RGD peptides have received a lot of attention over the two last decades, in particular to improve tumor therapy through the targeting of the αVβ3 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.

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 Fc_Z-(Fab_X)-Fab_Y 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, Fc_CD20-(Fab_HER2)-Fab_CD3, 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.

Melanoma is a highly metastatic and deadly form of cancer. Invasive melanoma cells overexpress integrin α_vβ₃, 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.

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).

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 α_Vβ₃ 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 α_Vβ₃ integrin and NRP1 induces NRP1 stabilization at the cell membrane surface which is not observed with the co-injection of the controls.

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 α_vβ₃ 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 α_vβ₃ overexpressing tumor cell lines, while significantly reduced activity was observed in the integrin‐negative cell line.