Tumor burden limits bispecific antibody efficacy through T cell exhaustion averted by concurrent cytotoxic therapy.
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ABSTRACT:
SUBMITTER: Meermeier EW
PROVIDER: S-EPMC8266040 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Outcome in high-risk patients with refractory or relapsed germ cell tumours (GCT) remains poor. Novel strategies enhancing therapeutic efficacy whilst limiting therapeutic burden are warranted, yet immunotherapy approaches geared towards activating endogenous antitumor responses have not been successful thus far. Redirection of cytotoxic effector cells by bispecific antibodies represents a promising approach in this setting. We demonstrate that the Epithelial Cell Adhesion Molecule (EpCAM) is broadly expressed in GCT cell lines of different histologic origin including seminoma, choriocarcinoma (CHC), and embryonal carcinoma (EC). In these GCT lines of variable EpCAM surface expression, targeting T cells by the prototypic bispecific EpCAM/CD3-antibody (bAb) Catumaxomab together with natural killer (NK) cell engagement via the Fc domain promotes profound cytotoxicity across a broad range of antibody dilutions. In contrast, tumor cell lysis mediated by either immune cell subset alone is influenced by surface density of the target antigen. In the CHC line JAR, NK cell-dependent cytotoxicity dominates, which may be attributed to differential surface expression of immunomodulatory proteins such as MHC-I, CD24, and Fas receptors on CHC and EC. In view of redirecting T cell therapy mediated by bispecific antibodies, such differences in GCT immunophenotype potentially favoring immune escape are worth further investigation.
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Project description:Among different cancer immunotherapy approaches, bispecific antibodies (BsAbs) are of great interest due to their ability to recruit immune cells to kill tumor cells directly. Various BsAbs against Her2 tumor cells have been proposed with potent cytotoxic activities. However, most of these formats require extensive processing to obtain heterodimeric bispecific antibodies. In this study, we describe a bispecific antibody, BiHC (bispecific Her2-CD3 antibody), constructed with a single-domain anti-Her2 and a single-chain Fv (variable fragment) of anti-CD3 in an IgG-like format. In contrast to most IgG-like BsAbs, the two arms in BiHC have different molecular weights, making it easier to separate hetero- or homodimers. BiHC can be expressed in Escherichia coli and purified via Protein A affinity chromatography. The purified BiHC can recruit T cells and induce specific cytotoxicity of Her2-expressing tumor cells in vitro. The BiHC can also efficiently inhibit the tumor growth in vivo. Thus, BiHC is a promising candidate for the treatment of Her2-positive cancers.
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Project description:Bispecific antibodies (bsAb) that bridge tumor cells and CD3-positive effector T cells are being developed against many tumor cell targets. While tumor cell factors other than target expression level appear to play a role in determining the efficacy of CD3 bsAb, the identity of such factors remains largely unknown. Using a co-culture system of primary human T cells and B lymphoma cell lines, we demonstrate a range of sensitivities to CD20xCD3 bsAb that is independent of CD20 surface expression. To identify genes that modulate tumor cell sensitivity to CD3 bsAb, we employed a genome-scale CRISPR activation screen in a CD20xCD3-sensitive human B lymphoma cell line. Among the most highly enriched sgRNAs were those targeting genes with predicted effects on cell-cell adhesion, including sialophorin (SPN). Increased expression of SPN impeded tumor cell clustering with T cells, thereby limiting CD3 bsAb-mediated tumor cell lysis. This inhibitory effect of SPN appeared to be dependent on sialylated core 2 O-glycosylation of the protein. While SPN is not endogenously expressed in the majority of B cell lymphomas, it is highly expressed in acute myeloid leukemia. CRISPR-mediated SPN knockout in AML cell lines facilitated T cell-tumor cell clustering and enhanced CD3 bsAb-mediated AML cell lysis. In sum, our data establish that the cell cross-linking mechanism of CD3 bsAb is susceptible to subversion by anti-adhesive molecules expressed on the tumor cell surface. Further evaluation of anti-adhesive pathways may provide novel biomarkers of clinical response and enable the development of effective combination regimens for this promising therapeutic class.
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Project description:Activation of the programmed cell death protein 1 and programmed cell death ligand 1 (PD-1/PD-L1) signaling axis plays important roles in intrinsic or acquired resistance to human epidermal growth factor receptor 2 (HER2)-directed therapies in the clinic. Therefore, therapies simultaneously targeting both HER2 and PD-1/PD-L1 signaling pathways are of great significance. Here, aiming to direct the anti-PD-L1 responses toward HER2-expressing tumor cells, we constructed a humanized bispecific IgG1 subclass antibody targeting both HER2 and PD-L1 (HER2/PD-L1; BsAb), which displayed satisfactory purity, thermostability, and serum stability. We found that BsAb showed enhanced antibody-dependent cell-mediated cytotoxicity (ADCC) activity in vitro. In the late phase of peripheral blood mononuclear cell (PBMC)-humanized HER2+ tumor xenograft models, BsAb showed superior therapeutic efficacies as compared with monoclonal antibodies (mAbs) or combination treatment strategies. In cynomolgus monkeys, BsAb showed favorable pharmacokinetics and toxicity profiles when administered at a 10 mg/kg dosage. Thus, HER2/PD-L1 BsAb was demonstrated as a potentially effective option for managing HER2+ and trastuzumab-resistant tumors in the clinic. We propose that the enhanced antitumor activities of BsAb in vivo may be due to direct inhibition of HER2 signaling or activation of T cells.
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Project description:Tumor necrosis factor (TNF) and interleukin (IL)-17A are pleiotropic cytokines implicated in the pathogenesis of several autoimmune diseases including rheumatoid arthritis (RA) and psoriatic arthritis (PsA). JNJ-61178104 is a novel human anti-TNF and anti-IL-17A monovalent, bispecific antibody that binds to both human TNF and human IL-17A with high affinities and blocks the binding of TNF and IL-17A to their receptors in vitro. JNJ-61178104 also potently neutralizes TNF and IL-17A-mediated downstream effects in multiple cell-based assays. In vivo, treatment with JNJ-61178104 resulted in dose-dependent inhibition of cellular influx in a human IL-17A/TNF-induced murine lung neutrophilia model and the inhibitory effects of JNJ-61178104 were more potent than the treatment with bivalent parental anti-TNF or anti-IL-17A antibodies. JNJ-61178104 was shown to engage its targets, TNF and IL-17A, in systemic circulation measured as drug/target complex formation in normal cynomolgus monkeys (cyno). Surprisingly, quantitative target engagement assessment suggested lower apparent in vivo target-binding affinities for JNJ-61178104 compared to its bivalent parental antibodies, despite their similar in vitro target-binding affinities. The target engagement profiles of JNJ-61178104 in humans were in general agreement with the predicted profiles based on cyno data, suggesting similar differences in the apparent in vivo target-binding affinities. These findings show that in vivo target engagement of monovalent bispecific antibody does not necessarily recapitulate that of the molar-equivalent dose of its bivalent parental antibody. Our results also offer valuable insights into the understanding of the pharmacokinetics/pharmacodynamics and target engagement of other bispecific biologics against dimeric and/or trimeric soluble targets in vivo.
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Project description:Tumor-targeted antibody therapy has had a major impact on reducing morbidity and mortality in a wide range of cancers. Antibodies mediate their antitumor activity in part by activating immune effector cells; however, the tumor microenvironment (TME) is enriched with cellular and soluble mediators that actively suppress generation of antitumor immunity. Here, we investigate the potential of prospectively identifying and neutralizing an immunomodulatory soluble mediator within the TME to enhance therapeutic efficacy of the HER2-directed antibody trastuzumab. Using the D5-HER2 cell line and an immunocompetent human HER2 transgenic animal (hmHER2Tg) in which human HER2 is a self-antigen, we determined that IL4 was present in the TME and produced by both tumor and stromal cells. A siRNA-based screening approach identified STAT5A as a novel negative regulator of IL4 production by D5-HER2 tumor cells. Furthermore, IL4 neutralization using the anti-IL4 antibody 11B11 enhanced the efficacy of trastuzumab and modulated the TME. For example, IL4 neutralization resulted in reduced levels of myeloid chemoattractants CCL2, CCL11, and CXCL5 in the TME. Combination therapy with 11B11 and trastuzumab resulted in a reduction of tumor-infiltrating CD11b(+)CD206(+) myeloid cells compared with monotherapy. These data suggest that IL4 neutralization enhances the efficacy of trastuzumab by influencing the phenotype of myeloid cells within the TME and provide further rationale for combining tumor-targeted antibody therapy with agents that neutralize factors in the TME that suppress generation of productive antitumor immune responses.
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Project description:Bispecific antibodies (BsAbs) are a sort of dual functional proteins with specific binding to two distinct targets, which have become a focus of interest in antibody engineering and drug development research and have a promising future for wide applications in cancer immunotherapy and autoimmune disease. The key of clinical application and commercial-scale manufacturing of BsAbs is the amenability to assembly and purification of desired heterodimers. Advances in genetic engineering technology had resulted in the development of diverse BsAbs. Multiple recombinant strategies have been used to solve the mispairing problem between light and heavy chains, as well as to enforce accurate dimerization of heterologous heavy chains. There are 23 platforms available to generate 62 BsAbs which can be further divided into IgG-like ones and fragment-based ones, and more than 50 molecules are undergoing clinical trials currently. BsAbs with IgG-like architecture exhibit superior advantages in structure (similar to natural antibodies), pharmacokinetics, half-life, FcR-mediated function, and biological activity. This review considers various IgG-like BsAb generation approaches, summarizes the clinical applications of promising new BsAbs, and describes the mechanism of BsAbs in tumor therapy.
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Project description:The CD20 mAb ofatumumab (OFA) induces complement-mediated lysis of B cells. In an investigator-initiated phase II trial of OFA plus chemotherapy for chronic lymphocytic leukemia (CLL), OFA treatment promoted partial CLL B cell depletion that coincided with reduced complement titers. Remaining CLL B cells circulated with bound OFA and covalently bound complement breakdown product C3d, indicative of ongoing complement activation. Presumably, neither complement- nor effector cell-based mechanisms were sufficiently robust to clear these remaining B cells. Instead, almost all of the bound OFA and CD20 was removed from the cells, in accordance with previous clinical studies that demonstrated comparable loss of CD20 from B cells after treatment of CLL patients with rituximab. In vitro experiments with OFA and rituximab addressing these observations suggest that host effector mechanisms that support mAb-mediated lysis and tumor cell clearance are finite, and they can be saturated or exhausted at high B cell burdens, particularly at high mAb concentrations. Interestingly, only a fraction of available complement was required to kill cells with CD20 mAbs, and killing could be tuned by titrating the mAb concentration. Consequently, maximal B cell killing of an initial and secondary B cell challenge was achieved with intermediate mAb concentrations, whereas high concentrations promoted lower overall killing. Therefore, mAb therapies that rely substantially on effector mechanisms subject to exhaustion, including complement, may benefit from lower, more frequent dosing schemes optimized to sustain and maximize killing by cytotoxic immune effector systems.
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Project description:T-cell-recruiting bispecific antibody therapy has yielded promising results in patients with hematologic malignancies, however, resistance and subsequent relapse remains a major challenge. T-cell exhaustion induced by persistent antigen stimulation or tonic receptor signaling has been reported to compromise outcomes of T-cell based immunotherapies. The impact of continuous exposure to bispecifics on T-cell function, however, remains poorly understood. In relapsed/refractory B-cell precursor acute lymphoblastic leukemia patients, 28-day continuous infusion with the CD19xCD3 bispecific antibody blinatumomab led to declining T-cell function. In an in vitro model system, mimicking 28-day continuous infusion with the half-life-extended CD19xCD3 bispecific AMG 562, we identified hallmark features of exhaustion arising over time. Continuous AMG 562 exposure induced progressive loss of T-cell function (day 7 vs day 28 mean specific lysis: 88.4% vs 8.6%; n = 6; p = .0003). Treatment-free intervals (TFIs), achieved by AMG 562 withdrawal, were identified as powerful strategy for counteracting exhaustion. TFIs induced strong functional reinvigoration of T cells (continuous vs TFI specific lysis on day 14: 34.9% vs 93.4%; ± SEM; n = 6; p < .0001) and transcriptional reprogramming. Furthermore, use of a TFI led to improved T-cell expansion and tumor control in vivo. Our data demonstrate the relevance of T-cell exhaustion in bispecific antibody therapy and highlight that T cells can be functionally and transcriptionally rejuvenated with TFIs. In view of the growing number of bispecific molecules being evaluated in clinical trials, our findings emphasize the need to consider and evaluate TFIs in application schedules to improve clinical outcomes.
2022-07-26 | GSE196463 | GEO
Project description:Anti-programmed cell death-1 (PD-1)/PD-ligand-1 (PD-L1) treatments are effective in a fraction of patients with advanced malignancies. However, the majority of patients do not respond to it. Resistance to cancer immunotherapy can be mediated by additional immune checkpoints. We hypothesized that co-targeting of PD-L1 and lymphocyte-activation gene 3 (LAG-3) could provide an alternative therapeutic approach. Here, we developed IBI323, a dual blockade bispecific antibody targeting PD-L1 and LAG-3. We assessed the binding affinity, blocking activity, cell bridging effect, and immunomodulation function of IBI323 using in vitro assays. We also evaluated, in two humanized mouse models, anti-tumor effects and antitumor T cell immunity induced by IBI323. IBI323 bound to PD-L1 and LAG-3 with similar potency as its parental antibodies and blocked the interaction of PD-1/PD-L1, CD80/PD-L1, and LAG-3/MHC-II. Moreover, IBI323 mediated the bridging of PD-L1+ cells and LAG-3+ cells and demonstrated superior immune stimulatory activity compared to each parent antibody in mixed leukocyte reaction. In PD-L1/LAG-3 double knock-in mice bearing human PD-L1 knock-in MC38 tumors, IBI323 showed stronger anti-tumor activity compared to each parental antibody. The better antitumor response correlated with increased tumor-specific CD8+ and CD4+ T cells. IBI323 also induced stronger anti-tumor effect against established A375 tumors compared with combination in mice reconstituted with human immune cells. Collectively, these data demonstrated that IBI323 preserved the blockade activities of parental antibodies while processing a novel cell bridging function. Based on the encouraging preclinical results, IBI323 has significant value in further clinical development.
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