How to choose the subtype of antibody for tumor immunotherapy?

• Facebook
• Twitter
• Reddit
• Tumblr
• LinkedIn
• Pinterest
• RSS Feed
• Link

How to choose the subtype of antibody for tumor immunotherapy?

How to choose the subtype of antibody for tumor immunotherapy? Monoclonal antibody (mab) has become an increasingly important class of drugs, and its clinical application has completely changed the field of cancer treatment.

Different monoclonal antibodies have different anti-tumor mechanisms, including blocking tumor-specific growth factor receptors or immunomodulatory molecules, and complement and cell-mediated tumor cell lysis.

Therefore, for many monoclonal antibodies, Fc-mediated effector functions are critical to the therapeutic efficacy. Since immunoglobulin subtypes differ in their ability to bind to FCR on immune cells and their ability to activate complement, the immune responses they activate are also different.

Therefore, the choice of the antibody subtype of a therapeutic monoclonal antibody depends on its expected mechanism of action. Considering that the clinical efficacy of many monoclonal antibodies is only achieved in patient subgroups, the selection of the best subtype and Fc optimization in the antibody development process may be an important step towards improving the prognosis of patients.

Next, we will discuss the choice of antibody subtypes from three aspects: tumor antigen-targeting antibodies, immune checkpoint inhibitor antibodies, and TNFR family agonistic antibodies.

The mechanism of action of tumor antigen targeting antibody

The first generation of therapeutic antibodies approved for clinical use is still the most common type of monoclonal antibody in cancer treatment, consisting of antibodies directed against tumor antigens.

These tumor antigens are more or less important for tumor growth, survival and invasion (such as anti-HER2, anti-EGFR).

However, some observations in humans and mice indicate that Fc-mediated immune cell activation is an important additional mechanism of action for many of these monoclonal antibodies.

The Fc part of the antibody can activate effector cells, such as FCR on NK cells, macrophages or neutrophils, and then mediate tumor cell lysis. This is mediated by cytotoxicity (antibody-dependent cell-mediated cytotoxicity-ADCC) or phagocytosis of tumor cells (antibody-dependent cell-mediated phagocytosis-ADCP). In addition, through its Fc tail, antibodies can activate the complement cascade by binding to C1q, leading to tumor cell lysis through several different mechanisms. This includes the formation of membrane attack complex (MAC), which directly induces the lysis of target cells (CDC) or attracts immune cells through the chemotaxis of complement components C3a and C5a.

In addition, C3b and C4b mediate complement-dependent cell-mediated cytotoxicity (CDCC) of NK cells, macrophages/monocytes and granulocytes, or complement-dependent cell-mediated phagocytosis of myeloid cells (CDCP) ). Antibody-mediated cell death can also lead to the release of tumor antigens and the formation of immune complexes (IC), thereby promoting the initiation of anti-tumor T cell responses and maintaining tumor control and rejection. In this process, the binding of FcγRs and the activation of complement play a key role in the uptake of IC by dendritic cells (DC) and the presentation of tumor antigens.

Optimize IgG effector function

IgG-Fc effector functions are mediated by complement and FcγRs, which are classified into activating receptors (FcγRI, FcγRIIa/IIc, FcγRIIIa, FcγRIIIb) or inhibitory receptors (FcγRIIb). Since most effector cells express activating and inhibiting FcγRs at the same time, the result of IgG binding is the result of a relatively comprehensive affinity, receptor availability, and signaling ability. The relative affinity of an antibody to its receptor is defined as the activation inhibition ratio (A/I).

The concept of A/I ratio is based on the observation of mice. The results show that the A/I of mIgG2a is higher, the A/I of mIgG1 is lower, and the A/I of mIgG2b is in the middle. Therefore, in many in vivo models, therapeutic antibodies of the mIgG2a subclass have been shown to be more effective in clearing tumors. Although the difference in A/I ratio between human IgG subtypes is not obvious, because of their different FcR binding profiles, their ability to induce immune responses is also different. IgG1 and IgG3 bind to all FCRs, but show higher affinity for activated FCRs.

Therefore, they are defined as Ig subtypes with strong Fc effector functions. On the other hand, IgG4 has similar affinity to most activated FcRs and inhibits FcγRIIb binding, and is considered low activity. Finally, except for the high-affinity H131 FcγRIIa allele, IgG2 binds poorly to most FCRs, and Fc effector functions are limited. Therefore, IgG1 and IgG3 can exert effective effector functions to deplete antibodies, and IgG2 and IgG4 are the first choices when avoiding Fc-mediated cell depletion.

1. Optimize activation inhibition ratio

A common method to improve IgG-Fc effector function is to optimize the A/I ratio by increasing the affinity of activated FcγRs and reducing the binding to inhibitory FcγRIIb. A method to increase the A/I ratio is successfully achieved through glycoengineering. The most relevant modification is the defucosylation of N297 polysaccharide, which significantly increases the affinity for FcγRIIIa and improves the ADCC effect. Two defucosylated monoclonal antibodies have been approved for marketing (mogamulizumab against CCR4 and obinutuzumab against CD20), and some others are currently undergoing clinical trials.

Another common strategy to increase the A/I ratio is to introduce point mutations in the Fc tail. The most promising monoclonal antibody in this category is margetuximab, an anti-HER2 antibody with 5-point mutations in the Fc tail, which improves the binding to FcγRIIIa and FcγRIIa, and reduces the binding to FcγRIIb.

2. Optimize complement dependent cytotoxicity

CDC is considered to be an important mechanism of action for some therapeutic monoclonal antibodies (such as anti-CD20). Optimizing Fc-mediated complement activation is an effective strategy.

Due to its naturally occurring pentameric and hexameric forms, IgM exhibits the greatest complement activation ability. However, IgM has not received much attention in the development of therapeutic monoclonal antibodies, and only a few tumor-targeted IgM monoclonal antibodies have been evaluated in clinical trials. Among them, PAT-SM6 has obtained the orphan drug qualification for multiple myeloma by EMA and FDA.

Among the IgG subtypes, IgG1 and IgG3 are good complement activators, and IgG3 seems to be the more effective subtype. However, although the inherent problems of IgG3, such as the short half-life in the body, have been successfully solved, its specific manufacturing problems still make it less attractive for drug development.

In addition, by constructing IgG1/IgG3 chimeric antibodies, the advantages of both IgG1 (favorable manufacturing characteristics) and IgG3 (enhanced CDC) can be combined. The best structure is called 113F, which combines CH1 and hinge region of IgG1 with CH2 of IgG3 and CH3 partly from IgG3 and partly from IgG1. The deglycosylated version of this chimeric antibody shows that, in addition to retaining protein A binding, the enhancement of CDC and ADCC is comparable to that of defucosylated IgG1. In vivo, the anti-CD20 113F antibody showed greater B cell depletion compared to IgG1 (both antibodies are defucosylated to improve ADCC). This study shows that the combination of optimized complement activation and A/I ratio is a promising strategy for improving tumor clearance antibodies.

Other strategies to enhance complement activation include introducing point mutations to improve the binding of IgG1 to C1q. Importantly, CDC-enhancing mutations can be combined with ADCP and ADCC-enhancing mutations in a single IgG1 to expand the effector functions of these antibodies. Finally, mutations that favor the formation of IgG hexamers also significantly enhance the binding of C1q, thereby enhancing the CDC effect. However, it remains to be seen whether these Fc mutations can be transformed into improved clinical efficacy.

3. Application of other subtypes of Ig

IgE: IgE can mediate its Fc effector function through two activation receptors (high-affinity FcεRI and low-affinity FcεRII). Although FcεRI is mainly expressed by mast cells (MC) and basophils, FcεRI is also expressed on eosinophils, dendritic cells, and myeloid cells. Compared with the IgG class, IgE has many advantages. For example, its affinity for its receptor FcεRI is two orders of magnitude higher than that of IgG’s high-affinity receptor FcγRI. With such a high FcɛRI affinity, IgE is locally retained on the cells expressing FcɛRI and has good bioavailability in tissues, which is of great significance for the treatment of solid tumors. In addition, IgE lacks inhibitory Fc receptors that can cause immunosuppression, such as FcγRIIb in IgG47.

One potential concern regarding IgE treatment is that degranulation of MC or basophils leads to the risk of potentially life-threatening allergic reactions. Fortunately, however, no signs of allergic reactions were found in preclinical models. The safety data in rodents and monkeys is satisfactory, supporting the first use of tumor-targeted anti-folate receptor α-IgE monoclonal antibody MOv18 (NCT02546921). Clinical Trials. Phase 1 data from 24 patients support the safety and potential efficacy of MOv18-IgE. Urticaria, which is easy to control, is the most common side effect, with only one patient experiencing allergic reactions. In addition, an anti-tumor effect was observed in one patient.

IgA: Another very promising tumor-clearing monoclonal antibody Ig subtype is IgA, which mediates its effector function through FcaRI. FcαRI is highly expressed on polymorphonuclear cells (PMNs), making neutrophils the most relevant cell type for IgA monoclonal antibody therapy. Neutrophils are the most abundant cytotoxic cell type in humans. They have a variety of powerful cell destruction mechanisms, including releasing cytotoxic molecules, inducing apoptosis and necrosis. In addition, similar to IgG1/IgG3 chimeras, people tried to construct IgG1/IgA chimeras, with the goal of combining the advantages of these two different subtypes. Research on these aspects is still in the early stages.

Antibodies against immune checkpoints

In theory, checkpoint blocking antibodies do not require Fc-mediated effects because their main effector function comes from blocking receptor-ligand interactions. However, in mouse models, functional Fc was found to contribute to the therapeutic effect of anti-CTLA4 checkpoint inhibitors. These studies show that although the number of effector T cells (Teff) and regulatory T cells (Treg) in the lymph nodes increased after treatment, the number of tumors, especially Treg, rather than Teff, decreased.

This decrease was only observed in anti-CTLA4 of the IgG2a subtype (the subtype with the highest A/I ratio in mice), and it was mFcγRIV dependent. The underlying mechanism may be caused by selective and abundant macrophages expressing high levels of FcγRIV in tumors. In addition, the level of CTLA4 expressed by Tregs is much higher than that of Teff cells, so they are preferentially depleted. These findings indicate the importance of TME for the efficacy of therapeutic monoclonal antibodies.

There are indications that the human anti-CTLA4 monoclonal antibody also shows the same effect. In patients with advanced melanoma with high NEO epitope load, studies have found that there is a positive correlation between the presence of the high-affinity V158 FcγRIIIa allele and the enhanced response of the CTLA-4 targeting antibody ipilimumab, which is the importance of Fc-mediated function Provides further clinical evidence.

Similarly, in the mouse model, the combination of anti-PD-L1 monoclonal antibody with activated FcγRs enhanced its therapeutic effect due to the depletion of immunosuppressive myeloid cell subpopulations in TME mediated by Fc. Currently, there are three clinically approved anti-PD-L1 monoclonal antibodies, two of which have a mutant Fc tail that eliminates the binding to FcγR (atezolizumab, durvalumab), and one is wild-type IgG1 (avelumab). Hundreds of clinical trials for these antibodies are currently underway, and future results may help to understand whether functional Fc can improve the clinical efficacy of PD-L1 targeting antibodies in humans. If so, further Fc effector function optimization may be an attractive direction.

Compared with anti-CTLA4 and anti-PD-L1, functional Fc impairs the activity of anti-PD-1 monoclonal antibodies in vivo. The underlying mechanism of this harmful effect is CD8+ T cells infiltrated by tumor cells, which are characterized by high PD-1 expression. Not surprisingly, the two clinical anti-PD-1 monoclonal antibodies belong to the IgG4 subclass and have poor Fc effector function. However, because IgG4 can still bind to activating FcγRs to a certain extent, it is meaningful to compare the efficacy of mutant monoclonal antibodies that completely abolish FcγR binding. Similarly, antibodies that target CD47 do not require Fc effector function.

In summary, these findings strongly indicate that the cellular composition of TME and the relative expression of target molecules on different immune cell populations can greatly affect the results of checkpoint blocking monoclonal antibody therapy. These factors determine the FC-mediated mechanism’s need for the best therapeutic effect, and thus determine the subtype selection of checkpoint inhibitors.

Agonist antibodies targeting TNFR

The Fc part of agonistic monoclonal antibodies targeting specific members of the tumor necrosis factor receptor (TNFR) family has been shown to play a key role in its therapeutic effect. The purpose of these monoclonal antibodies is to activate death receptors on tumor cells, such as DR4, DR5 and FAS, to induce cell death, or to activate costimulatory receptors on immune cells, such as CD40, 4-1BB, OX40, GITR and CD27 to improve the anti-tumor immune response.

TNFR requires trimerization to initiate its associated signaling cascade. Therefore, the bivalent binding of these receptors to the Fab arms is usually not sufficient to activate them, requiring additional cross-linking. For these antibodies, the interaction with FcγRs is an effective scaffold for aggregation. Specifically, FcγRIIb is the main scaffold for antibody-mediated TNFR cross-linking and downstream signal activation due to its relatively high expression. Therefore, in vivo studies have found that the activity of agonist antibodies is highly dependent on the successful participation of FcγRIIb in mice, and the Fc engineered antibody with improved FcγRIIb binding shows stronger anti-tumor activity. However, the expression of FcyRIIb is dynamic and can be down-regulated by specific cytokines, making FcyRIIb-mediated receptor aggregation and cross-linking unpredictable.

In addition, the effective participation of FcR through agonistic antibodies was found to be associated with severe liver toxicity, which may be due to the high expression of FcγRIIb on certain hepatocyte subpopulations. Therefore, it is necessary to explore new strategies to improve the agonistic activity of these monoclonal antibodies without relying on the participation of FcγR. One of the strategies is to use hIgG2 (B). This compact and highly excited conformation of hIgG2 is the result of a unique disulfide bond rearrangement in the hinge region.

Compared with hIgG2 (A), the Fab arms of hIgG2 (A) are not connected to the hinge by disulfide bonds. In hIgG2 (B), there are two disulfide bonds between each Fab arm and the hinge, making them more rigid. And it is possible to bring TNFR molecules closer together. Therefore, the use of hIgG2 (B) is a feasible strategy to improve the activity of the monoclonal antibody FcγR-independent agonist against members of the TNFR family. In addition, the subtype switching from hIgG1 to hIgG2 is sufficient to convert the immunosuppressive anti-CD40 antagonist antibody into a potent agonist with anti-tumor activity. These findings are one of the most significant examples where the selection of subtypes can completely change the activity of mAbs.

Another way to improve the agonistic activity of TNFR family-targeted monoclonal antibodies is the recently developed HERA platform. HERA is an artificial chimeric molecule that has two trimer TNFR binding domains fused to the IgG1-Fc backbone that does not bind to FcγR. The obtained hexavalent molecule can exert its agonistic activity without FcγR-mediated cross-linking. So far, two HERA molecules targeting CD27 and CD40 have shown promising anti-tumor activity, and there is no obvious toxicity in preclinical mouse models.

The strategies described to increase agonist activity in an FcγR-independent manner have an additional advantage because they can prevent unnecessary consumption of immune cells expressing target molecules. However, experiments in mice have shown that the therapeutic effects of certain TNFR family targeted agonist antibodies (such as anti-GITR, anti-OX40, or anti-4-1BB) also involve Treg depletion, which shows that, similar to anti-CTLA4, the function Sexual Fc may also be advantageous.

Summary

The clinical application of monoclonal antibodies has fundamentally changed the treatment of tumors. However, it has become increasingly apparent that monoclonal antibodies modulate their effects through a variety of different mechanisms of action. It is very important to choose the correct Ig subtype, so people have invested a lot of energy to understand the Fc-mediated effects and Fc modification of different antibody subtypes in order to further improve the efficacy of antibodies. In order to optimize Fc-mediated effector functions, a variety of strategies have been developed to provide brand new opportunities for improving antibody-based cancer treatments. In addition, by considering patient-related factors, such as their immune status, TME or FcγR polymorphism characteristics, Ig subtype selection can allow the development of antibodies that are active in a wider range of patients, or allow selective use of antibodies tailored to individual needs. Antibody. These considerations may enable us to move towards tailor-made drugs and more effective monoclonal antibody treatments in the future.

(source:internet, reference only)

Disclaimer of medicaltrend.org

Important Note: The information provided is for informational purposes only and should not be considered as medical advice.