SGN-B7H4V, an investigational vedotin ADC directed to the immune checkpoint ligand B7-H4, shows promising activity in preclinical models.

BACKGROUND: SGN-B7H4V is a novel investigational vedotin antibody-drug conjugate (ADC) comprising a B7-H4-directed human monoclonal antibody conjugated to the cytotoxic payload monomethyl auristatin E (MMAE) via a protease-cleavable maleimidocaproyl valine citrulline (mc-vc) linker. This vedotin linker-payload system has been clinically validated in multiple Food and Drug Administration approved agents including brentuximab vedotin, enfortumab vedotin, and tisotumab vedotin. B7-H4 is an immune checkpoint ligand with elevated expression on a variety of solid tumors, including breast, ovarian, and endometrial tumors, and limited normal tissue expression. SGN-B7H4V is designed to induce direct cytotoxicity against target cells by binding to B7-H4 on the surface of target cells and releasing the cytotoxic payload MMAE upon internalization of the B7-H4/ADC complex. METHODS: B7-H4 expression was characterized by immunohistochemistry across multiple solid tumor types. The ability of SGN-B7H4V to kill B7-H4-expressing tumor cells in vitro and in vivo in a variety of xenograft tumor models was also evaluated. Finally, the antitumor activity of SGN-B7H4V as monotherapy and in combination with an anti-programmed cell death-1 (PD-1) agent was evaluated using an immunocompetent murine B7-H4-expressing Renca tumor model. RESULTS: Immunohistochemistry confirmed B7-H4 expression across multiple solid tumors, with the highest prevalence in breast, endometrial, and ovarian tumors. In vitro, SGN-B7H4V killed B7-H4-expressing tumor cells by MMAE-mediated direct cytotoxicity and antibody-mediated effector functions including antibody-dependent cellular cytotoxicity and antibody-dependent cellular phagocytosis. In vivo, SGN-B7H4V demonstrated strong antitumor activity in multiple xenograft models of breast and ovarian cancer, including xenograft tumors with heterogeneous B7-H4 expression, consistent with the ability of vedotin ADCs to elicit a bystander effect. In an immunocompetent murine B7-H4-expressing tumor model, SGN-B7H4V drove robust antitumor activity as a monotherapy that was enhanced when combined with an anti-PD-1 agent. CONCLUSION: The immune checkpoint ligand B7-H4 is a promising molecular target expressed by multiple solid tumors. SGN-B7H4V demonstrates robust antitumor activity in preclinical models through multiple potential mechanisms. Altogether, these preclinical data support the evaluation of SGN-B7H4V as a monotherapy in the ongoing phase 1 study of SGN-B7H4V in advanced solid tumors (NCT05194072) and potential future clinical combinations with immunotherapies.

Proteomic analysis of the androgen receptor via MS-compatible purification of biotinylated protein on streptavidin resin.

The strength of the streptavidin/biotin interaction poses challenges for the recovery of biotinylated molecules from streptavidin resins. As an alternative to high-temperature elution in urea-containing buffers, we show that mono-biotinylated proteins can be released with relatively gentle heating in the presence of biotin and 2% SDS/Rapigest, avoiding protein carbamylation and minimizing streptavidin dissociation. We demonstrate the utility of this mild elution strategy in two studies of the human androgen receptor (AR). In the first, in which formaldehyde cross-linked complexes are analyzed in yeast, a mass spectrometry-based comparison of the AR complex using SILAC reveals an association between the androgen-activated AR and the Hsp90 chaperonin, while Hsp70 chaperonins associate specifically with the unliganded complex. In the second study, the endogenous AR is quantified in the LNCaP cell line by absolute SILAC and MRM-MS showing approximately 127,000 AR copies per cell, substantially more than previously measured using radioligand binding.

Suppression of the immune response to FVIII in hemophilia A mice by transgene modified tolerogenic dendritic cells.

Current methods for eradicating clinically significant inhibitory antibodies to human factor VIII (hFVIII) in patients with hemophilia A rely on repeated delivery of high doses of factor concentrates for a minimum of many months. We hypothesize that tolerance can be induced more efficiently and reliably through hFVIII antigen presentation by tolerogenic dendritic cells (tDCs). In this study, we generated tDCs from hemophilia A mice and modified them with a foamy virus vector expressing a bioengineered hFVIII transgene. Naive and preimmunized mice infused with hFVIII expressing tDCs showed suppression of the T cell and inhibitor responses to recombinant hFVIII (rhFVIII). Treatment with hFVIII expressing tDCs was also associated with a higher percentage of splenocytes demonstrating a regulatory T cell phenotype in immunized mice. Furthermore, CD4(+) T cells harvested from recipients of hFVIII expression vector-modified tDCs were able to mediate antigen-specific immune suppression in naive secondary recipients. We also demonstrated a trend for improved suppression of inhibitor formation by coexpressing interleukin-10 (IL-10) and hFVIII from a bicistronic vector. These preclinical results demonstrate the potential for employing vector modified ex vivo generated tDCs to treat high titer inhibitors in patients with hemophilia A.

Suppression of FVIII inhibitor formation in hemophilic mice by delivery of transgene modified apoptotic fibroblasts.

The development of inhibitory antibodies to factor VIII (FVIII) is currently the most significant complication of FVIII replacement therapy in the management of patients with severe hemophilia A. Immune tolerance protocols for the eradication of inhibitors require daily delivery of intravenous FVIII for at least 6 months and are unsuccessful in 20-40% of treated patients. We hypothesize that tolerance can be induced more efficiently and reliably by delivery of FVIII antigen within autologous apoptotic cells (ACs). In this study, we demonstrated suppression of the T cell and inhibitor responses to FVIII by infusion of FVIII expression vector modified apoptotic syngeneic fibroblasts in both naive and preimmunized hemophilia A mice. ACs without FVIII antigen exerted modest generalized immune suppression mediated by anti-inflammatory signals. However, FVIII expressing apoptotic syngeneic fibroblasts produced much stronger antigen-specific immune suppression. Mice treated with these fibroblasts generated CD4+ T cells that suppressed the immune response to FVIII after adoptive transfer into naive recipients and antigen-specific CD4+CD25+ regulatory T cells (Tregs) that inhibited the proliferation of FVIII responsive effector T cells in vitro. These preclinical results demonstrate the potential for using FVIII vector modified autologous ACs to treat high-titer inhibitors in patients with hemophilia A.

Incomplete restoration of Mpl expression in the mpl-/- mouse produces partial correction of the stem cell-repopulating defect and paradoxical thrombocytosis.

Expression of Mpl is restricted to hematopoietic cells in the megakaryocyte lineage and to undifferentiated progenitors, where it initiates critical cell survival and proliferation signals after stimulation by its ligand, thrombopoietin (TPO). As a result, a deficiency in Mpl function in patients with congenital amegakaryocytic thrombocytopenia (CAMT) and in mpl(-/-) mice produces profound thrombocytopenia and a severe stem cell-repopulating defect. Gene therapy has the potential to correct the hematopoietic defects of CAMT by ectopic gene expression that restores normal Mpl receptor activity. We rescued the mpl(-/-) mouse with a transgenic vector expressing mpl from the promoter elements of the 2-kb region of DNA just proximal to the natural gene start site. Transgene rescued mice exhibit thrombocytosis but only partial correction of the stem cell defect. Furthermore, they show very low-level expression of Mpl on platelets and megakaryocytes, and the transgene-rescued megakaryocytes exhibit diminished TPO-dependent kinase phosphorylation and reduced platelet production in bone marrow chimeras. Thrombocytosis is an unexpected consequence of reduced Mpl expression and activity. However, impaired TPO homeostasis in the transgene-rescued mice produces elevated plasma TPO levels, which serves as an unchecked stimulus to drive the observed excessive megakaryocytopoiesis.