July 25, 2024

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How to select the subtype for Antibody Oncology Therapy?

How to select the subtype for Antibody Oncology Therapy?



 

How to select the subtype for Antibody Oncology Therapy?

Monoclonal antibodies ( mabs ) have become an increasingly important class of drugs whose clinical application has revolutionized the field of cancer treatment.

Different mAbs have different anti-tumor mechanisms, including blocking tumor-specific growth factor receptors or immune regulatory molecules, as well as complement and cell-mediated tumor cell lysis.

Thus, for many mAbs, Fc-mediated effector functions are critical for therapeutic efficacy. Because immunoglobulin subtypes differ in their ability to bind to FCRs on immune cells and activate complement, they activate different immune responses.

Therefore, the choice of antibody isotype for a therapeutic mAb depends on its intended mechanism of action.

Considering that the current clinical efficacy of many monoclonal antibodies is only achieved in a subset of patients, optimal isotype selection and Fc optimization during antibody development could be an important step towards improving patient outcomes.

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

 

 


Mechanism of action of tumor antigen targeting antibodies


The first generation of therapeutic antibodies approved for clinical use remains the most common class of monoclonal antibodies in cancer treatment and consists 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, several observations in humans and mice suggest that Fc-mediated activation of immune cells is an important additional mechanism of action for many of these mAbs.

 

How to select the subtype for Antibody Oncology Therapy?

 

 

The Fc portion of the antibody can activate the FCR on effector cells, such as 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 ).

Furthermore, antibodies, through their Fc tails, can activate the complement cascade by binding to C1q, leading to tumor cell lysis through several different mechanisms.

This includes the formation of the membrane attack complex ( MAC ), direct induction of lysis of target cells ( CDC ) or the attraction of immune cells through 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 (CDCP) of myeloid cells ).

Antibody-mediated cell death also leads to the release of tumor antigens and the formation of immune complexes ( ICs ), which promote the initiation of anti-tumor T cell responses and maintain tumor control and rejection.

In this process, the binding to 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.

 

 


Optimizing IgG effector function


IgG-Fc effector functions are mediated through complement and FcγRs, which are classified as activating receptors ( FcγRI, FcγRIIa/IIc, FcγRIIIa, FcγRIIIb ) or inhibitory receptors ( FcγRIIb ).

Since most effector cells express both activating and inhibitory FcγRs, the outcome of IgG binding is the result of a relative combination of affinity, receptor availability, and signaling capacity.

The relative affinity of an antibody for its receptor is defined as the activation-inhibition ratio ( A/I ).

 

How to select the subtype for Antibody Oncology Therapy?

 

The concept of the A/I ratio is based on observations in mice showing that mIgG2a has a higher A/I, mIgG1 has a lower A/I, and mIgG2b has an intermediate A/I.

Thus, therapeutic antibodies of the mIgG2a subclass have been shown to be more effective in eradicating tumors in a number of in vivo models.

Although the differences in A/I ratios between human IgG subtypes are less pronounced, they differ in their ability to induce immune responses due to their different FcR binding profiles. IgG1 and IgG3 bind to all FCRs, but display higher affinity for activated FCRs. Therefore, they are defined as Ig subtypes with strong Fc effector functions.

IgG4, on the other hand, binds with similar affinity to most activating FcRs and inhibits FcγRIIb binding and is considered to be less active. Finally, IgG2 binds poorly to most FCRs with limited Fc effector function, except for the high-affinity H131 FcγRIIa allele.

Thus, IgG1 and IgG3 are capable of efficient effector functions to deplete antibodies, whereas IgG2 and IgG4 are preferred in avoiding Fc-mediated depletion of cells.

 

 

1. Optimizing the activation-inhibition ratio

A common approach to improve IgG-Fc effector function is to optimize the A/I ratio by increasing the affinity of activating FcγRs and decreasing the binding to inhibitory FcγRIIb.

One approach to increase the A/I ratio was successfully achieved through glycoengineering, the most relevant modification being defucosylation of the N297 polysaccharide, which significantly increased the affinity for FcγRIIIa and improved the ADCC effect.

Two afucosylated mAbs have been approved ( mogamulizumab against CCR4 and obinutuzuma b against CD20 ), and others are currently in clinical trials.

 

Another common strategy to increase the A/I ratio is to introduce point mutations in the Fc tail.

The most promising mAb in this class is margetuximab, an anti-HER2 antibody with a 5-point mutation in its Fc tail, resulting in improved binding to FcγRIIIa and FcγRIIa and reduced binding to FcγRIIb.

 

2. Optimizing Complement-Dependent Cytotoxicity

CDC is considered to be an important mechanism of action of 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 capacity.

However, IgM has not received much attention in the development of therapeutic mAbs, and only a few tumor-targeting IgM mAbs have been evaluated in clinical trials. Among them, PAT-SM6 has obtained the orphan drug qualification for multiple myeloma from EMA and FDA.

 

Of the IgG subclasses, IgG1 and IgG3 are good complement activators, with IgG3 appearing to be the more potent subclass.

However, although the inherent problems of IgG3, such as its short in vivo half-life, have been successfully addressed, its specific manufacturing issues still make it less attractive for drug development.

 

In addition, by constructing IgG1/IgG3 chimeric antibodies, the combination of the advantages of both IgG1 ( favorable manufacturing characteristics ) and IgG3 ( enhanced CDC ) can be realized.

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 showed an enhancement of CDC and ADCC comparable to defucosylated IgG1, in addition to retaining protein A binding.

In vivo, anti-CD20 113F antibody exhibited greater B cell depletion compared to IgG1 ( both antibodies were afucosylated to improve ADCC ).

This study demonstrates 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 IgG1 binding to C1q.

Importantly, CDC-enhancing mutations can be combined with ADCP and ADCC-enhancing mutations in individual IgG1s to amplify the effector functions of these antibodies.

Finally, mutations favoring IgG hexamer formation also significantly enhanced C1q binding, thereby enhancing CDC action. However, whether these Fc mutations translate into improved clinical efficacy remains to be seen.

 

3. Application of other subtypes of Ig

IgE :

IgE can mediate its Fc effector functions through two activating receptors ( high-affinity FcεRI and low-affinity FcεRII ).

Although FcεRI is primarily expressed by mast cells ( MC ) and basophils, FcεRI is also expressed on eosinophils, dendritic cells, and myeloid cells.

IgE has many advantages over the IgG class, for example, its affinity for its receptor FcεRI is two orders of magnitude higher than IgG’s high-affinity receptor FcγRI.

With such a high affinity for FcɛRI, IgE is locally retained on FcɛRI-expressing cells and has good bioavailability in tissues, which has important implications for the treatment of solid tumors.

In addition, IgE lacks inhibitory Fc receptors, such as FcγRIIb in IgG47, that cause immunosuppression.

 

How to select the subtype for Antibody Oncology Therapy?

 

A potential concern with IgE therapy is the risk of potentially life-threatening anaphylaxis from degranulation of MCs or basophils.

Fortunately, however, no signs of hypersensitivity reactions were seen in preclinical models, and the safety data in rodents and monkeys were satisfactory, supporting the first trial using the tumor-targeting anti-folate receptor α-IgE monoclonal antibody MOv18 (NCT02546921 ) .

Clinical Trials. Phase 1 data from 24 patients supported the safety and potential efficacy of MOv18-IgE.

Easily manageable urticaria was the most common side effect, and only 1 patient experienced anaphylaxis. In addition, an antitumor effect was observed in one patient.

 

IgA :

Another rather promising tumor-clearing mAb Ig subtype is IgA, which mediates its effector function through FcαRI.

FcαRI is highly expressed on polymorphonuclear cells ( PMNs ), making neutrophils the most relevant cell type for IgA mAb therapy.

Neutrophils are the most abundant cytotoxic cell type in humans. They possess multiple powerful mechanisms of cell destruction, including release of cytotoxic molecules, induction of apoptosis and necrosis.

Furthermore, similar to IgG1/IgG3 chimeras, attempts have been made to construct IgG1/IgA chimeras with the aim of combining the advantages of these two distinct subtypes.

Research in these areas is still in its early stages.

 

 

 


Antibodies against immune checkpoints


In theory, checkpoint-blocking antibodies do not require Fc-mediated effectors because their primary effector function comes from blocking receptor-ligand interactions.

However, in a mouse model, a functional Fc was found to contribute to the therapeutic effect of anti-CTLA4 checkpoint inhibitors.

These studies showed that while the numbers of both effector T cells ( Teff ) and regulatory T cells ( Treg ) were increased in lymph nodes after treatment, in tumors specifically Treg, but not Teff, decreased.

This decrease was only observed with anti-CTLA4 of the IgG2a subtype ( the subtype with the highest A/I ratio in mice ) and showed mFcγRIV dependence.

The underlying mechanism may be caused by selectively abundant macrophages expressing high levels of FcγRIV in tumors.

Furthermore, Tregs express much higher levels of CTLA4 than Teff cells and thus are preferentially depleted. These findings demonstrate the importance of the TME for the efficacy of therapeutic mAbs.

 

There are indications that human anti-CTLA4 monoclonal antibodies have shown the same effect.

In advanced melanoma patients with a high NEO epitope burden, we found a positive correlation between the presence of a high-affinity V158 FcγRIIIa allele and enhanced responses to the CTLA-4-targeting antibody ipilimumab, suggesting importance of Fc-mediated function Further clinical evidence is provided.

 

Likewise, in mouse models, the combination of anti-PD-L1 mAbs with activating FcγRs enhanced their therapeutic effect due to Fc-mediated depletion of immunosuppressive myeloid subsets in the TME.

Currently, there are three clinically approved anti-PD-L1 monoclonal antibodies, two of which have a mutated Fc tail that abolishes binding to FcγRs ( atezolizumab, durvalumab ), and one is wild-type IgG1 ( avelumab ).

Hundreds of clinical trials targeting these antibodies are currently underway, and future results may help to understand whether a functional Fc can improve the clinical efficacy of PD-L1-targeting antibodies in humans. If so, further optimization of Fc effector function may be an attractive direction.

 

Functional Fc compromised the activity of anti-PD-1 mAbs in vivo compared with anti-CTLA4 and anti-PD-L1.

The underlying mechanism for this deleterious effect is tumor cell-infiltrating CD8+ T cells, which are characterized by high PD-1 expression.

Not surprisingly, two clinical anti-PD-1 mAbs were of the IgG4 subclass with poor Fc effector function. However, since IgG4 can still bind to activating FcγRs to some extent, it is interesting to compare the efficacy of mutant mAbs that completely abolish FcγR binding. Likewise, antibodies targeting CD47 do not require Fc effector function.

 

Taken together, these findings strongly suggest that the cellular composition of the TME and the relative expression of target molecules on different immune cell populations can greatly influence the outcome of checkpoint-blockade mAb therapy.

These factors determine the need for optimal therapeutic efficacy of FC-mediated mechanisms and thus the subtype selection of checkpoint inhibitors.

 

 


Agonist antibodies targeting TNFR


The Fc portion of agonistic mAbs targeting specific members of the tumor necrosis factor receptor ( TNFR ) family has been shown to play a key role in their therapeutic efficacy.

The purpose of this type of mAb 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 enhance the anti-tumor immune response.

 

How to select the subtype for Antibody Oncology Therapy?

 

 

TNFR requires trimerization to initiate its associated signaling cascade. Therefore, bivalent binding of these receptors to the Fab arms is often not sufficient to activate them and additional crosslinking is required.

For these antibodies, the interaction with FcγRs is an efficient scaffold for aggregation. Specifically, FcγRIIb is a major scaffold for antibody-mediated TNFR crosslinking and downstream signaling activation because of its relatively high expression.

Thus, in vivo studies found that the activity of agonist antibodies was highly dependent on the successful engagement of FcγRIIb in mice, and Fc-engineered antibodies with improved FcγRIIb binding showed stronger antitumor activity.

However, FcγRIIb expression is dynamic and can be downregulated by specific cytokines, making FcγRIIb-mediated crosslinking of receptor aggregation unpredictable.

 

Furthermore, efficient FcR engagement by agonistic antibodies was found to be associated with severe hepatotoxicity, possibly due to high expression of FcγRIIb on certain hepatocyte subsets.

Therefore, new strategies need to be explored to enhance the agonistic activity of these mAbs independent of FcγR involvement.

One such strategy is to use hIgG2 (B). This compact and highly agonistic 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, hIgG2(B) has two disulfide bonds between each Fab arm and the hinge, making them more rigid, And potentially bring TNFR molecules closer together.

Therefore, the use of hIgG2 (B) is a viable strategy to increase the FcγR-independent agonist activity of mAbs against TNFR family members.

Furthermore, subtype switching from hIgG1 to hIgG2 is sufficient to convert immunosuppressive anti-CD40 antagonistic antibodies into potent agonists with antitumor activity.

These findings are one of the most striking examples of how subtype selection can completely alter mAb activity.

 

Another approach to enhance the agonistic activity of TNFR family-targeted mAbs is the recently developed HERA platform.

HERA is an artificial chimeric molecule with two trimeric TNFR-binding domains fused to an IgG1-Fc backbone that does not bind FcγRs.

The resulting hexavalent molecule is able to exert its agonistic activity without FcγR-mediated cross-linking.

So far, two HERA molecules targeting CD27 and CD40 have shown promising antitumor activity without significant toxicity in preclinical mouse models.

 

The described strategies to increase agonist activity in an FcγR-independent manner have an additional advantage, since they prevent unnecessary depletion of immune cells expressing the target molecule.

However, experiments in mice have shown that the depletion of Tregs is also involved in the therapeutic effects of certain TNFR family-targeted agonist antibodies such as anti-GITR, anti-OX40, or anti-4-1BB , suggesting that, similar to anti-CTLA4, functional Sexual Fc may also be beneficial.

 

 

 


Summary


The clinical application of monoclonal antibodies has fundamentally changed the treatment of tumors.

However, it is becoming increasingly apparent that mAbs mediate their effects through a number of different mechanisms of action.

The selection of the correct Ig subtype is crucial , so much effort has been devoted to understanding the Fc-mediated effects of different antibody subtypes as well as Fc modifications to further improve antibody efficacy.

To optimize Fc-mediated effector functions, multiple strategies have been developed, opening entirely new opportunities for improved antibody-based cancer therapy.

Furthermore, by considering patient-related factors, such as their immune status, TME, or signature of FcγR polymorphisms, Ig subtype selection can allow the development of antibodies that are active in a wider range of patients, or allow the selective use of antibodies tailored to individual needs.

These considerations may bring us one step closer to patient-tailored drugs and more effective mAb therapy in the future.

 

 

 

 

 

 

 

references:

1. Isotype selection for antibody-based cancer therapy. Clin ExpImmunol. 2020 Nov 5.

2. clinicaltrials.gov

How to select the subtype for Antibody Oncology Therapy?

(source:internet, reference only)


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