Enabling the next steps in cancer immunotherapy: from antibody-based bispecifics to multispecifics, with an evolving role for bioconjugation chemistry

Thoreau, Fabien; Chudasama, Vijay

doi:10.1039/d1cb00082a

“…It follows that a modular method which can rapidly produce conjugates from a pool of components for initial testing would be advantageous. 4 The CiTEs envisaged here have an anti-CD3/anti-HER2 core with a checkpoint inhibitory Fab (anti-PD-1 Fab) or a checkpoint modulating enzyme (Salmonella typhimurium Sialidase, ST Sia) attached. To understand the significance of ST Sia, the role of sialic acid in immune regulation will be briefly examined.…”

Section: Checkpoint-inhibitory T Cell Engagers (Cites)mentioning

confidence: 99%

“…A promising class of such molecules combines T cell re-directing bsAb technology with immunomodulating proteins for additional therapeutic benefit. 4 Here we report a first-in-class chemical method for the synthesis of functionalized three-protein conjugates and then test their efficacy in vitro.…”

Section: Introductionmentioning

confidence: 99%

Chemical generation of checkpoint inhibitory T cell engagers (CiTEs) for the treatment of cancer

Szijj

¹

,

Gray

²

,

Ribi

³

et al. 2022

Preprint

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Bispecific antibodies (bsAbs) provide enticing therapeutic opportunities in the area of immunotherapy, especially in the field of immuno-oncology. These constructs can bind two separate antigenic epitopes and thus provide access to unique mechanisms of action (MoAs). A key MoA is unlocked by bispecific T cell engagers (BiTEs), which cause T cells to be cross-linked with a targeted cancer cell, ultimately leading to death of the targeted cell. It has been shown that the combination of a BiTE with checkpoint inhibition, such as blockade of the PD-1/PD-L1 pathway, can lead to a synergistic effect and greater efficacy. Constructs built from a BiTE core (anti-CD3/anti-cancer antigen) with an immunomodulatory protein added, have been dubbed checkpoint-inhibitory T cell-engagers (CiTEs). Both bsAbs and CiTEs have traditionally been generated via protein engineering. However, recently, improved chemical methods for the construction of bsAbs have been reported. This includes a strategy developed by the Chudasama and Baker groups to synthesize homogenous fragment-based bsAbs from antibodies’ fragments antigen binding (Fabs), utilising click-enabled pyridazinediones (PDs) for functional disulfide re-bridging, followed by strain-promoted inverse electron-demand Diels-Alder cycloaddition (SPIEDAC) click chemistry to attach the two Fabs to each other. In this paper, we describe a first-in-class chemical method to generate biotin-functionalized three-protein conjugates, building significantly on the previously described PD-method. The three-protein constructs generated here include two such CiTE molecules, one containing an anti-PD-1 Fab, the other containing an immunomodulatory enzyme Salmonella typhimurium sialidase; FabCD3-FabHER2-FabPD-1-Biotin and FabHER2-FabCD3-Sia-Biotin. These constructs (along with suitable controls) were tested for their biological activity, and each of their protein components were shown to retain their function. Their efficacy was also compared to a simpler BiTE scaffold and was shown to be superior, with the sialidase-containing CiTE especially showing significantly enhanced potency in vitro. The chemical method described here has the potential to enable the rapid generation of a plethora of multi-protein constructs, which we envisage would be especially useful in hit-identification screening but could also potentially be scaled up for drug-development after further optimization.

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“…Limited examples of such threeprotein or four-protein conjugates, all generated via protein engineering, have been reported in the context of immunotherapy -these have been reviewed recently. 4 For the purposes of this work, formats where T cell engagement are combined with immunomodulation should be discussed in more detail.…”

Section: Checkpoint-inhibitory T Cell Engagers (Cites)mentioning

confidence: 99%

Chemical generation of checkpoint inhibitory T cell engagers (CiTEs) for the treatment of cancer

Szijj

¹

,

Gray

²

,

Ribi

³

et al. 2022

Preprint

Self Cite

0

View full text Add to dashboard Cite

Bispecific antibodies (bsAbs) provide enticing therapeutic opportunities in the area of immunotherapy, especially in the field of immuno-oncology. These constructs can bind two separate antigenic epitopes and thus provide access to unique mechanisms of action (MoAs). A key MoA is unlocked by bispecific T cell engagers (BiTEs), which cause T cells to be cross-linked with a targeted cancer cell, ultimately leading to death of the targeted cell. It has been shown that the combination of a BiTE with checkpoint inhibition, such as blockade of the PD-1/PD-L1 pathway, can lead to a synergistic effect and greater efficacy. Constructs built from a BiTE core (anti-CD3/anti-cancer antigen) with an immunomodulatory protein added, have been dubbed checkpoint-inhibitory T cell-engagers (CiTEs). Both bsAbs and CiTEs have traditionally been generated via protein engineering. However, recently, improved chemical methods for the construction of bsAbs have been reported. This includes a strategy developed by the Chudasama and Baker groups to synthesize homogenous fragment-based bsAbs from antibodies’ fragments antigen binding (Fabs), utilising click-enabled pyridazinediones (PDs) for functional disulfide re-bridging, followed by strain-promoted inverse electron-demand Diels-Alder cycloaddition (SPIEDAC) click chemistry to attach the two Fabs to each other. In this paper, we describe a first-in-class chemical method to generate biotin-functionalized three-protein conjugates, building significantly on the previously described PD-method. The three-protein constructs generated here include two such CiTE molecules, one containing an anti-PD-1 Fab, the other containing an immunomodulatory enzyme Salmonella typhimurium sialidase; FabCD3-FabHER2-FabPD-1-Biotin and FabHER2-FabCD3-Sia-Biotin. These constructs (along with suitable controls) were tested for their biological activity, and each of their protein components were shown to retain their function. Their efficacy was also compared to a simpler BiTE scaffold and was shown to be superior, with the sialidase-containing CiTE especially showing significantly enhanced potency in vitro. The chemical method described here has the potential to enable the rapid generation of a plethora of multi-protein constructs, which we envisage would be especially useful in hit-identification screening but could also potentially be scaled up for drug-development after further optimization.

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“…Bispecific antibodies (bsAbs) are (usually) artificial proteins containing two different binding elements (not necessarily Fab moieties) enabling their interaction with two epitopes of the same target or, in most cases, interaction with epitopes of two different targets. 1 The capacity of bsAbs to simultaneously interact with two targets/receptors offers various applications: 1) redirection of immune cells such as T cells, NK cells or macrophages toward tumour cells in order to trigger or improve immunosuppression of the tumour (such bsAbs are referred to as "immune cell engagers", and have had a huge impact on the immunotherapy landscape); 2 2) the simultaneous modulation of two different pathways in pathogenesis; 3 3) increasing selectivity and/or avidity for a target cell by interacting with two different antigens at the cell surface (or two epitopes of the same antigen); 4 4) holding effector proteins together as a substitute for an inactivated or faulty scaffold protein. 5,6 The majority of reported bsAbs in literature and clinical trials are T cell engagers, termed "Bispecific T cell Engagers" (BiTEs).…”

Section: Introductionmentioning

confidence: 99%

“…5,6 The majority of reported bsAbs in literature and clinical trials are T cell engagers, termed "Bispecific T cell Engagers" (BiTEs). 2 These BiTEs are designed to recruit immune cells to the tumour site by combining affinity for a receptor on the surface of T cells (usually CD3, a T cell co-receptor involved in T cell activation) and a tumour associated antigen (TAA). While one side of the bsAb interacts with a T cell, activating it, the other side can interact with a target cell, bringing them into close vicinity and leading to formation of an immunological synapse which allows target cell destruction by the activated T cell.…”

Section: Introductionmentioning

confidence: 99%

Modular chemical construction of IgG-like mono- and bispecific synthetic antibodies (SynAbs)

Thoreau

¹

,

Szijj

²

,

Greene

³

et al. 2022

Preprint

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In recent years there has been rising interest in the field of protein–protein conjugation, especially related to bispecific antibodies (bsAbs) and their therapeutic applications. These constructs contain two paratopes capable of binding two distinct epitopes on target molecules and are thus able to perform complex biological functions (mechanisms of action) not available to monospecific mAbs. Traditionally these bsAbs have been constructed through protein engineering, but recently chemical methods for their construction have started to (re-)emerge. While these have been shown to offer increased modularity, speed and for some methods, even the inherent capacity for further functionalization (e.g., with small molecule cargo), most of these approaches lacked the ability to include a fragment crystallizable (Fc) modality. The Fc component of IgG antibodies offers effector function and increased half-life. Here we report a first-in-class disulfide re-bridging and click-chemistry-based method for the generation of Fc-containing, IgG-like mono- and bispecific antibodies. These are in the FcZ (FabX)-FabY format, i.e., two distinct Fabs and an Fc, potentially all from different antibodies, attached in a homogeneous and covalent manner. We have dubbed these molecules synthetic antibodies (SynAbs). We have constructed a bispecific T cell-engager (BiTE) SynAb, FcCD20-(FabHER2)-FabCD3, and have confirmed that it exhibits the expected biological functions, including the ability to kill HER2+ target cells in a co-culture assay with T cells.

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Enabling the next steps in cancer immunotherapy: from antibody-based bispecifics to multispecifics, with an evolving role for bioconjugation chemistry

Abstract: In the last two decades, immunotherapy has established itself as one of the leading strategy for cancer treatment, as illustrated by the exponentially growing number of related clinical trials. This...

Cited by 12 publications

References 219 publications

Chemical generation of checkpoint inhibitory T cell engagers (CiTEs) for the treatment of cancer

Chemical generation of checkpoint inhibitory T cell engagers (CiTEs) for the treatment of cancer

Chemical generation of checkpoint inhibitory T cell engagers (CiTEs) for the treatment of cancer

Modular chemical construction of IgG-like mono- and bispecific synthetic antibodies (SynAbs)

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