G6b-B antibody-based cis-acting platelet receptor inhibitors (CAPRIs) as a new family of anti-thrombotic therapeutics

Alexandra Mazharian, Ophélie Bertin, Amrita Sarkar, Johanna Augros, Alicia Bornert, Cécile Loubière, Friederike Jönsson, Juli Warwicker, W. Abbott Mark, Omer Dushek, Caroline Vayne, Lubica Rauova, Klaus Fütterer, Jérôme Rollin, Mortimer Poncz, Yotis A. Senis

Posted on: 3 June 2024 , updated on: 5 June 2024

Preprint posted on 14 May 2024

The CAPRI sun rises: new bispecific antiplatelet agents for targeted inhibition of signalling through GPVI or CD32A

Selected by Simon Cleary

Categories: cell biology, immunology, pharmacology and toxicology

Background

A medicine that prevents thrombosis without increasing bleeding risk is the ‘holy grail’ of platelet research. Excitingly, researchers have now identified multiple platelet receptors that are important for thrombosis, but only seem to play supporting roles in haemostasis.

One pro-thrombotic platelet receptor is the collagen receptor glycoprotein VI (GPVI). Platelets come in to contact with collagen when blood vessels are damaged by atherosclerosis, and the GPVI-mediated platelet responses that ensue contribute to thrombotic strokes. Promisingly, a recent trial indicates that glenzocimab, an antibody fragment that blocks GPVI binding to collagen, might decrease mortality without increasing bleeding risk when given in combination with alteplase following ischaemic stroke (1).

Another platelet-expressed receptor involved in thrombotic responses is FcγRIIA (FCGR2A, also referred to as CD32A), a low affinity immunoglobulin Fcγ receptor that mediates responses to IgG antibodies. CD32A drives the devastating thrombosis and bleeding experienced by people affected by antibody responses that cause antiphospholipid syndrome (APS), heparin-induced thrombocytopaenia (HIT) or vaccine-induced thrombotic thrombocytopenia (VITT).

Both GPVI and CD32A signal through immunoreceptor tyrosine-based activation motifs (ITAMs) to trigger platelet activation. Physiologically, ITAMs are counteracted by inhibitory phosphatases under the control of receptors containing immunoreceptor tyrosine-based inhibitory motifs (ITIMs). One ITIM-containing receptor, G6B (also known as G6b-B and MPIG6B), is exclusively expressed on megakaryocytes and platelets. It has been speculated that the natural function of G6B might be enhanced to suppress harmful platelet responses (2), but achieving this is not easy because the ITIM domains of G6B need to be positioned immediately adjacent to target ITAMs for effective inhibition mediated by SHP-1 or SHP-2 tyrosine phosphatases.

Key findings

Bispecific single chain variable fragments (scFvs) are fusion proteins built from fragments of two different antibodies that are used in the form of bispecific T cell engagers (BiTEs) to treat forms of leukaemia. BiTEs act by bringing T-cell receptors onto cancer cell-expressed antigens to promote T cell-mediated anticancer immune responses.

In this preprint (3), Mazharian and colleagues tested an alternative use for bispecific scFvs – using them to bring G6B together with GPVI or CD32A on the surfaces of platelets (Figure 1). The authors named these bispecific agents cis-acting platelet receptor inhibitors (CAPRIs). Structural modelling indicated that CAPRIs were sufficiently small to bring ITIMs close enough to ITAMs to enable targeted inhibition. CAPRIs elicited their intended cell signalling effects as well as agonist-specific inhibition in functional assays. Excitingly, the G6B-CD32A CAPRI displayed protective effects in models using human blood, endothelial cells and antibodies to reproduce APS, HIT and VITT pathophysiology in vitro.

Figure 1: Proposed mechanism of action of cis-acting platelet receptor inhibitors (CAPRIs).

Why this work is interesting

We live in an era of modular pharmacology. Drug hunters around the world are designing agents that bind multiple different targets to make molecular glues, proteolysis targeting chimeras (PROTACs), bispecific T cell engagers (BiTEs), and antibody products that can be bispecific, trispecific or even tetraspecific. This preprint caught my eye because it describes pioneering work applying modular therapeutic development approaches to platelets. To my knowledge, only one bispecific antibody is actively under investigation that intentionally targets platelets (HMB-001, bringing the clotting factor FVIIa together with TLT-1 on the surface of activated platelets to treat inherited bleeding disorders (4)), and only one study has used PROTACs for targeted protein degradation in platelets (5).

I also find the concept of alleviating disease by increasing inhibition an attractive approach. Rational design of a therapeutic to selectively increase activity of a protein used to be near-impossible. This study is a nice example of how expertise in cell signalling can be combined with modular, recombinant approaches to expand possibilities for development of therapeutics.

CD32A is interesting to me as a target because of its implication in multiple antibody-mediated diseases and the lack of evidence that it plays an important role in either immunity or haemostasis, with mice entirely lacking an equivalent receptor (conflict of interest disclosure: I have worked on CD32A in the context of responses to alloantibodies (6)). Notably, the CD32A antibody used in this preprint was developed into a humanised, effector-dead therapeutic candidate (VIB9600, formerly MEDI9600). A phase I clinical trial was registered for VIB9600, but this trial was terminated at some point during a series of spinouts and acquisitions (from Medimmune to Viela Bio, Horizon Therapeutics and then Amgen), making it hard to tell whether this therapeutic is still under investigation. It is therefore exciting to see CD32A revisited as a target for therapeutics with a novel approach.

Questions I would like to ask the authors about their work

Is there an assay that could test whether the G6B-GPVI CAPRI accelerates alteplase-mediated dissolution of an established thrombus or makes the platelets released during thrombolysis less likely to cause further harm? Superiority of a CAPRI relative to glenzocimab with this type of test would be very encouraging.
Demonstrating that the G6B-CD32A CAPRI has greater efficacy in the APS, HIT or VITT models compared to a therapeutic in current use (e.g. high-dose intravenous immunoglobulins) would be interesting. Do you have plans/enough patient IgGs to do this?
I imagine that this is difficult to test, but is it possible that the greater extent of inhibition of platelet adhesion by G6B-GPVI CAPRIs relative to glenzocimab might end up increasing bleeding risk?
I was concerned that CAPRIs might cross-link and agglutinate different platelets (becoming ‘TAPRIs’?), so it was good to read assurance that this does not seem to happen in the discussion. CD32A is expressed on leukocytes as well as on platelets. Did you look at whether the G6B-CD32A CAPRI causes platelet-leukocyte complexes to form in the whole blood assays?

References

Mazighi M, et al. Safety and efficacy of platelet glycoprotein VI inhibition in acute ischaemic stroke (ACTIMIS): a randomised, double-blind, placebo-controlled, phase 1b/2a trial. The Lancet Neurology. 2024;23(2):157–167.
Soriano Jerez EM, Gibbins JM, Hughes CE. Targeting platelet inhibition receptors for novel therapies: PECAM-1 and G6b-B. Platelets. 2021;32(6):761–769.
Mazharian A, et al. G6b-B antibody-based cis-acting platelet receptor inhibitors (CAPRIs) as a new family of anti-thrombotic therapeutics. bioRxiv. 2024;2024.05.10.593500.
Gandhi PS, et al. A bispecific antibody approach for the potential prophylactic treatment of inherited bleeding disorders. Nat Cardiovasc Res. 2024;3(2):166–185.
Trory JS, et al. Chemical degradation of BTK/TEC as a novel approach to inhibit platelet function. Blood advances. [published online ahead of print: November 7, 2022]. https://doi.org/10.1182/BLOODADVANCES.2022008466.
Cleary SJ, et al. IgG hexamers initiate complement-dependent acute lung injury. J Clin Invest. 2024;e178351.

Tags: platelets, thrombosis

doi: https://doi.org/10.1242/prelights.37541

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Author's response

Yotis Senis shared

Is there an assay that could test whether the G6B-GPVI CAPRI accelerates alteplase-mediated dissolution of an established thrombus or makes the platelets released during thrombolysis less likely to cause further harm? Superiority of a CAPRI relative to glenzocimab with this type of test would be very encouraging.
Yes, we are gearing up to do those sorts of experiments, both in vitro using microfluidic assays, and in vivo, using a humanized G6B/GPVI mouse model that we now have, and a human platelet xeno-infusion mouse model developed in the Poncz lab.

Head-to-head comparisons with glenzocimab thus far show an advantage of the bispecific CAPRIs at inhibiting thrombus formation in microfluidic studies. We show region sequences of anti-GPVI antibodies that bind inside or outside the collagen binding site of GPVI become more potent inhibitors of GPVI-mediated platelet activation and function.
Demonstrating that the G6B-CD32A CAPRI has greater efficacy in the APS, HIT or VITT models compared to a therapeutic in current use (e.g. high-dose intravenous immunoglobulins) would be interesting. Do you have plans/enough patient IgGs to do this?
That is the plan. This work will be done collaboratively with Morty Poncz, who is a leading expert in these prothrombotic conditions. The beauty of collaboration.
I imagine that this is difficult to test, but is it possible that the greater extent of inhibition of platelet adhesion by G6B-GPVI CAPRIs relative to glenzocimab might end up increasing bleeding risk?
Bleeding is a potential risk with any anti-platelet therapeutic. However, I do not anticipate it being an issue with G6B-GPVI CAPRIs, because we know that GPVI deficiency does not lead to profound bleeding in mice or humans.
I was concerned that CAPRIs might cross-link and agglutinate different platelets (becoming ‘TAPRIs’?), so it was good to read assurance that this does not seem to happen in the discussion. CD32A is expressed on leukocytes as well as on platelets. Did you look at whether the G6B-CD32A CAPRI causes platelet-leukocyte complexes to form in the whole blood assays?
Indeed. Trans-mediated agglutination of platelets was a concern that kept coming up. We took the plunge nonetheless and it turns out not to be an issue. It comes down to biophysics and stoichiometries. It appears that there is a higher probability that CAPRIs will co-cluster receptors on the same platelet (cis interactions), rather than binding receptors on adjacent platelets (trans interactions).

We have not checked whether the G6B-CD32A CAPRI glues platelets to leukocytes. The fact that the G6B-GPVI CAPRIs do not stick platelets together suggests this will not be an issue with platelets and leukocytes, but we need to check.

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