What is Immune-Stimulating Antibody Conjugate (ISAC)?

Immune-stimulating antibody conjugate (ISAC) consist of three components, including a tumor-targeting antibody, an immune agonist and a non-cleavable linker binding them together. The immune agonists or modulators as payloads of ISACs enable the ability to convert “cold” tumors into immune “hot” tumors where immunosuppressed but potentially tumor-reactive T cells are present within the tumor microenvironment (TME). ISACs can achieve activation of immune response and tumor killing by modulating immune stimulation and TME.

Definition of hot and cold tumors

It has been first described that three major immune coordination profiles (hot, altered and cold) within primary colorectal cancer (CRC) are observed. Due to the observation that the type, density and location of immune cells within the tumor site could predict survival in CRC, a concept for patient stratification is proposed, which leads to the development and implementation of the Immunoscore. Immunoscore is a standardized scoring system through the quantification of two lymphocyte populations both at the tumor center and the invasive margin.

The Immunoscore ranges from Immunoscore 0 (I0, absence of these two lymphocytes in both locations) to I4 (high immune cell densities in both locations). By classifying cancers according to their immune infiltration rather than the cancer-based, introducing the notion of cold tumor (non-infiltrated, I0) and hot tumor (highly infiltrated, I4). The classification of immune hot, altered and cold tumors can be used to predict the risk of recurrence and survival. Study suggested that of these three types of CRCs, hot tumors have the lowest risk of recurrence, while cold tumors have the highest risk of that.

Effect of immune agonist

Given that antigen-presenting cells (APCs) can elicit widespread T-cell immunity and tend to be more frequent than T cells within the TME, APCs have emerged as an attractive target for therapeutic intervention. Toll-like receptor (TLR) agonists are a class of immune stimulants that can activate APCs, leading to direct tumor cell killing via phagocytosis and enhanced T-cell mediated immunity against tumor neoantigens. It has been demonstrated that antitumor immune responses can be greatly enhanced when immune stimulants such as TLR agonists are used in conjunction with tumor-targeting antibodies which promote phagocytosis after engagement of Fcγ receptors (FcγRs). Therefore, the combination of immune agonist and antibody targeting tumor leads to enhanced uptake of tumor antigens by phagocytosed tumor-associated APCs, resulting in T cell activation, immune response and eradication of primary tumors and metastatic lesions.

Technology of ISAC

Intratumoral delivery of combination of antibodies and immune agonists such as TLR 7/8 agonists has been shown to activate tumor resident APCs, driving uptake, processing, and presentation of tumor neoantigens to T cells. This ISAC strategy makes progress in the management of patients with HER2-expressing or HER2-driven solid tumors, with HER2 as a prognostic indicator for clinical treatment monitoring and an important target for tumor-targeting drugs.

Antibody conjugation

In general, the ISACs can be produced through a two-step conjugation method. First, lysine residues containing reactive group primary amine of monoclonal antibody can be modified with a bifunctional cross-linker, such as N-succinimidyl-S-acetylthioacetate (SATA). The primary amine of lysine residue reacts with the electron-deficient ester group of SATA by nucleophilic substitution to generate the amide compound. Second, deprotection of the acetylated thiol that this functional group comes from SATA to generate a reactive thiol, following by reaction between a thiol with an immune agonist to produce the ISAC. In addition, ISACs also can be produced by a one-step conjugation method, which using an amine-reactive linker agonist reacted directly with the lysine residues of a monoclonal antibody. In this method, polyethylene glycol (PEG) can be used as the cross-linker.

In conclusion

There are a number of immunosuppressive factors present in tumor microenvironment, so it is necessary that trying to utilize various molecules to stimulate the immune system. The innate immune agonists for recognizing receptors such as TLR can alter the tumor microenvironment and stimulate adaptive antitumor immunity. The challenge of delivering these agonists is that they must be delivered intratumorally, due to agonists present toxicities associated with widespread immune activation after systemic administration. Therefore, the immune-stimulating antibody conjugates (ISACs) are developed to solve this issue. The ISACs used in the tumor therapy with capable of safely eliciting tumor-targeted responses though systemic delivery.


  1. Ackerman, S. E., Alonso, M. N., et al., Immune-stimulating antibody conjugates elicit robust myeloid activation and durable antitumor immunity, Nature Cancer, 2021, 2, 18-33.
  2. Urban-Wojciuk, Z., et al., The role of TLRs in anti-cancer immunity and tumor rejection, Front. Immunol., 2019, 10, 2388.
  3.  Galon, J., and Bruni, D., Approaches to treat immune hot, altered and cold tumours with combination immunotherapies, Nature Reviews Drug Discovery, 2019, 18, 197–218.
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