Isotypes and patient-tailored medicine
With so many different strategies to improve the downstream effector functions of tumour-targeting antibodies, the question arises which approach to follow. The answer may depend on patient-related factors and tumour intrinsic characteristics. For example, two FcγR polymorphisms that affect the binding of IgG antibodies have been described: H131R in FcγRIIa and V158F in FcγRIIIa. The R131 variant shows lower affinity for IgG2 while the F158 variant shows lower affinity for IgG1 and IgG3. The clinical implication of these variants has not yet been fully resolved, with some studies finding negative correlation with therapeutic efficacy while others do not60,61. Nevertheless, if larger and better designed studies confirm the negative correlation between lower affinity FcRs variants and response to IgG antibody treatment, these patients may benefit more from IgE, IgA or mAbs optimised for complement activation. When it comes to complement optimised mAbs it may further be important to consider tumour microenvironment (TME) factors such as pH that can affect CDC62 or the expression level of complement regulatory proteins which allow complement evasion by cancer cells63. Furthermore, it has been shown that C reactive protein (CRP) shares its binding site on FcγRs (I and II) and FcαRI with IgG and IgA, respectively, whereas it can also bind to C1q64,65. Whether CRP can act as a competitive inhibitor for FcRs and complement binding of those antibodies in vivo has not been studied yet, but it could have important implications. For instance, patients suffering from chronic inflammatory and neurodegenerative diseases, such as atherosclerosis, type 2 diabetes mellitus or Parkinson’s disease have chronically elevated CRP levels66 which may interfere with antibody treatment. Thus, for those patients, IgE-based antibody treatment might be an attractive choice. In conclusion, antibody engineering offers a wide range of opportunities to improve effector functions of mAbs. but patient-related factors should also be taken into consideration for optimal isotype selection. This multilevel approach could result in a more effective personalised treatment.
Antibodies targeting immunological checkpoint proteins
A recently identified class of mAbs for cancer therapy are the so-called checkpoint inhibitors. These antibodies do not target the tumour directly but enhance anti-tumour immune responses by targeting immunological checkpoint proteins, such as PD-1 or CTLA-4, or their ligands such as PD-L1. These checkpoint proteins are expressed on activated T cells and limit excessive T cell responses. As a means of immune resistance, the ligands of PD-1 are often expressed by tumour cells67 as well as by myeloid cells infiltrating the TME68,69. Checkpoint blockade leads to enhanced T cell activation67,70 and, consequently, the clinical introduction of checkpoint inhibitors led to a tremendous improvement of cancer therapy for several different types of cancers.
In theory, checkpoint blocking antibodies do not require Fc-mediated effects, as their main effector function is expected to be derived from blocking the receptor-ligand interaction (Fab-mediated). However, it was found that a functional Fc tail contributed to the therapeutic efficacy of anti-CTLA4 checkpoint inhibitors in mouse models71,72. These studies revealed that whereas both effector T cell (Teff) and regulatory T cell (Treg) populations were increased in lymph nodes after therapy, within tumours, specifically the Treg but not the Teff population was decreased. This decrease was only observed with anti-CTLA4 of the IgG2a isotype (the isotype with highest A/I ratio in mouse) and appeared mFcγRIV dependent. The underlying mechanism was found to be caused by a selectively high abundance of macrophages expressing high levels of FcγRIV in tumours but not in lymph nodes71. Furthermore, Tregs express much higher levels of CTLA4 than Teff cells and were therefore preferentially depleted13. These findings point to the importance of the TME for therapeutic mAbs efficacy.
There are indications that anti-CTLA4 mAbs show the same effect in humans. A recent study confirmed the importance of Treg depletion for human anti-CTLA4 antibody in a hFcγR mouse model73. In addition, in advanced melanoma patients with high neoepitope burden the authors found a positive correlation between the presence of the high-affinity V158 FcγRIII allele and increased response to the CTLA-4 targeting antibody ipilimumab, providing further clinical evidence for the importance of Fc-mediated function. These findings may be relevant to explain why only some patients respond to anti-CTLA4 therapy and provide further rationale to optimise CTLA4 mAbs by improving their A/I ratio74 or switching to IgA or IgE isotypes given the microenvironmental requirements for selective tumour Treg depletion are met.
Similarly, it was shown that the binding of anti-PD-L1 mAb to activating FcγRs enhances its therapeutic efficacy in mouse models, due to Fc-mediated depletion of immunosuppressive myeloid cell subsets in the TME68. However, although another study confirmed that Fc-mediated depletion of myeloid cells in the TME contributes to the therapeutic effect of anti-PD-L1 antibodies, this effect was found to be dependent on the mouse genetic background as it occurred in CT26 tumours transplanted in BALB/c but not MC38 tumours in C57BL/6 mice69. The depleted myeloid cell subset was the one with the highest PD-L1 expression whereas PD-L1 expression on the tumour cells did not contribute to the therapeutic effect of anti-PD-L1 antibody69. Currently, there are three clinically approved anti-PD-L1 mAbs, two of which have a mutated Fc tail with abrogated FcγR binding (atezolizumab, durvalumab) and one is a wild-type IgG1 (avelumab). Since hundreds of clinical trials with these antibodies are currently ongoing, future results might help to resolve the question whether a functional Fc tail improves clinical efficacy of PD-L1 targeting antibodies in humans. If so, a further Fc-effector function optimisation might be an appealing step forward.
In contrast to anti-CTLA4 and anti-PD-L1, a functional Fc tail compromised the activity of anti-PD-1 mAbs in vivo. The underlying mechanism of this detrimental effect was the depletion of tumour-infiltrating CD8+ T cells, which are characterised by high PD-1 expression68. Not surprisingly, two clinically approved anti-PD-1 mAbs are of the IgG4 subclass, the human IgG subclass with the lowest A/I ratio. However, since IgG4 still binds to activating FcγRs to some extent, it would be interesting to compare its therapeutic efficacy with that of a mutated mAb with completely abolished FcγR binding75. Similarly, antibodies targeting CD47, a ‘don’t eat me signal’ often upregulated by tumour cells to avoid elimination by myeloid cells as part of CD47/SIRP-α checkpoint pathway, do not require Fc-effector function either76.
In conclusion, these findings strongly suggest that the cellular composition of the TME as well as the relative expression of the target molecule on different immune cell populations can greatly affect the outcome of checkpoint blocking mAb therapy. These factors dictate the need for Fc-mediated mechanisms for an optimal therapeutic effect and, thus, the isotype selection for checkpoint inhibitors. (fig.4b ).