Qa-1b, the murine ortholog of the nonclassical MHC-E family, contains minimal polymorphism and exhibits reduced surface stability compared with classical MHC class I molecules. To investigate Qa-1b conformations and their immunological relevance, we employed two antibodies: EXX-1, which selectively recognizes Qa-1b bound to the canonical leader peptide Qdm, and 6A8.6F10, a broadly used Qa-1b-reactive antibody. Genome-wide CRISPR screens revealed that Qdm presentation was induced by interferon-γ and required the components of the peptide-loading complex (PLC) and endoplasmic reticulum quality control. EXX-1 binding thus reflected broad cellular integrity and mirrored CD94/NKG2x receptor engagement. In contrast, 6A8.6F10 staining occurred independently of PLC components such as ERAP1 and tapasin, and intriguingly increased in their absence. Accordingly, exogenous pulsing with Qa-1b-binding peptides markedly reduced 6A8.6F10 antibody binding and resonance shift assays revealed that 6A8.6F10 selectively recognizes peptide-deficient Qa-1b complexes. These findings suggest an additional layer of regulation beyond the immune checkpoint NKG2x/CD94, involving peptide-free MHC-E.

HLA-E's function as an immune checkpoint in cancer depends on its display of the canonical peptide (VL9), yet direct profiling of these complexes has been stymied by lack of specific reagents. We now introduce ABX002, a fully human TCR-mimic antibody capable of recognizing all tested VL9/HLA-E complexes with high affinity and specificity in situ. Using ABX002, we reveal that canonical VL9/HLA-E surface expression is tightly controlled by inflammatory cues, remarkably infrequent on tumors without stimulation, and almost absent from immune cells except myeloid-lineage cells. ABX002 unlocks cell-type and context-specific quantification of HLA-E antigen presentation, providing unprecedented insight into immune evasion and regulation. It additionally disrupts the NKG2A checkpoint, restoring cytotoxic lymphocyte function and enabling mechanistic and therapeutic mapping of HLA-E restricted peptide presentation. Together, these findings position ABX002 as a transformative tool for dissecting the landscape and biology of canonical peptide restriction in cancer immunity.

HLA-E’s function as an immune checkpoint in cancer depends on its display of the canonical peptide (VL9), yet direct profiling of these complexes has been stymied by lack of specific reagents. We now introduce ABX002, a fully human TCR-mimic antibody capable of recognizing all tested VL9/HLA-E complexes with high affinity and specificity in situ. Using ABX002, we reveal that canonical VL9/HLA-E surface expression is tightly controlled by inflammatory cues, remarkably infrequent on tumors without stimulation, and almost absent from immune cells except myeloid-lineage cells. ABX002 unlocks cell-type and context-specific quantification of HLA-E antigen presentation, providing unprecedented insight into immune evasion and regulation. It additionally disrupts the NKG2A checkpoint, restoring cytotoxic lymphocyte function and enabling mechanistic and therapeutic mapping of HLA-E restricted peptide presentation. Together, these findings position ABX002 as a transformative tool for dissecting the landscape and biology of canonical peptide restriction in cancer immunity.

Abstract Canonical leader peptides, derived from the signal sequences of classical MHC class I molecules, are presented by HLA-E in humans and Qa1 in mice. These peptides serve as ligands for the NKG2A/CD94 receptor complex on NK cells and CD8+ T cells, promoting immune homeostasis. Disruptions of this inhibitory axis—such as during viral infection—can lead to the display of novel peptides that override this inhibition and activate immune responses. However, the mechanisms controlling canonical peptide presentation remain unclear. To better understand how Qa1-restricted inhibitory peptide presentation is regulated, we targeted Signal Peptide Peptidase (SPP), an ER-resident protease that cleaves signal peptides. SPP was knocked out in two tumor models: YUMMER melanoma and KPC pancreatic adenocarcinoma. Changes in Qa1-bound peptides were evaluated using mass spectrometry, and tumor growth was assessed in vivo. Loss of SPP markedly reduced canonical peptide loading in both tumor models. SPP-deficient melanoma tumors were strongly rejected in vivo, while pancreatic tumors lacking SPP showed no such reduction, despite similar decreases in canonical inhibitory peptide levels. Interestingly, the dominant Qa1-bound peptide in both models remained the canonical peptide, indicating that alternative, SPP-independent mechanisms can liberate and generate this peptide for presentation. In the KPC model, novel peptides with potential inhibitory properties were identified. Ongoing studies aim to determine whether these peptides can sustain immune suppression in the absence of the canonical peptide. Our findings demonstrated that even in the absence of SPP, alternative mechanisms maintain inhibitory peptide presentation in tumor cells. Ongoing studies targeting other SPP family members and newly identified candidate inhibitory peptides aim to elucidate these compensatory pathways. A clearer definition of the Qa1/HLA-E inhibitory pathway will help determine whether this axis can be more effectively disrupted to improve anti-tumor immune responses. Citation Format: Roya Solhi, Clara Wolfe, Hu Chen, Achintya Perumal, Kyle Ockerman, Grant Brennan, Jiayao Ye, Adrienne H. Long, Marc Schwartz, Susan Klaeger, Steven A. Carr, Thorbald van Hall, Jon A. Weidanz, Soroush Ghaffari, Kathleen B. Yates, Robert T. Manguso, Qin Ma, Hakimeh Ebrahimi-Nik. Investigating the role of signal peptide peptidase in tumor immune evasion via Qa-1-mediated peptide presentation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 1 (Regular Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(7 Suppl):Abstract nr 6579.

CD3 bispecific antibodies (bsAbs) are emerging as an important treatment option in the arsenal of oncologists. There are numerous FDA-approved CD3 bsAbs for both hematological and solid tumors. Despite these recent advances, the success of CD3 bsAbs in solid cancer has been hampered by hurdles like limited intratumoral T cell numbers, immunosuppressive tumor microenvironments (TME), and poor memory T-cell induction. Furthermore, tumor surface antigen selection for an optimal therapeutic window and acceptable collateral damage to normal tissues is challenging. In this review, we discuss recent research investigating combination approaches aimed at improving CD3 bsAb efficacy in solid cancer.

BackgroundCD3 bispecific antibody (CD3 bsAb) therapy has become an established treatment modality for some cancer types and exploits endogenous T cells irrespective of their specificity. However, durable clinical responses are hampered by immune escape through loss of tumor target antigen expression. Induction of long-lasting tumor-specific immunity might therefore improve therapeutic efficacy, but has not been studied in detail yet for CD3 bsAbs. Here, we examined multiple combination strategies aiming to improve survival rates in solid tumors and, simultaneously, install endogenous immunity capable of protection to tumor rechallenge.MethodsTwo syngeneic mouse tumor models were employed: The immunologically “cold” B16F10 melanoma and the immunologically “hot” MC38.TRP1 colon carcinoma model. Mice were treated with CD3xTRP1 bsAb (murine Fc-inert immunoglobulin G2a) as monotherapy, or in combination with agonistic costimulatory antibodies, Fc-active tumor-opsonizing antibodies, or tumor-(non)specific vaccines. Treatment efficacy of primary tumors and protection from rechallenge was monitored, as well as induction of tumor-specific T-cell responses.ResultsIn the immunologically “cold” B16F10 model, all combination therapies improved antitumor activity compared with CD3 bsAb monotherapy and induced systemic tumor-specific T-cell responses. However, this endogenous T-cell immunity swiftly waned and failed to protect mice from subsequent tumor rechallenge, except for combination therapy with tumor-specific vaccination. These vaccines strongly improved the therapeutic efficacy of CD3 bsAb against primary tumors and led to long-term immunological protection. In the immunologically “hot” MC38.TRP1 model, CD3 bsAb combined with only the vaccine adjuvant was sufficient to generate protective T-cell immunity and, moreover, prevented tumor escape via antigen loss.ConclusionsThese results demonstrate the impact of tumor antigenicity on the induction of protective endogenous antitumor immunity during CD3 bsAb treatment and, importantly, show that the combination with tumor-specific vaccines improves therapeutic efficacy and installs long-term immunological memory in both “hot” and “cold” tumors.

Antibody conjugates are the foundation of a wide range of diagnostic and therapeutic applications. Although many antibody-conjugation techniques are robust and efficient, obtaining homogeneous multimeric conjugation products remains challenging. Here we report a modular and versatile technique for the site-directed multivalent conjugation of antibodies via the small-protein ubiquitin. Specifically, multiple ubiquitin fusions with antibodies, antibody fragments, nanobodies, peptides or small molecules such as fluorescent dyes can be conjugated to antibodies and nanobodies within 30 min. The technique, which we named ‘ubi-tagging’, allowed us to efficiently generate a bispecific T-cell engager as well as nanobodies conjugated to dendritic-cell-targeted antigens that led to potent T-cell responses. Using both recombinant ubi-tagged proteins and synthetic ubiquitin derivatives allows for the iterative, site-directed and multivalent conjugation of antibodies and nanobodies to a plethora of molecular moieties. A technique for the site-directed conjugation of antibodies via the small-protein ubiquitin allows for the efficient multivalent conjugation of antibodies and nanobodies to fusions of ubiquitin with molecular or proteinic moieties.

Monoclonal antibodies are important modalities in the treatment of cancer. Post-translational modifications of proteins, such as glycosylation, can affect the binding affinity of therapeutic antibodies. Whether other PTMs modulate therapeutic antibody binding to different surface proteins is currently underexplored. Pyroglutamation is the post-translational cyclization of an N-terminal glutamine or glutamic acid residue into a pyroglutamate by glutaminyl cyclase. In this study, we investigated the impact of pyroglutamation on the binding affinity of three therapeutic antibodies targeting CD47 and TRP1. Here, we show that pyroglutamation on CD47 and TRP1 modulates the binding of anti(α)-CD47 magrolimab and αTRP1 TA99 and flanvotumab. Furthermore, the N-terminal glutamine on CD47 is crucial for effective antibody recognition, while pyroglutamation of TRP1 is involved in trafficking to the cell surface. These findings highlight that the pyroglutamation by glutaminyl cyclase can modulate the binding affinity of antibodies with therapeutic potential.

Figure S15. Reo preexposure induces Reo-specific NAbs and CD8+ T cells.

Figure S9. Intratumoral T-cell influx after Reo treatment in μMT or C57BL/6J mice.

Figure S10. Preexposure affects the specificity of Reo-specific CD8+ T cells.

AbstractReovirus type 3 Dearing (Reo), manufactured for clinical application as pelareorep, is an attractive anticancer agent under evaluation in multiple phase 2 clinical trials for the treatment of solid tumors. It elicits its anticancer efficacy by inducing both oncolysis and intratumoral T-cell influx. Because most people have been preexposed to Reo, neutralizing antibodies (NAb) are prevalent in patients with cancer and might present a barrier to effective Reo therapy. Here, we tested serum of patients with cancer and healthy controls (n = 100) and confirmed that Reo NAbs are present in >80% of individuals. To investigate the effect of NAbs on both the oncolytic and the immunostimulatory efficacy of Reo, we established an experimental mouse model with Reo preexposure. The presence of preexposure-induced NAbs reduced Reo tumor infection and prevented Reo-mediated control of tumor growth after intratumoral Reo administration. In B cell–deficient mice, the lack of NAbs provided enhanced tumor growth control after Reo monotherapy, indicating that NAbs limit the oncolytic capacity of Reo. In immunocompetent mice, intratumoral T-cell influx was not affected by the presence of preexposure-induced NAbs and consequently, combinatorial immunotherapy strategies comprising Reo and T-cell engagers or checkpoint inhibitors remained effective in these settings, also after a clinically applied regimen of multiple intravenous pelareorep administrations. Altogether, our data indicate that NAbs hamper the oncolytic efficacy of Reo, but not its immunotherapeutic capacity. Given the high prevalence of seropositivity for Reo in patients with cancer, our data strongly advocate for the application of Reo as part of T cell–based immunotherapeutic strategies.

Table S1. List of antibodies used for flow cytometric analysis. Table S2. Sequences of Reo-derived peptides tested using intracellular cytokine staining. Table S3. List of primers used for RT-qPCR analysis.

Figure S3. Kinetics of neutralizing antibodies after preexposure.

Purpose: Pigmentation in uveal melanoma is associated with increased malignancy and is known as a barrier for photodynamic therapy. We investigated the role of pigmentation in tumor behavior and the response to light-activated Belzupacap sarotalocan (Bel-sar) treatment in a pigmented (wild type) and nonpigmented (tyrosinase knock-out [TYR knock-out]) cell line in vitro and in a murine model. Methods: The B16F10 (TYR knock-out) was developed using CRISPR/Cas9. After the treatment with light-activated Bel-sar, cytotoxicity and exposure of damage-associated molecular patterns (DAMPs) were measured by flow cytometry. Treated tumor cells were co-cultured with bone marrow-derived macrophages (BMDMs) and dendritic cells (DCs) to assess phagocytosis and activation. Both cell lines were injected subcutaneously in syngeneic C57BL/6 mice. Results: Knock-out of the tyrosinase gene in B16F10 led to loss of pigmentation and immature melanosomes. Pigmented tumors contained more M1 and fewer M2 macrophages compared with amelanotic tumors. Bel-sar treatment induced near complete cell death, accompanied with enhanced exposure of DAMPs in both cell lines, resulting in enhanced phagocytosis of BMDMs and maturation of DCs. Bel-sar treatment induced a shift to M1 macrophages and delayed tumor growth in both in vivo tumor models. Following treatment, especially the pigmented tumors and their draining lymph nodes contained IFN-gamma positive CD8+T cells. Conclusions: Pigmentation influenced the type of infiltrating macrophages in the tumor, with more M1 macrophages in pigmented tumors. Belzupacap sarotalocan treatment induced immunogenic cell death and tumor growth delay in pigmented as well as in nonpigmented models and stimulated M1 macrophage influx in both models.

CD3 bispecific antibody (CD3 bsAb) therapy is clinically approved for refractory hematological malignancies, but responses in solid tumors have been limited so far. One of the main hurdles in solid tumors is the lack of sufficient T-cell infiltrate. Here, we show that pre-treatment vaccination, even when composed of tumor-unrelated antigens, induces CXCR3-mediated T-cell influx in immunologically ‘cold’ tumor models in male mice. In the absence of CD3 bsAb, the infiltrate is confined to the tumor invasive margin, whereas subsequent CD3 bsAb administration induces infiltration of activated effector CD8 T cells into the tumor cell nests. This combination therapy installs a broadly inflamed Th1-type tumor microenvironment, resulting in effective tumor eradication. Multiple vaccination formulations, including synthetic long peptides and viruses, empower CD3 bsAb therapy. Our results imply that eliciting tumor infiltration with vaccine-induced tumor-(un)related T cells can greatly improve the efficacy of CD3 bsAbs in solid tumors.

Figure S2. Gating strategy used for flow cytometric analysis of immune cell composition.

Figure S8. Neutralization capacity of plasma used for the transfer to NSG mice.