Asymmetric anti-CLL-1×CD3 bispecific antibody, ABL602 2+1, with attenuated CD3 affinity endows potent antitumor activity but limited cytokine release.

BACKGROUND: Acute myeloid leukemia (AML) is a type of leukemia in adults with a high mortality rate and poor prognosis. Although targeted therapeutics, chemotherapy, and hematopoietic stem cell transplantation can improve the prognosis, the recurrence rate is still high, with a 5-year survival rate of approximately 40%. This study aimed to develop an IgG-based asymmetric bispecific antibody that targets CLL-1 and CD3 for treating AML. METHODS: ABL602 candidates were compared in terms of binding activity, T-cell activation, and tumor-killing activities. ABL602-mediated T-cell activation and tumor-killing activities were determined by measuring the expression of activation markers, cytokines, cytolytic proteins, and the proportion of dead cells. We evaluated in vivo tumor growth inhibitory activity in two mouse models bearing subcutaneously and orthotopically engrafted human AML. Direct tumor-killing activity and T-cell activation in patient-derived AML blasts were also evaluated. RESULTS: ABL602 2+1 showed a limited CD3 binding in the absence of CLL-1, suggesting that steric hindrance on the CD3 binding arm could reduce CLL-1 expression-independent CD3 binding. Although the CD3 binding activity was attenuated compared with that of 1+1, ABL602 2+1 exhibited much stronger T-cell activation and potent tumor-killing activities in AML cell lines. ABL602 2+1 efficiently inhibited tumor progression in subcutaneously and orthotopically engrafted AML mouse models. In the orthotopic mouse model, tumor growth inhibition was observed by gross measurement of luciferase activity, as well as a reduced proportion of AML blasts in the bone marrow, as determined by flow cytometry and immunohistochemistry (IHC) staining. ABL602 2+1 efficiently activated T cells and induced the lysis of AML blasts, even at very low effector:target (E:T) ratios (eg, 1:50). Compared with the reference 1+1 antibody, ABL602 did not induce the release of cytokines including interleukin-6 and tumor necrosis factor-α in the healthy donor-derived peripheral blood mononuclear cell. CONCLUSIONS: With its potent tumor-killing activity and reduced cytokine release, ABL602 2+1 is a promising candidate for treating patients with AML and warrants further study.

LAG-3xPD-L1 bispecific antibody potentiates antitumor responses of T cells through dendritic cell activation.

Several preclinical studies demonstrate that antitumor efficacy of programmed cell death-1 (PD-1)/programmed death-ligand 1 (PD-L1) blockade can be improved by combination with other checkpoint inhibitors. Lymphocyte-activation gene 3 (LAG-3) is an inhibitory checkpoint receptor involved in T cell exhaustion and tumor immune escape. Here, we describe ABL501, a bispecific antibody targeting LAG-3 and PD-L1 in modulating immune cell responses against tumors. ABL501 that efficiently inhibits both LAG-3 and PD-L1 pathways enhances the activation of effector CD4+ and CD8+ T cells with a higher degree than a combination of single anti-LAG-3 and anti-PD-L1. The augmented effector T cell responses by ABL501 resulted in mitigating regulatory-T-cell-mediated immunosuppression. Mechanistically, the simultaneous binding of ABL501 to LAG-3 and PD-L1 promotes dendritic cell (DC) activation and tumor cell conjugation with T cells that subsequently mounts effective CD8+ T cell responses. ABL501 demonstrates its potent in vivo antitumor efficacy in a humanized xenograft model and with knockin mice expressing human orthologs. The immune profiling analysis of peripheral blood reveals an increased abundance of LAG-3hiPD-1hi memory CD4+ T cell subset in relapsed cholangiocarcinoma patients after gemcitabine plus cisplatin therapy, which are more responsive to ABL501. This study supports the clinical evaluation of ABL501 as a novel cancer immunotherapeutic, and a first-in-human trial has started (NCT05101109).

Novel anti-4-1BB×PD-L1 bispecific antibody augments anti-tumor immunity through tumor-directed T-cell activation and checkpoint blockade.

BACKGROUND: Stimulation of 4-1BB with agonistic antibodies is a promising strategy for improving the therapeutic efficacy of immune checkpoint inhibitors (ICIs) or for overcoming resistance to ICIs. However, dose-dependent hepatotoxicity was observed in clinical trials with monoclonal anti-4-1BB agonistic antibodies due to the activation of 4-1BB signaling in liver resident Kupffer cells. METHODS: To avoid this on-target liver toxicity, we developed a novel bispecific antibody (4-1BB×PD-L1 bispecific antibody, termed "ABL503") uniquely designed to activate 4-1BB signaling only in the context of PD-L1, while also blocking PD-1/PD-L1 signaling. RESULTS: Functional evaluation using effector cells expressing both 4-1BB and PD-1 revealed superior biological activity of ABL503 compared with the combination of each monoclonal antibody. ABL503 also augmented T-cell activation in in vitro assays and further enhanced the anti-PD-L1-mediated reinvigoration of tumor-infiltrating CD8+ T cells from patients with cancer. Furthermore, in humanized PD-L1/4-1BB transgenic mice challenged with huPD-L1-expressing tumor cells, ABL503 induced superior anti-tumor activity and maintained an anti-tumor response against tumor rechallenge. ABL503 was well tolerated, with normal liver function in monkeys. CONCLUSION: The novel anti-4-1BB×PD-L1 bispecific antibody may exert a strong anti-tumor therapeutic efficacy with a low risk of liver toxicity through the restriction of 4-1BB stimulation in tumors.

N-terminal selective conjugation method widens the therapeutic window of antibody-drug conjugates by improving tolerability and stability.

Antibody-drug conjugates (ADCs) are targeted therapeutic agents that treat cancers by selective delivery of highly potent cytotoxic drugs to tumor cells via cancer-specific antibodies. However, their clinical benefit is limited by off-target toxicity and narrow therapeutic windows. To overcome these limitations, we have applied reductive alkylation to develop a new type of ADC that has cytotoxic drugs conjugated to the N-terminal of an antibody through amine bonds introduced via reductive alkylation reactions (NTERM). To test whether the NTERM-conjugated ADCs can widen therapeutic windows, we synthesized three different ADCs by conjugating trastuzumab and monomethyl auristatin-F using three different methods, and compared their stability, efficacy, and toxicity. The NTERM-conjugated ADC was more stable in vitro and in vivo than the thiol-conjugated and the lysine-conjugated ADCs. The NTERM-conjugated ADC showed lower toxicity compared to other ADCs, whereas its efficacy was comparable to that of the thiol-conjugated ADC and better than that of the lysine-conjugated ADC. These results suggest that the NTERM conjugation method could widen the therapeutic window of ADCs by enhancing its stability and reducing toxicity.

B7-H3×4-1BB bispecific antibody augments antitumor immunity by enhancing terminally differentiated CD8+ tumor-infiltrating lymphocytes.

Cancer immunotherapy with 4-1BB agonists has limited further clinical development because of dose-limiting toxicity. Here, we developed a bispecific antibody (bsAb; B7-H3×4-1BB), targeting human B7-H3 (hB7-H3) and mouse or human 4-1BB, to restrict the 4-1BB stimulation in tumors. B7-H3×m4-1BB elicited a 4-1BB-dependent antitumor response in hB7-H3-overexpressing tumor models without systemic toxicity. BsAb primarily targets CD8 T cells in the tumor and increases their proliferation and cytokine production. Among the CD8 T cell population in the tumor, 4-1BB is solely expressed on PD-1+Tim-3+ "terminally differentiated" subset, and bsAb potentiates these cells for eliminating the tumor. Furthermore, the combination of bsAb and PD-1 blockade synergistically inhibits tumor growth accompanied by further increasing terminally differentiated CD8 T cells. B7-H3×h4-1BB also shows antitumor activity in h4-1BB-expressing mice. Our data suggest that B7-H3×4-1BB is an effective and safe therapeutic agent against B7-H3-positive cancers as monotherapy and combination therapy with PD-1 blockade.

Development, validation, and application of ELISA for detection of anti-HD105 antibodies in pre-clinical safety evaluation using monkeys.

Unwanted immunogenicity of protein therapeutics can result in severe side effects and should be assessed in animals before applying the treatment to humans. Monkeys are the most relevant choice for pre-clinical toxicity testing of antibody-based therapeutics. To assess the immunogenicity of HD105, a novel antibody therapeutic that targets both vascular endothelial growth factor and Delta-like-ligand 4, a bridging enzyme-linked immunosorbent assay was developed as an anti-drug antibody (ADA) assay and validated for use in pre-clinical studies using non-human primates. This method was found to have suitable assay sensitivity, intra- and inter-assay precision, confirmation, drug tolerance, recovery, and sample stability for measuring ADA in monkey serum samples. The results showed that ADA elevation occurred following repeated doses of HD105, and that ADA production was negatively associated with serum HD105 concentration. These results suggest that intravenous administration of HD105 induces production of ADA in monkeys and that the detection of ADA may be negatively influenced by free HD105 in serum.

Silibinin sensitizes human glioma cells to TRAIL-mediated apoptosis via DR5 up-regulation and down-regulation of c-FLIP and survivin.

Silibinin, a flavonoid isolated from Silybum marianum, has been reported to have cancer chemopreventive and therapeutic effects. Here, we show that treatment with subtoxic doses of silibinin in combination with tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) induces rapid apoptosis in TRAIL-resistant glioma cells, but not in human astrocytes, suggesting that this combined treatment may offer an attractive strategy for safely treating gliomas. Although the proteolytic processing of procaspase-3 by TRAIL was partially blocked in glioma cells, cotreatment with silibinin efficiently recovered TRAIL-induced caspase activation in these cells. Silibinin treatment up-regulated DR5, a death receptor of TRAIL, in a transcription factor CHOP-dependent manner. Furthermore, treatment with silibinin down-regulated the protein levels of the antiapoptotic proteins FLIP(L), FLIP(S), and survivin through proteasome-mediated degradation. Taken together, our results show that the activity of silibinin to modulate multiple components in the death receptor-mediated apoptotic pathway is responsible for its ability to recover TRAIL sensitivity in TRAIL-resistant glioma cells.