, which limits the in vivo efficacy of cancer therapies. Targeting immune checkpoint molecules such as CTLA-4, PD-1, and PD-L1 with therapeutic antibodies can activate antitumor immune responses. IBI315 is an anti-PD-1 × HER2 BsAb, which connects PD-1 expressing T cells with HER2 expressing tumor cells, blocks the PD-1/PD-L1 signaling pathway in a HER2-dependent manner, and inhibits the activation of HER2 signaling pathway. IBI315 combines targeted therapy with immunotherapy to enhance antitumor activity through multiple mechanisms of action.

Targeting CD47 to enhance macrophage-mediated phagocytosis: CD47 is widely expressed in healthy and cancer cells and transmits a ""do not eat me"" signal when interacting with SIRPα on bone marrow cells. Anti-cd47 mAb enhances antitumor activity by blocking the CD47-SIRRP-α axis through release of macrophage-mediated phagocytosis, but it also damages normal CD47-positive cells such as erythrocytes. Therefore, to protect erythrocytes while maximizing CD47 blocking potency. The CD47-based BsAb TG-1801 (also known as NI-1701), an IgG1 bispecific κλ antibody (CD19 × CD47), was introduced to simultaneously target malignant B lymphocytes and selectively block CD47. A Phase I clinical trial of TG-1801 is currently underway.

clinical-stage multi-specific antibodies

Table 3 The FDA-approved and clinical-stage multi-specific antibodies


In recent years, novel therapeutic antibodies have made remarkable progress in cancer treatment. Studies have shown that the miniaturization and multifunctionalization of antibodies is a flexible and feasible strategy for the diagnosis or treatment of malignant tumors in complex tumor environments. The rapid development of next-generation sequencing, single-cell RNA sequencing, spatial omics, and comprehensive bioinformatics analysis has enabled people to have a more in-depth and comprehensive understanding of malignant tumor tissues. The era of antibody-based intracellular antigen targeting and tumor precision therapy has broad development prospects.

As a leading PEG supplier, Biopharma PEG provides GMP standard PEG derivatives and bulk orders via custom synthesis, offering the opportunity to match customers' special quality requirements. We provide a wide array of different ADC linkers to empower our customer's advanced research. ADC linkers with molecular weights, branching, and functional groups not listed in our online catalog may be available by custom synthesis.

[1]. Jin S, Sun Y, Liang X, et al.
Emerging new therapeutic antibody derivatives for cancer treatment[J]. Signal Transduction and Targeted Therapy, 2022, 7(1): 1-28.

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Emerging Multiple Myeloma Therapies: mAbs, ADCs, CAR-T Cells & Bispecific Antibodies
[2].ADC Drugs Global Sales of 2021 and Future Prospects
[3].Bispecific Antibodies - Current Status and Prospects
[4].Global Antibody-drug Conjugates (ADCs): Approvals & Clinical Trails Review

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