ABSTRACT: T-cell engaging bispecific antibodies (biAbs) can mediate potent and specific tumor cell eradication in liquid cancers. Substantial effort has been invested in expanding this concept to solid cancers. To explore their utility in the treatment of ovarian cancer, we built a set of asymmetric biAbs in IgG1-like format that bind CD3 on T cells with a conventional scFv arm and folate receptor 1 (FOLR1) on ovarian cancer cells with a conventional or a chemically programmed Fab arm. For avidity engineering, we also built an asymmetric biAb format with a tandem Fab arm. We show that both conventional and chemically programmed CD3 × FOLR1 biAbs exert specific in vitro and in vivo cytotoxicity toward FOLR1-expressing ovarian cancer cells by recruiting and activating T cells. While the conventional T-cell engaging biAb was curative in an aggressive mouse model of human ovarian cancer, the potency of the chemically programmed biAb was significantly boosted by avidity engineering. Both conventional and chemically programmed CD3 × FOLR1 biAbs warrant further investigation for ovarian cancer immunotherapy.
Project description:Chemically programmed bispecific antibodies (biAbs) endow target cell-binding small molecules with the ability to recruit and activate effector cells of the immune system. Here we report a platform of chemically programmed biAbs aimed at redirecting cytotoxic T cells to eliminate cancer cells. Two different antibody technologies were merged together to make a novel chemically programmed biAb. This was achieved by combining the humanized anti-hapten monoclonal antibody (mAb) h38C2 with the humanized anti-human CD3 mAb v9 in a clinically investigated diabody format known as Dual-Affinity Re-Targeting (DART). We show that h38C2 × v9 DARTs can readily be equipped with tumor-targeting hapten-derivatized small molecules without causing a systemic response harming healthy tissues. As a proof of concept, we chemically programmed h38C2 × v9 with hapten-folate and demonstrated its selectivity and potency against folate receptor 1 (FOLR1)-expressing ovarian cancer cells in vitro and in vivo Unlike conventional biAbs, chemically programmed biAbs in DART format are highly modular with broad utility in terms of both target and effector cell engagement. Most importantly, they provide tumor-targeting compounds access to the power of cancer immunotherapy.
Project description:Bispecific antibodies (biAbs) that mediate cytotoxicity by recruiting and activating endogenous immune cells are an emerging class of next-generation antibody therapeutics. Of particular interest are biAbs of relatively small size (?50 kDa) that can redirect cytotoxic T cells through simultaneous binding of tumor cells. Here we describe a conceptually unique class of biAbs in which the tumor cell specificity of a humanized antibody fragment that recognizes CD3 on T cells is chemically programmed through a C-terminal selenocysteine (Sec) residue. We demonstrate that through chemically programmed specificity for integrin ?(4)?(1) or folate receptor 1 (FOLR1), and common specificity for CD3, these hybrid molecules exert potent and specific in vitro and ex vivo cytotoxicity toward tumor cell lines and primary tumor cells in the presence of primary T cells. Importantly, the generic nature of chemical programming allows one to apply our approach to virtually any specificity, promising a broad utility of chemically programmed biAbs in cancer therapy.
Project description:Head and neck cancers (HNCs) are the sixth most common type of cancer in the world. Despite the development of refined surgical techniques and precise targeted radiation, patients with HNCs have a dismal prognosis. Here, we examine the expression profile of B7-H3 in HNCs and verify whether B7-H3 can serve as a novel therapeutic target for HNCs via anti-B7-H3×CD3 bispecific antibodies (biAbs). We analyzed the expression level of B7-H3 in 274 HNC samples and evaluated the association between B7-H3 expression and clinicopathological parameters. Anti-B7-H3×CD3 biAbs were constructed, and the efficacy of these biAbs in targeting HNCs was assessed in vitro and in vivo. As a result, high expression of B7-H3 was detected in 66.1% of clinical HNC samples and was correlated with poor survival. Specific antitumor effects of anti-B7-H3×CD3 biAbs were confirmed in vitro using HNC cell lines. In xenograft HNC mouse model, anti-B7-H3×CD3 biAbs delayed tumor growth and prolonged survival. In conclusion, B7-H3 is frequently overexpressed in HNCs and could be a promising therapeutic target for biAb therapy.
Project description:CD47 serves as an anti-phagocytic receptor that is upregulated by cancer to promote immune escape. As such, CD47 is the focus of intense immuno-oncology drug development efforts. However, as CD47 is expressed ubiquitously, clinical development of conventional drugs, e.g., monoclonal antibodies, is confronted with patient safety issues and poor pharmacology due to the widespread CD47 "antigen sink". A potential solution is tumor-directed blockade of CD47, which can be achieved with bispecific antibodies (biAbs). Using mouse CD47-blocking biAbs in a syngeneic tumor model allowed us to evaluate the efficacy of tumor-directed blockade of CD47 in the presence of the CD47 antigen sink and a functional adaptive immune system. We show here that CD47-targeting biAbs inhibited tumor growth in vivo, promoting durable antitumor responses and stimulating CD8+ T cell activation in vitro. In vivo efficacy of the biAbs could be further enhanced when combined with chemotherapy or PD-1/PD-L1 immune checkpoint blockade. We also show that selectivity and pharmacological properties of the biAb are dependent on the affinity of the anti-CD47 arm. Taken together, our study validates the approach to use CD47-blocking biAbs either as a monotherapy or part of a multi-drug approach to enhance antitumor immunity.
Project description:Bispecific antibodies (BiAbs) offer a unique opportunity to redirect immune effector cells to kill cancer cells. BiAbs combine the benefits of different binding specificities of two monoclonal antibodies (mAbs) into a single construct. This unique feature of BiAbs enables approaches that are not possible with single mAbs. Advances in antibody engineering and antigen profiling of malignant cells have led to the development of a number of BiAb formats and their combinations for redirecting effector cells to tumor targets. There have been significant advances in the design and application of BiAbs for intravenous and local injection.The initial barrier of cytokine storm has been partially overcome by more recent constructs that have improved clinical effectiveness without dose-limiting toxicities. Since the recent revival of BiAbs, there has been multiple, ongoing, phase I/II and III trials, and some promising clinical outcomes have been reported in completed clinical studies. This review focuses on arming T cells with BiAbs to create the 'poor man's cytotoxic lymphocyte'.
Project description:Passive transfer of broadly neutralizing human antibodies against HIV-1 protects macaques against infection. However, HIV-1 uses several strategies to escape antibody neutralization, including mutation of the gp160 viral surface spike, a glycan shield to block antibody access to the spike, and expression of a limited number of viral surface spikes, which interferes with bivalent antibody binding. The latter is thought to decrease antibody apparent affinity or avidity, thereby interfering with neutralizing activity. To test the idea that increasing apparent affinity might enhance neutralizing activity, we engineered bispecific anti-HIV-1 antibodies (BiAbs) that can bind bivalently by virtue of one scFv arm that binds to gp120 and a second arm to the gp41 subunit of gp160. The individual arms of the BiAbs preserved the binding specificities of the original anti-HIV IgG antibodies and together bound simultaneously to gp120 and gp41. Heterotypic bivalent binding enhanced neutralization compared with the parental antibodies. We conclude that antibody recognition and viral neutralization of HIV can be improved by heteroligation.
Project description:Antibodies to the cysteine-rich domain II of Plasmodium vivax Duffy binding protein (PvDBP) can inhibit binding of this parasite ligand to its receptor on red blood cells, the Duffy antigen/receptor for chemokines. These binding-inhibitory antibodies (BIAbs) also inhibit P. vivax invasion of reticulocytes in vitro.To investigate whether naturally acquired anti-PvDBP antibodies are associated with reduced risk of clinical malaria in a population exposed to low levels of P. vivax transmission, we measured total levels of immunoglobulin G antibodies to 5 PvDBP variants and used a functional in vitro assay to quantify their binding-inhibitory activity in a cohort of 466 rural Amazonians followed up for up to 37 months.No association between total immunoglobulin G antibody responses to any PvDBP variant and risk of symptomatic, laboratory-confirmed vivax malaria was observed in this cohort. However, a Cox proportional hazards model, adjusted for age, sex, and genotype for the Duffy antigen/receptor for chemokines, showed a >40% decrease in the prospective risk of clinical vivax malaria in subjects with the strongest BIAb responses (upper and middle terciles). High BIAb responses were mostly PvDBP variant transcending and stable over time.Strong naturally acquired BIAb responses are associated with a reduced risk of clinical P. vivax malaria in rural Amazonians.
Project description:T-cell bispecific antibodies (TCBs) crosslink tumor and T-cells to induce tumor cell killing. While TCBs are very potent, on-target off-tumor toxicity remains a challenge when selecting targets. Here, we describe a protease-activated anti-folate receptor 1?TCB (Prot-FOLR1-TCB) equipped with an anti-idiotypic anti-CD3 mask connected to the anti-CD3 Fab through a tumor protease-cleavable linker. The potency of this Prot- FOLR1-TCB is recovered following protease-cleavage of the linker releasing the anti-idiotypic anti-CD3 scFv. In vivo, the Prot-FOLR1-TCB mediates antitumor efficacy comparable to the parental FOLR1-TCB whereas a noncleavable control Prot-FOLR1-TCB is inactive. In contrast, killing of bronchial epithelial and renal cortical cells with low FOLR1 expression is prevented compared to the parental FOLR1-TCB. The findings are confirmed for mesothelin as alternative tumor antigen. Thus, masking the anti-CD3 Fab fragment with an anti-idiotypic mask and cleavage of the mask by tumor-specific proteases can be applied to enhance specificity and safety of TCBs.
Project description:Targeting cancer antigens by T cell-engaging bispecific antibody (BiAb) or chimeric antigen receptor T cell therapy has achieved successes in hematological cancers, but attempts to use it to fight solid cancers have been disappointing, in part due to antigen escape. MEK inhibitor had limited activity as a single agent, but enhanced antitumor activity when combined with other therapies, such as targeted drugs or immunotherapy agents. This study aimed to analyze the expression of B7-H3 in non-small-cell lung cancer (NSCLC) and bladder cancer (BC) and to evaluate the combinatorial antitumor effect of B7-H3 × CD3 BiAb with MEK inhibitor trametinib. We found B7-H3 was highly expressed in NSCLC and BC compared with normal samples and its increased expression was associated with poor prognosis. Treatment with trametinib alone could induce apoptosis in tumor cell, while has no effect on T cell proliferation, and a noticeable elevation of B7-H3 expression in tumor cells was also observed following treatment. B7-H3 × CD3 BiAb specifically and efficiently redirected their cytotoxicity against B7-H3 overexpressing tumor cells both in vitro and in xenograft mouse models. While trametinib treatment alone affected tumor growth, the combined therapy increased T cell infiltration and significantly suppressed tumor growth. Together, these data suggest that combination therapy with B7-H3 × CD3 BiAb and MEK inhibitor may serve as a new therapeutic strategy in the future clinical practice for the treatment of NSCLC and BC.
Project description:In this retrospective study we evaluated the respective correlations and clinical relevance of FOLR1 mRNA expression, FOLR1 promoter specific methylation and global DNA hypomethylation in type I and type II ovarian cancer.Two hundred fifty four ovarian cancers, 13 borderline tumours and 60 samples of healthy fallopian epithelium and normal ovarian epithelium were retrospectively analysed for FOLR1 expression with RT-PCR. FOLR1 DNA promoter methylation and global DNA hypomethylation (measured by means of LINE1 DNA hypomethylation) were evaluated with MethyLight technique.No correlation between FOLR1 mRNA expression and its specific promoter DNA methylation was found neither in type I nor in type II cancers, however, high FOLR1 mRNA expression was found to be correlated with global DNA hypomethylation in type II cancers (p = 0.033). Strong FOLR1 mRNA expression was revealed for Grades 2-3, FIGO stages III-IV, residual disease > 0, and serous histotype. High FOLR1 expression was found to predict increased platinum sensitivity in type I cancers (odds ratio = 3.288; 1.256-10.75; p = 0.020). One-year survival analysis showed in type I cancers an independent better outcome for strong expression of FOLR1 in FIGO stage III and IV. For the entire follow up period no significant independent outcome for FOLR1 expression was revealed. In type I cancers LINE 1 DNA hypomethylation was found to exhibit a worse PFS and OS which were confirmed to be independent in multivariate COX regression model for both PFS (p = 0.026) and OS (p = 0.012).No correlations were found between FOLR1 expression and its specific promoter methylation, however, high FOLR1 mRNA expression was associated with DNA hypomethylation in type II cancers. FOLR1 mRNA expression did not prove to predict clinical outcome in type II cancers, although strong FOLR1 expression generally denotes ovarian cancers with highly aggressive phenotype. In type I cancers, however, strong FOLR1 expression has been found to be a reliable indicator of improved platinum responsiveness reflecting a transient better one-year follow up outcome in highly FOLR1 expressing type I cancers. An independent prognostic role of global DNA hypomethylation was demonstrated in type I tumours.