Structural analysis of light chain-driven bispecific antibodies targeting CD47 and PD-L1.

In contrast to natural antibodies that rely mainly on the heavy chain to establish contacts with their cognate antigen, we have developed a bispecific antibody format in which the light chain (LC) drives antigen binding and specificity. To better understand epitope-paratope interactions in this context, we determined the X-ray crystallographic structures of an antigen binding fragment (Fab) in complex with human CD47 and another Fab in complex with human PD-L1. These Fabs contain a κ-LC and a λ-LC, respectively, which are paired with an identical heavy chain (HC). The structural analysis of these complexes revealed the dominant contribution of the LCs to antigen binding, but also that the common HC provides some contacts in both CD47 and PD-L1 Fab complexes. The anti-CD47 Fab was affinity optimized by diversifying complementary-determining regions of the LC followed by phage display selections. Using homology modeling, the contributions of the amino acid modification to the affinity increase were analyzed. Our results demonstrate that, despite a less prominent role in natural antibodies, the LC can mediate high affinity binding to different antigens and neutralize their biological function. Importantly, Fabs containing a common variable heavy (VH) domain enable the generation of bispecific antibodies retaining a truly native structure, maximizing their therapeutic potential.

ATOR-1017 (evunzekibart), an Fc-gamma receptor conditional 4-1BB agonist designed for optimal safety and efficacy, activates exhausted T cells in combination with anti-PD-1.

BACKGROUND: 4-1BB (CD137) is a co-stimulatory receptor highly expressed on tumor reactive effector T cells and NK cells, which upon stimulation prolongs persistence of tumor reactive effector T and NK cells within the tumor and induces long-lived memory T cells. 4-1BB agonistic antibodies have been shown to induce strong anti-tumor effects that synergize with immune checkpoint inhibitors. The first generation of 4-1BB agonists was, however, hampered by dose-limiting toxicities resulting in suboptimal dose levels or poor agonistic activity. METHODS: ATOR-1017 (evunzekibart), a second-generation Fc-gamma receptor conditional 4-1BB agonist in IgG4 format, was designed to overcome the limitations of the first generation of 4-1BB agonists, providing strong agonistic effect while minimizing systemic immune activation and risk of hepatoxicity. The epitope of ATOR-1017 was determined by X-ray crystallography, and the functional activity was assessed in vitro and in vivo as monotherapy or in combination with anti-PD1. RESULTS: ATOR-1017 binds to a unique epitope on 4-1BB enabling ATOR-1017 to activate T cells, including cells with an exhausted phenotype, and NK cells, in a cross-linking dependent, FcγR-conditional, manner. This translated into a tumor-directed and potent anti-tumor therapeutic effect in vivo, which was further enhanced with anti-PD-1 treatment. CONCLUSIONS: These preclinical data demonstrate a strong safety profile of ATOR-1017, together with its potent therapeutic effect as monotherapy and in combination with anti-PD1, supporting further clinical development of ATOR-1017.

Targeting platelet GPVI with glenzocimab: a novel mechanism for inhibition.

Platelet glycoprotein VI (GPVI) is attracting interest as a potential target for the development of new antiplatelet molecules with a low bleeding risk. GPVI binding to vascular collagen initiates thrombus formation and GPVI interactions with fibrin promote the growth and stability of the thrombus. In this study, we show that glenzocimab, a clinical stage humanized antibody fragment (Fab) with a high affinity for GPVI, blocks the binding of both ligands through a combination of steric hindrance and structural change. A cocrystal of glenzocimab with an extracellular domain of monomeric GPVI was obtained and its structure determined to a resolution of 1.9 Å. The data revealed that (1) glenzocimab binds to the D2 domain of GPVI, GPVI dimerization was not observed in the crystal structure because glenzocimab prevented D2 homotypic interactions and the formation of dimers that have a high affinity for collagen and fibrin; and (2) the light variable domain of the GPVI-bound Fab causes steric hindrance that is predicted to prevent the collagen-related peptide (CRP)/collagen fibers from extending out of their binding site and preclude GPVI clustering and downstream signaling. Glenzocimab did not bind to a truncated GPVI missing loop residues 129 to 136, thus validating the epitope identified in the crystal structure. Overall, these findings demonstrate that the binding of glenzocimab to the D2 domain of GPVI induces steric hindrance and structural modifications that drive the inhibition of GPVI interactions with its major ligands.

The Bispecific Tumor Antigen-Conditional 4-1BB x 5T4 Agonist, ALG.APV-527, Mediates Strong T-Cell Activation and Potent Antitumor Activity in Preclinical Studies.

4-1BB (CD137) is an activation-induced costimulatory receptor that regulates immune responses of activated CD8 T and natural killer cells, by enhancing proliferation, survival, cytolytic activity, and IFNγ production. The ability to induce potent antitumor activity by stimulating 4-1BB on tumor-specific cytotoxic T cells makes 4-1BB an attractive target for designing novel immuno-oncology therapeutics. To minimize systemic immune toxicities and enhance activity at the tumor site, we have developed a novel bispecific antibody that stimulates 4-1BB function when co-engaged with the tumor-associated antigen 5T4. ALG.APV-527 was built on the basis of the ADAPTIR bispecific platform with optimized binding domains to 4-1BB and 5T4 originating from the ALLIGATOR-GOLD human single-chain variable fragment library. The epitope of ALG.APV-527 was determined to be located at domain 1 and 2 on 4-1BB using X-ray crystallography. As shown in reporter and primary cell assays in vitro, ALG.APV-527 triggers dose-dependent 4-1BB activity mediated only by 5T4 crosslinking. In vivo, ALG.APV-527 demonstrates robust antitumor responses, by inhibiting growth of established tumors expressing human 5T4 followed by a long-lasting memory immune response. ALG.APV-527 has an antibody-like half-life in cynomolgus macaques and was well tolerated at 50.5 mg/kg. ALG.APV-527 is uniquely designed for 5T4-conditional 4-1BB-mediated antitumor activity with potential to minimize systemic immune activation and hepatotoxicity while providing efficacious tumor-specific responses in a range of 5T4-expressing tumor indications as shown by robust activity in preclinical in vitro and in vivo models. On the basis of the combined preclinical dataset, ALG.APV-527 has potential as a promising anticancer therapeutic for the treatment of 5T4-expressing tumors.

Preoperative Ultrasonographic Evaluation of Subclavian Vein and Inferior Vena Cava for Predicting Hypotension Associated with Induction of General Anesthesia.

Introduction: Induction of general anesthesia is often associated with hypotension and is a common scenario faced by anesthesiologists. Intraoperative hypotension can have detrimental effects and cause various adverse effects leading to an extended hospital stay. Patients' preinduction volume status can have an effect on postinduction blood pressure. Ultrasonography is a useful tool for measuring intravascular volume status. We studied the ability of ultrasonographic measurement of subclavian vein (SCV) and inferior vena cava (IVC) diameter, collapsibility index (CI) to predict hypotension after induction of general anesthesia. Materials and Methods: We included 120 patients in our study. SCV measurements during spontaneous and deep inspiration and IVC measurements were taken before induction and postinduction blood pressure was monitored. Patients with mean arterial blood pressure <60 mmHg or with a 30% decrease from baseline were considered to be having hypotension. Results: The CI of IVC with a cutoff 37% showed sensitivity of 94% and specificity of 84% which was statistically significant. The CI of 36% of SCV during deep breathing was found to have high sensitivity and specificity of 90% and 87%. Conclusion: Our study in spontaneously breathing preoperative patients shows that SCV CI in deep breathing and IVC CI is very sensitive and reliable in predicting postinduction hypotension. Bedside ultrasound measurements can be easily done to obtain valuable information to recognize patients who could be at risk from postinduction hypotension.