Combination of Targeted Therapies for Colorectal Cancer Treatment.

The design of innovative therapeutic strategies enabling the selective destruction of tumor cells while sparing healthy tissues remains highly challenging in cancer therapy. Here, we show that the combination of two targeted therapies, including bevacizumab (Bev), and a ß-glucuronidase-responsive albumin-binding prodrug of monomethyl auristatin E (MMAE), is efficient for the treatment of colorectal cancer implanted in mice. This combined therapy produces a therapeutic activity superior to that of the association of FOLFOX and Bev currently used to treat patients with this pathology. The increased anticancer efficacy is due to either a synergistic or an additive effect between Bev and MMAE selectively released from the glucuronide prodrug in the tumor microenvironment. Since numerous drug delivery systems such as antibody-drug conjugates employ MMAE as a cytotoxic payload, this finding may be of great interest for improving their therapeutic index by combining them with Bev, particularly for the therapy of colorectal cancer.

A Novel Family of Acid-Cleavable Linker Based on Cyclic Acetal Motifs for the Production of Antibody-Drug Conjugates with High Potency and Selectivity.

Cleavable linkers have become the subject of intense study in the field of chemical biology, particularly because of their applications in the construction of antibody-drug conjugates (ADC), where they facilitate lysosomal cleavage and liberation of drugs from their carrier protein. Due to lysosomes' acidic nature, acid-labile motifs have attracted much attention, leading to the development of hydrazone and carbonate linkers among several other entities. Continuing our efforts in designing new moieties, we present here a family of cyclic acetals that exhibit excellent plasma stability and acid lability, notably in lysosomes. Incorporated in ADC, they led to potent constructs with picomolar potency in vitro and similar in vivo efficacy as the commercially available ADC Kadcyla in mouse xenograft models.

Synthesis and Biological Characterization of Monomeric and Tetrameric RGD-Cryptophycin Conjugates.

The effective delivery of cytotoxic agents to tumor cells is a key challenge in anticancer therapy. Multivalent integrinspecific ligands are considered a promising tool to increase the binding affinity, selectivity, and internalization efficiency of small-molecule drug conjugates. Herein, we report the synthesis and biological evaluation of a multimeric conjugate containing the high-affinity integrin αv ß3 binding ligand RAFT-c(RGDfK)4 , a lysosomally cleavable Val-Cit linker, and cryptophycin-55 glycinate, a potent inhibitor of tubulin polymerization. In vitro cytotoxicity assays verified that the multimeric RGD-cryptophycin conjugate displays improved potency compared to the monomeric analogue in integrin αv ß3 overexpressing tumor cell lines, while significantly reduced activity was observed in the integrin-negative cell line.

Design of RGD-ATWLPPR peptide conjugates for the dual targeting of αVß3 integrin and neuropilin-1.

Targeting the tumour microenvironment is a promising strategy to detect and/or treat cancer. The design of selective compounds that co-target several receptors frequently overexpressed in solid tumours may allow a reliable and selective detection of tumours. Here we report the modular synthesis of compounds encompassing ligands of αVß3 integrin and neuropilin-1 that are overexpressed in the tumour microenvironment. These compounds were then evaluated through cellular experiments and imaging of tumours in mice. We observed that the peptide that displays both ligands is more specifically accumulating in the tumours than in controls. Simultaneous interaction with αVß3 integrin and NRP1 induces NRP1 stabilization at the cell membrane surface which is not observed with the co-injection of the controls.

Recent, non-classical, approaches to antibody lysine modification.

This review will discuss recent development in the bioconjugation of lysine residues on antibodies. As several chemoselective reagents have already been developed for modifying amine groups, recent strategies now tend to aim at being site-specific. Four general methods have been listed: kinetically controlled, template-directed or enzymatic strategies as well as the use of chemically programmed antibodies.

Systemic Delivery of Tumor-Targeted Bax-Derived Membrane-Active Peptides for the Treatment of Melanoma Tumors in a Humanized SCID Mouse Model.

Melanoma is a highly metastatic and deadly form of cancer. Invasive melanoma cells overexpress integrin αvß3, which is a well-known target for Arg-Gly-Asp-based (RGD) peptides. We developed a sophisticated method to synthetize milligram amounts of a targeted vector that allows the RGD-mediated targeting, internalization, and release of a mitochondria-disruptive peptide derived from the pro-apoptotic Bax protein. We found that 2.5 µM Bax[109-127] was sufficient to destabilize the mitochondria in ten different tumor cell lines, even in the presence of the anti-apoptotic Bcl2 protein, which is often involved in tumor resistance. This pore-forming peptide displayed antitumor activity when it was covalently linked by a disulfide bridge to the tetrameric RAFT-c[RGD]4-platform and after intravenous injection in a human melanoma tumor model established in humanized immuno-competent mice. In addition to its direct toxic effect, treatment with this combination induced the release of the immuno-stimulating factor monocyte chimoattractant protein 1 (MCP1) in the blood and a decrease in the level of the pro-angiogenic factor FGF2. Our novel multifunctional, apoptosis-inducing agent could be further customized and assayed for potential use in tumor-targeted therapy.

"Polymultivalent" Polymer-Peptide Cluster Conjugates for an Enhanced Targeting of Cells Expressing αvß3 Integrins.

A new class of "polymultivalent" ligands combining several ligand clusters and a water-soluble biocompatible polymer is introduced. These original conjugates bear two levels of multivalency. They are prepared by covalent coupling of a controlled number of tetrameric cRGD peptide clusters along a well-defined copolymer synthesized by RAFT polymerization. The presence of multiple copies of peptide clusters on the same polymer backbone resulted in a much-higher relative potency than the free cluster reference. Thanks to the "polymultivalency", up to ∼2 orders of magnitude potency enhancement was reached in a competitive cell adhesion assay (nanomolar-range IC50 values). In addition, confocal microscopy and flow cytometry demonstrated that fluorescent "polymultivalent" conjugates (emitting in the far-red/near-infrared region) were able to specifically and selectively label cells expressing αvß3-integrin, the natural receptor of cRGD.

Multimeric RGD-Based Strategies for Selective Drug Delivery to Tumor Tissues.

RGD peptides have received a lot of attention over the two last decades, in particular to improve tumor therapy through the targeting of the αVß3 integrin receptor. This review focuses on the molecular design of multimeric RGD compounds, as well as the design of suitable linkers for drug delivery. Many examples of RGD-drug conjugates have been developed, and we show the importance of RGD constructs to enhance binding affinity to tumor cells, as well as their drug uptake. Further, we also highlight the use of RGD peptides as theranostic systems, promising tools offering dual modality, such as tumor diagnosis and therapy. In conclusion, we address the challenging issues, as well as ongoing and future development, in comparison with large molecules, such as monoclonal antibodies.

Enabling the formation of native mAb, Fab' and Fc-conjugates using a bis-disulfide bridging reagent to achieve tunable payload-to-antibody ratios (PARs).

Either as full IgGs or as fragments (Fabs, Fc, etc.), antibodies have received tremendous attention in the development of new therapeutics such as antibody-drug conjugates (ADCs). The production of ADCs involves the grafting of active payloads onto an antibody, which is generally enabled by the site-selective modification of native or engineered antibodies via chemical or enzymatic methods. Whatever method is employed, controlling the payload-antibody ratio (PAR) is a challenge in terms of multiple aspects including: (i) obtaining homogeneous protein conjugates; (ii) obtaining unusual PARs (PAR is rarely other than 2, 4 or 8); (iii) using a single method to access a range of different PARs; (iv) applicability to various antibody formats; and (v) flexibility for the production of heterofunctional antibody-conjugates (e.g. attachment of multiple types of payloads). In this article, we report a single pyridazinedione-based trifunctional dual bridging linker that enables, in a two-step procedure (re-bridging/click), the generation of either mAb-, Fab'-, or Fc-conjugates from native mAb, (Fab')2 or Fc formats, respectively. Fc and (Fab')2 formats were generated via enzymatic digestion of native mAbs. Whilst the same reduction and re-bridging protocols were applied to all three of the protein formats, the subsequent click reaction(s) employed to graft payload(s) drove the generation of a range of PARs, including heterofunctional PARs. As such, exploiting click reactivity and/or orthogonality afforded mAb-conjugates with PARs of 6, 4, 2 or 4 + 2, and Fab'- and Fc-conjugates with a PAR of 3, 2, 1 or 2 + 1 on-demand. We believe that the homogeneity, novelty and variety in accessible PARs, as well as the applicability to various antibody-conjugate formats enabled by our non-recombinant method could be a suitable tool for antibody-drug conjugates optimisation (optimal PAR value, optimal payloads combination) and boost the development of new antibody therapeutics (Fab'- and Fc-conjugates).

Modular Chemical Construction of IgG-like Mono- and Bispecific Synthetic Antibodies (SynAbs).

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 rebridging 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 T cell-engager (TCE) SynAb, FcCD20-(FabHER2)-FabCD3, and have confirmed that it exhibits the expected biological functions, including the ability to kill HER2+ target cells in a coculture assay with T cells.

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

In the past two decades, immunotherapy has established itself as one of the leading strategies for cancer treatment, as illustrated by the exponentially growing number of related clinical trials. This trend was, in part, prompted by the clinical success of both immune checkpoint modulation and immune cell engagement, to restore and/or stimulate the patient's immune system's ability to fight the disease. These strategies were sustained by progress in bispecific antibody production. However, despite the decisive progress made in the treatment of cancer, toxicity and resistance are still observed in some cases. In this review, we initially provide an overview of the monoclonal and bispecific antibodies developed with the objective of restoring immune system functions to treat cancer (cancer immunotherapy), through immune checkpoint modulation, immune cell engagement or a combination of both. Their production, design strategy and impact on the clinical trial landscape are also addressed. In the second part, the concept of multispecific antibody formats, notably MuTICEMs (Multispecific Targeted Immune Cell Engagers & Modulators), as a possible answer to current immunotherapy limitations is investigated. We believe it could be the next step to take for cancer immunotherapy research and expose why bioconjugation chemistry might play a key role in these future developments.

A novel thiol-labile cysteine protecting group for peptide synthesis based on a pyridazinedione (PD) scaffold.

Herein we report a thiol-labile cysteine protecting group based on an unsaturated pyridazinedione (PD) scaffold. We establish compatibility of the PD in conventional solid phase peptide synthesis (SPPS), showcasing this in the on-resin synthesis of biologically relevant oxytocin. Furthermore, we establish the applicability of the PD protecting group towards both microwave-assisted SPPS and native chemical ligation (NCL) in a model system.