TNFR family targeting agonistic antibodies
Recently, the Fc tail of agonistic mAbs that target specific members of
the Tumour Necrosis Factor Receptor (TNFR) family has been shown to play
a critical role in their therapeutic efficacy. This class of mAbs is
designed to either activate death receptors such as DR4, DR5 and FAS on
cancer cells in order to induce cell death, or to activate
co-stimulatory receptors such as CD40, 41BB, OX40, GITR and CD27 on
immune cells in order to improve anti-tumour immune responses.
TNFRs require trimerisation in order to initiate their associated
signalling cascade77. Therefore, bivalent engagement
of these receptors with Fab arms is usually not sufficient for their
activation and additional cross-linking is required. For these
antibodies, the interaction with FcγRs functions as an effective
scaffold for clustering. Specifically, it has been shown that FcγRIIb
represents a dominant scaffold for antibody mediated TNFR crosslinking
and activation of downstream signalling because of its relatively high
expression78,79. Consequently, in vivoagonistic antibody activity was found to be highly dependent on
successful FcγRIIb engagement80,81 and Fc-engineered
antibodies with improved FcγRIIb binding showed stronger anti-tumour
activity82,83. However, the expression of FcgRIIb is
dynamic and can be downregulated by particular
cytokines84, leaving the success of FcγRIIb-mediated
cross-linking for receptor clustering unpredictable. In addition,
effective FcR-engagement by agonistic antibodies was found to be
associated with serious hepatotoxicity85–87, which
could potentially be explained by the high expression of FcγRIIb on
certain subsets of liver cells88. Therefore, new
strategies have been explored to improve the agonistic activity of these
mAbs independent of FcγR engagement. One of these strategies is the use
of hIgG2(B), whose unique disulphide bonds rearrangement in the hinge
region89 provides it with a compact and highly
agonistic conformation90. In line with this finding,
the agonistic effect of anti-CD40 hIgG2 antibodies was demonstrated to
be FcγR-independent both in vitro and in vivo , confirming
that the use of hIgG2(B) is a viable strategy for improving the
agonistic activity of mAbs targeting TNFR family
members91. Furthermore, isotype switching from
hIgG1 to hIgG2 was sufficient to convert an immunosuppressive anti-CD40
antagonistic antibody into a potent agonist with anti-tumour
activity92. These findings constitute one of the most
striking examples of how the choice of the isotype can completely change
the activity of a mAb.
Another approach to improve the agonistic activity of TNFR family
targeting mAbs, independent of FcγR engagement, is the recently
developed HERA platform. HERA is an artificial chimeric molecule which,
instead of Fab-arms, has two trimeric TNFR binding domains, fused to an
IgG1 Fc backbone with abrogated FcγR binding. The resulting hexavalent
molecule is capable of exerting its agonistic activity without
FcγR-mediated crosslinking. So far, two HERA molecules targeting CD27
and CD40 have shown promising anti-tumour activity, without significant
toxicological signs in pre-clinical mouse models93,94.
These findings suggest that agonistic HERA molecules may offer improved
safety combined with unaltered efficacy and thus an advantageous
clinical profile.
The strategies described to improve agonistic activity in a
FcγR-independent manner could have an additional advantage as they
prevent unwanted depletion of immune cells expressing the target
molecule. However, experiments in mice suggest that the therapeutic
effect of some TNFR family targeting agonistic antibodies (such as
anti-GITR95, anti-OX4096 or
anti-4-1BB97) also involved Treg depletion, suggesting
that, analogous to anti-CTLA4, a functional Fc tail might be
advantageous. Similarly, some Fc-mediated downstream effector functions
may be useful for agonistic mAbs targeting death receptors on cancer
cells, as Fc-mediated cytotoxicity and ADCP would act as an additional
tumour cell depleting mechanism and might facilitate cross-presentation
inducing an adaptive anti-tumour response.
A few solutions have been proposed to combine the divergent properties,
as mentioned above, in a single Ig molecule. For instance, a pentameric
IgM antibody with high complement activation capacity has been used to
successfully induce DR5 clustering via multivalent interaction, inducing
tumour regression in preclinical models98. An
alternative approach which takes advantage of Ig multimerisation, but
avoids IgM manufacturing issues, is the so called HexaBody technology.
It is based on a single point mutation (E430G) in the Fc domain of IgG1
that enhances Fc-Fc interactions upon binding to membrane-bound
targets99. Consequently, these antibodies have a
strong tendency to form hexamers on the target cell, ultimately leading
to both high agonistic activity and improved CDC100. A
combination of different HexaBodies targeting two different epitopes on
DR5 is currently in an early clinical testing (NCT03576131). Given this
enhanced complement activation of HexaBodies, this antibody form could
furthermore be attractive whenever tumour cell lysis is intended, such
as for classical tumour antigen-targeting antibodies, such as anti-CD20;
suggesting for the design of an entirely novel type of tumour
antigen-targeting antibodies.
In addition to HexaBodies, a highly agonistic anti-4-1BB recombinant Ig
with potent Fc-effector function was achieved by combining human IgG2
CH1 and hinge locked in B conformation, with murine IgG2a CH2 and CH3
(the IgG subclass with the highest A/I ratio in the
mouse)97. In mice, tumour treatment with this chimeric
construct induced both Teff stimulation in lymph nodes (strong 4-1BB
agonism) and Fc-mediated Treg depletion within tumours, leading to
increased intra-tumoural Teff/Treg ratio and enhanced survival compared
to a wild-type mIgG2a construct97. By analogy with the
mouse example, a chimera of hIgG2(B) and hIgG1 might be applicable in
humans.
In conclusion, important breakthroughs have been made in the design of
TNFR agonistic antibodies by making their activity FcγR independent. It
is precisely the FcγR independency that may overcome initial problems
seen in the clinic such as severe toxicity and modest efficacy. However,
the contribution of Fc-mediated cell-depletion to the therapeutic
efficacy represents an important consideration for the optimal design of
a specific agonistic antibody (fig.4c ).