Small, compact, and highly symmetric cube-octameric silsesquioxanes have recently attracted increased attention as scaffolds for tailor-made bioconjugates. The expanded arsenal of effective conjugation methods (CuAAC, TEC, oxime ligation) allows one to decorate these nanoparticles bearing up to eight addressable organic substituents, with a wide range of biorelevant ligands, among them carbohydrates, peptides, miniproteins, reporter molecules, and rare-earth chelates. Low toxicity of COSS-based molecules combined with solubility in aqueous systems and half-life sufficient for in vivo studies make these structures attractive targets for a number of applications, among them drug delivery, tumor diagnostics and therapy.

The immobilization of bioactive molecules onto nanocellulose leads to constructs that combine the properties of the grafted compounds with the biocompatibility and low cytotoxicity of cellulose carriers and the advantages given by their nanometer dimensions. However, the methods commonly used for protein grafting suffer from lack of selectivity, long reaction times, nonphysiological pH ranges and solvents, and the necessity to develop a tailor-made reaction strategy for each individual case. To overcome these restrictions, a generic two-step procedure was developed that takes advantage of the highly efficient oxime ligation combined with enzyme-mediated protein coupling onto the surface of peptide-modified crystalline nanocellulose. The described method is based on efficient and orthogonal transformations, requires no organic solvents, and takes place under physiological conditions. Being site-directed and regiospecific, it could be applied to a vast number of functional proteins.

The prostate‐specific membrane antigen (PSMA) has been demonstrated in numerous studies to be expressed specifically on prostate carcinoma cells and on the neovasculature of several other cancer entities. However, the simultaneous expression of PSMA on both, tumor cells as well as tumor vessels remains unclear, even if such “dual” expression would constitute an important asset to facilitate sufficient influx of effector cells to a given tumor site. We report here on the generation of a PSMA antibody, termed 10B3, which exerts superior dual reactivity on sections of prostate carcinoma and squamous cell carcinoma of the lung. 10B3 was used for the construction of T‐cell recruiting bispecific PSMAxCD3 antibodies in Fab‐ and IgG‐based formats, designated Fabsc and IgGsc, respectively. In vitro, both molecules exhibited comparable activity. In contrast, only the larger IgGsc molecule induced complete and durable elimination of established tumors in humanized mice due to favorable pharmacokinetic properties. Upon treatment of three patients with metastasized prostate carcinoma with the IgGsc reagent, marked activation of T cells and rapid reduction of elevated PSA levels were observed.

Bispecific antibodies (bsAb) and chimeric antigen receptor (CAR) T cells allow for antibody guided recruitment of T cells against tumors. Both are successfully used for treatment of CD19 expressing leukemias, but may cause cytokine release syndrome (CRS) as a major dose-limiting side effect. For CRS prevention, steroids are recommended prior to bsAb treatment, despite their well-known lymphotoxic activity. The IL-6 receptor antibody tocilizumab is established for treatment of CRS induced by CAR T cells, but was not considered for CRS prevention in bsAb therapy. We here compared the influence of dexamethasone and tocilizumab on bsAb-mediated T cell proliferation and tumor lysis in vitro and in vivo and found that dexamethasone profoundly inhibited T cell proliferation and antitumor activity as induced by two different bsAb, particularly at low effector:target ratios, whereas tocilizumab did not affect efficacy. When we applied tocilizumab early during treatment of three patients with a newly developed PSMAxCD3 bsAb, significant CRS attenuation despite high IL-6 serum levels was observed. Thus, early IL-6 blockade may reduce the undesired sequelae of CRS upon bsAb therapy without affecting therapeutic activity, allowing in turn for safe application of effective doses.

BackgroundIn lymphoid malignancies, the introduction of chimeric antigen receptor T (CAR-T) cells and bispecific antibodies (bsAbs) has achieved remarkable clinical success. However, such immunotherapeutic strategies are not yet established for acute myeloid leukemia (AML), the most common form of acute leukemia in adults. Common targets in AML such as CD33, CD123, and CLEC12A are highly expressed on both AML blasts and on normal myeloid cells and hematopoietic stem cells (HSCs), thereby raising toxicity concerns. In B-cell acute lymphoblastic leukemia (B-ALL), bsAbs and CAR-T therapy targeting CD19 and CD22 have demonstrated clinical success, but resistance via antigen loss is common, motivating the development of agents focused on alternative targets. An attractive emerging target is FLT3, a proto-oncogene expressed in both AML and B-ALL, with low and limited expression on myeloid dendritic cells and HSCs.MethodsWe developed and characterized CLN-049, a T cell-activating bsAb targeting CD3 and FLT3, constructed as an IgG heavy chain/scFv fusion. CLN-049 binds the membrane proximal extracellular domain of the FLT3 protein tyrosine kinase, which facilitates the targeting of leukemic blasts regardless of FLT3 mutational status. CLN-049 was evaluated for preclinical safety and efficacy in vitro and in vivo.ResultsCLN-049 induced target-restricted activation of CD4+ and CD8+ T cells. AML cell lines expressing a broad range of surface levels of FLT3 were efficiently lysed on treatment with subnanomolar concentrations of CLN-049, whereas FLT3-expressing hematopoietic progenitor cells and dendritic cells were not sensitive to CLN-049 killing. Treatment with CLN-049 also induced lysis of AML and B-ALL patient blasts by autologous T cells at the low effector-to-target ratios typically observed in patients with overt disease. Lysis of leukemic cells was not affected by supraphysiological levels of soluble FLT3 or FLT3 ligand. In mouse xenograft models, CLN-049 was highly active against human leukemic cell lines and patient-derived AML and B-ALL blasts.ConclusionsCLN-049 has a favorable efficacy and safety profile in preclinical models, warranting evaluation of its antileukemic activity in the clinic.

Over the past decade, DNA and RNA aptamers have attracted keen research interest due to their ability to specifically bind targets of therapeutic relevance. However, their application is often hampered by a short serum half-life and missing effector functions. Conjugation of aptamers to antibody Fc fragments could improve pharmacokinetics, enable immune effector mechanisms, and provide an option for the introduction of desired payloads (e.g., toxins or fluorescent dyes). We developed a modular scaffold-supported system based on human IgG1 Fc fragments, which allows for its dual functionalization with moieties of interest. In our approach, two bioorthogonal, enzyme-mediated reactions were used in combination with oxime ligation and self-assembly based on PNA-DNA base pairing. Thus, an engineered synthetic peptide nucleic acid (PNA) oligomer was coupled to the C-termini of the Fc dimer upon sequence-specific sortase A-mediated transpeptidation. Hybridization of the resulting Fc-PNA conjugate with a tailored DNA aptamer that binds cancer-related hepatocyte growth factor receptor (c-MET) led to a hybrid construct which showed strong and specific binding to c-MET and was readily internalized by c-MET-overexpressing cells. To install an additional orthogonally addressable site, aldehyde tag technology was applied followed by oxime ligation with an aminooxy-bearing fluorescent dye as model cargo. Delivery of fluorescent probe specifically to c-MET-overexpressing cells was confirmed by flow cytometry. Our approach can provide access to engineered aptamer-Fc conjugates with desired target specificity and cytotoxic payloads.

We report a comprehensive study on novel, highly efficient, and biodegradable hybrid molecular transporters. To this end, we designed a series of cell-penetrating, cube-octameric silsesquioxanes (COSS), and investigated cellular uptake by confocal microscopy and flow cytometry. A COSS with dense spatial arrangement of guanidinium groups displayed fast uptake kinetics and cell permeation at nanomolar concentrations in living HeLa cells. Efficient uptake was also observed in bacteria, yeasts, and archaea. The COSS-based carrier was significantly more potent than cell-penetrating peptides (CPPs) and displayed low toxicity. It efficiently delivered a covalently attached cytotoxic drug, doxorubicin, to living tumor cells. As the uptake of fluorescently labeled carrier remained in the presence of serum, the system could be considered particularly attractive for the in vivo delivery of therapeutics.

A modular approach combining inverse electron-demand Diels-Alder coupling (DARinv) and oxime ligation expands the toolbox of bioorthogonal peptide chemistry. Applicability of versatile site-specific bifunctional building blocks is demonstrated by generation of defined conjugates comprising linear, cystine-bridged and multi-disulfide functional peptides as well as their conjugation with hybrid silsesquioxane nanoparticles.