Szykowska A, Chen Y, Smith TB, Preger C, Yang J, Qian D, Mukhopadhyay SM, Wigren E, Neame SJ, Gräslund S, Persson H, Atkinson PJ, Di Daniel E, Mead E, Wang J, Davis JB, Burgess-Brown NA, Bullock AN. Selection and structural characterization of anti-TREM2 scFvs that reduce levels of shed ectodomain. Structure. 2021 Nov 4;29(11):1241-1252.e5. Epub 2021 Jul 6 PubMed.

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  1. This is a compelling study demonstrating the power of ASOs. The authors show ASOs can potently target gene expression within microglia (especially for microglial-enriched genes), allowing for nuanced experiments that have been difficult to perform before.

    Again, this study highlights that targeting microglia is sufficient to modulate many aspects of disease progression in mice, despite the pathological proteins being neuronally derived, hence it further validates microglia as a therapeutic target.

    I also think it is important to highlight the timing effects of the treatment, which point to the fact that microglia are doing different things at different disease stages. That needs to be considered when thinking about translation to humans, especially as pathology is occurring at different speeds and stages within the vastness of the human brain. 

    It is not clear if it is the knockdown of Trem2 and the reversal of a DAM gene expression signature and association with plaques that may be beneficial, or if it is a transient early compensatory switch to a more plaque-degrading phenotype. This can be further explored and compared to the mechanisms of action of TREM2 activating antibodies, which also seem to be similarly beneficial.

    View all comments by Kim Green

  2. The paper by Szykowska et al. is a valuable contribution to the community, as it provides the first publication of the three-dimensional structure of TREM2 antibody complexes. Such studies are needed to identify and understand the molecular mechanisms of how therapeutic antibodies modulate TREM2 function.

    The need for scFv dimerization in order to inhibit TREM2 shedding is very much in line with what we have reported in our 4D9 paper, where a Fab fragment of 4D9 failed to inhibit shedding in cell culture (Schlepckow et al., 2020). 

    Interestingly, the scFv antibody fragments reported in that paper do not bind to the stalk region as reported for previously published agonistic TREM2 Aβs (Schlepckow et al., 2020; Wang et al., 2020; Ibach et al., 2021), but rather to the Ig fold. It will be very important to investigate the effect of these scFvs on TREM2 downstream signaling, as previous studies have shown that agonistic TREM2 antibodies lead to increases in pSYK signaling, which again is dependent on the cross-linking activity of the antibody (Schlepckow et al., 2020; Ibach et al., 2021; Ellwanger et al., 2021).

    A valuable feature of these antibody fragments in regard to future applications is the fact that their binding epitopes do not overlap with the putative ligand binding site in the Ig fold, or with the sites of the two most prominent LOAD-associated TREM2 variants at positions 47 and 62.

    Future studies need to provide more insight into the mechanism of shedding inhibition, i.e., do the antibodies simply block access of ADAM proteases to the cleavage site or do they drive internalization leading to a reduction of cell surface TREM2 and thereby to a reduction of shed ectodomain, as discussed by the authors in their paper? Will that be dependent on where the antibody binds?

    References:

    Schlepckow K, Monroe KM, Kleinberger G, Cantuti-Castelvetri L, Parhizkar S, Xia D, Willem M, Werner G, Pettkus N, Brunner B, Sülzen A, Nuscher B, Hampel H, Xiang X, Feederle R, Tahirovic S, Park JI, Prorok R, Mahon C, Liang CC, Shi J, Kim DJ, Sabelström H, Huang F, Di Paolo G, Simons M, Lewcock JW, Haass C. Enhancing protective microglial activities with a dual function TREM2 antibody to the stalk region. EMBO Mol Med. 2020 Apr 7;12(4):e11227. Epub 2020 Mar 10 PubMed.

    Ibach M, Mathews M, Linnartz-Gerlach B, Theil S, Kumar S, Feederle R, Brüstle O, Neumann H, Walter J. A reporter cell system for the triggering receptor expressed on myeloid cells 2 reveals differential effects of disease-associated variants on receptor signaling and activation by antibodies against the stalk region. Glia. 2021 May;69(5):1126-1139. Epub 2020 Dec 14 PubMed.

    Wang S, Mustafa M, Yuede CM, Salazar SV, Kong P, Long H, Ward M, Siddiqui O, Paul R, Gilfillan S, Ibrahim A, Rhinn H, Tassi I, Rosenthal A, Schwabe T, Colonna M. Anti-human TREM2 induces microglia proliferation and reduces pathology in an Alzheimer's disease model. J Exp Med. 2020 Sep 7;217(9) PubMed.

    Ellwanger DC, Wang S, Brioschi S, Shao Z, Green L, Case R, Yoo D, Weishuhn D, Rathanaswami P, Bradley J, Rao S, Cha D, Luan P, Sambashivan S, Gilfillan S, Hasson SA, Foltz IN, van Lookeren Campagne M, Colonna M. Prior activation state shapes the microglia response to antihuman TREM2 in a mouse model of Alzheimer's disease. Proc Natl Acad Sci U S A. 2021 Jan 19;118(3) PubMed.

    View all comments by Kai Schlepckow

  3. Szykowska et al. have described four novel scFvs selected by phage display that bind to human TREM2, and the crystal structures of two with highest binding affinity/avidity. According to the reported structures of scFv-2- and scFv-4-TREM2 complexes, both scFv CDR-H3s bind to β strands A, F, and G of TREM2, where some hydrophobic residues may contribute to high binding affinity (e.g., Y104 and 105 on scFv-2 and Y102 and Y103 on scFv-4), although scFv-4 additionally binds to the C-C' loop on TREM2 (PDB: 6YYE, 6Y6C). This may explain the low affinity of scFv-1, since it lacks tyrosine at the N-terminal portion of CDR-H3.

    Interestingly, the epitopes of both scFvs sit at the opposite site of the CDR loops of the Ig-like domain of TREM2. Importantly, such binding sites would not be impacted by most AD risk variants, including R47H and R62H, suggesting a therapeutic potential of these scFvs to target TREM2 signaling.

    Previously, we demonstrated that an anti-mouse-TREM2 antibody (178) treatment has an anti-tumor effect similar to TREM2 deficiency (Molgora et al., 2020). This antibody may block TREM2 signaling after binding, in contrast to other anti-mouse/human-TREM2-activating antibodies tested in Alzheimer’s disease models including AL002a, AL002c, and 4D9 (Schlepckow et al., 2020; Wang et al., 2020; Ellwanger et al., 2021). 

    Although the details of molecular structures remain unclear, it is possible that epitope differences across those antibodies may contribute to opposite manipulations on TREM2 signaling. Therefore, it would be interesting to examine whether the similarity of the binding sites between scFv-2 and scFv-4 on the TREM2 Ig-like domain leads to similar functionality, and whether the extended interface between scFv-4 and C-C' loop of TREM2 completely alters the outcome.

    Another interesting observation from this paper is that the protective activity against natural shedding of TREM2 tends to be dimer-specific. It is plausible that the reduction of TREM2 shedding requires scFv dimerization and is paralleled by complex internalization in their optimized experiments.

    Two interesting questions await further demonstrations: (1) Does activation and shedding of TREM2 require oligomerization of the receptor? (2) Is the protection of shedding due to stabilization of TREM2 on the plasma membrane or promotion of antibody-TREM2 complex internalization? The second question is discussed by Szykowska et al., who suggest a role for internalization.

    The first question is also worth discussing. According to the TREM2-PS structure (PDB: 6B8O, Sudom et al., 2018), the ligand binding requires another adjacent TREM2 molecule to form one complete binding pocket. TREM2 also forms a trimeric complex according to their crystal packing. In the high-resolution scFv-4/TREM2 complex structure (PDB: 6Y6C), it turns out that TREM2 molecules form dimeric complexes in this crystal packing. The putative ligand binding pockets (R47, R62) are toward the surface, and another risk variant, T96K, seems to be in the interface. Given that the oligomerization of scFvs is essential for protecting shedding, it may be true that both signaling and shedding of TREM2 involve receptor clustering and the interaction between scFvs and TREM2 are in multimeric states. The mutation on T96K may contribute to high-affinity self-dimerization that could explain the increase of signaling activation with this variant in our previous results (Song et al., 2017). 

    References:

    Ellwanger DC, Wang S, Brioschi S, Shao Z, Green L, Case R, Yoo D, Weishuhn D, Rathanaswami P, Bradley J, Rao S, Cha D, Luan P, Sambashivan S, Gilfillan S, Hasson SA, Foltz IN, van Lookeren Campagne M, Colonna M. Prior activation state shapes the microglia response to antihuman TREM2 in a mouse model of Alzheimer's disease. Proc Natl Acad Sci U S A. 2021 Jan 19;118(3) PubMed.

    Molgora M, Esaulova E, Vermi W, Hou J, Chen Y, Luo J, Brioschi S, Bugatti M, Omodei AS, Ricci B, Fronick C, Panda SK, Takeuchi Y, Gubin MM, Faccio R, Cella M, Gilfillan S, Unanue ER, Artyomov MN, Schreiber RD, Colonna M. TREM2 Modulation Remodels the Tumor Myeloid Landscape Enhancing Anti-PD-1 Immunotherapy. Cell. 2020 Aug 20;182(4):886-900.e17. Epub 2020 Aug 11 PubMed.

    Schlepckow K, Monroe KM, Kleinberger G, Cantuti-Castelvetri L, Parhizkar S, Xia D, Willem M, Werner G, Pettkus N, Brunner B, Sülzen A, Nuscher B, Hampel H, Xiang X, Feederle R, Tahirovic S, Park JI, Prorok R, Mahon C, Liang CC, Shi J, Kim DJ, Sabelström H, Huang F, Di Paolo G, Simons M, Lewcock JW, Haass C. Enhancing protective microglial activities with a dual function TREM2 antibody to the stalk region. EMBO Mol Med. 2020 Apr 7;12(4):e11227. Epub 2020 Mar 10 PubMed.

    Song W, Hooli B, Mullin K, Jin SC, Cella M, Ulland TK, Wang Y, Tanzi RE, Colonna M. Alzheimer's disease-associated TREM2 variants exhibit either decreased or increased ligand-dependent activation. Alzheimers Dement. 2017 Apr;13(4):381-387. Epub 2016 Aug 9 PubMed.

    Sudom A, Talreja S, Danao J, Bragg E, Kegel R, Min X, Richardson J, Zhang Z, Sharkov N, Marcora E, Thibault S, Bradley J, Wood S, Lim AC, Chen H, Wang S, Foltz IN, Sambashivan S, Wang Z. Molecular basis for the loss-of-function effects of the Alzheimer's disease-associated R47H variant of the immune receptor TREM2. J Biol Chem. 2018 Aug 10;293(32):12634-12646. Epub 2018 May 24 PubMed.

    Wang S, Mustafa M, Yuede CM, Salazar SV, Kong P, Long H, Ward M, Siddiqui O, Paul R, Gilfillan S, Ibrahim A, Rhinn H, Tassi I, Rosenthal A, Schwabe T, Colonna M. Anti-human TREM2 induces microglia proliferation and reduces pathology in an Alzheimer's disease model. J Exp Med. 2020 Sep 7;217(9) PubMed.

    View all comments by Yun Chen

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