ABSTRACT
Regulatory T cells (Treg) are highly enriched within many tumors and suppress immune responses to cancer. There is intense interest in reprogramming Tregs to contribute to antitumor immunity. OX40 and CD137 are expressed highly on Tregs, activated and memory T cells, and NK cells. In this study, using a novel bispecific antibody targeting mouse OX40 and CD137 (FS120m), we show that OX40/CD137 bispecific agonism induces potent antitumor immunity partially dependent upon IFNγ production by functionally reprogrammed Tregs. Treatment of tumor-bearing animals with OX40/CD137 bispecific agonists reprograms Tregs into both fragile Foxp3+ IFNγ+ Tregs with decreased suppressive function and lineage-instable Foxp3- IFNγ+ ex-Tregs. Treg fragility is partially driven by IFNγ signaling, whereas Treg instability is associated with reduced IL2 responsiveness upon treatment with OX40/CD137 bispecific agonists. Importantly, conditional deletion of Ifng in Foxp3+ Tregs and their progeny partially reverses the antitumor efficacy of OX40/CD137 bispecific agonist therapy, revealing that reprogramming of Tregs into IFNγ-producing cells contributes to the anti-tumor efficacy of OX40/CD137 bispecific agonists. These findings provide insights into mechanisms by which bispecific agonist therapies targeting costimulatory receptors highly expressed by Tregs potentiate antitumor immunity in mouse models. SIGNIFICANCE: The bispecific antibody FS120, an immunotherapy currently being tested in the clinic, partially functions by inducing anti-tumor activity of Tregs, which results in tumor rejection.
Subject(s)
Antibodies, Bispecific , Interferon-gamma , Receptors, OX40 , T-Lymphocytes, Regulatory , Tumor Necrosis Factor Receptor Superfamily, Member 9 , Animals , T-Lymphocytes, Regulatory/drug effects , T-Lymphocytes, Regulatory/immunology , Receptors, OX40/agonists , Receptors, OX40/immunology , Mice , Tumor Necrosis Factor Receptor Superfamily, Member 9/agonists , Interferon-gamma/metabolism , Antibodies, Bispecific/pharmacology , Antibodies, Bispecific/therapeutic use , Mice, Inbred C57BL , Humans , Cell Line, Tumor , FemaleABSTRACT
The costimulatory receptor CD137 (also known as TNFRSF9 or 4-1BB) sustains effective cytotoxic T-cell responses. Agonistic anti-CD137 cancer immunotherapies are being investigated in clinical trials. Development of the first-generation CD137-agonist monotherapies utomilumab and urelumab was unsuccessful due to low antitumor efficacy mediated by the epitope recognized on CD137 or hepatotoxicity mediated by Fcγ receptors (FcγR) ligand-dependent CD137 activation, respectively. M9657 was engineered as a tetravalent bispecific antibody (mAb2) in a human IgG1 backbone with LALA mutations to reduce binding to FCγRs. Here, we report that M9657 selectively binds to mesothelin (MSLN) and CD137 with similar affinity in humans and cynomolgus monkeys. In a cellular functional assay, M9657 enhanced CD8+ T cell-mediated cytotoxicity and cytokine release in the presence of tumor cells, which was dependent on both MSLN expression and T-cell receptor/CD3 activation. Both FS122m, a murine surrogate with the same protein structure as M9657, and chimeric M9657, a modified M9657 antibody with the Fab portion replaced with an anti-murine MSLN motif, demonstrated in vivo antitumor efficacy against various tumors in wild-type and human CD137 knock-in mice, and this was accompanied by activated CD8+ T-cell infiltration in the tumor microenvironment. The antitumor immunity of M9657 and FS122m depended on MSLN expression density and the mAb2 structure. Compared with 3H3, a murine surrogate of urelumab, FS122m and chimeric M9657 displayed significantly lower on-target/off-tumor toxicity. Taken together, M9657 exhibits a promising profile for development as a tumor-targeting immune agonist with potent anticancer activity without systemic immune activation and associated hepatotoxicity.
Subject(s)
Chemical and Drug Induced Liver Injury , Neoplasms , Humans , Animals , Mice , Mesothelin , Inflammation , Tumor Necrosis Factor Receptor Superfamily, Member 9 , Tumor MicroenvironmentABSTRACT
PURPOSE: This phase 1 study (NCT03440437) evaluated the safety, tolerability, pharmacokinetics (PK), and activity of FS118, a bispecific antibody-targeting LAG-3 and PD-L1, in patients with advanced cancer resistant to anti-PD-(L)1 therapy. PATIENTS AND METHODS: Patients with solid tumors, refractory to anti-PD-(L)1-based therapy, received intravenous FS118 weekly with an accelerated dose titration design (800 µg to 0.3 mg/kg) followed by 3+3 ascending dose expansion (1 to 20 mg/kg). Primary objectives were safety, tolerability, and PK. Additional endpoints included antitumor activity, immunogenicity, and pharmacodynamics. RESULTS: Forty-three patients with a median of three prior regimens in the locally advanced/metastatic setting, including at least one anti-PD-(L)1 regimen, received FS118 monotherapy. FS118 was well tolerated, with no serious adverse events relating to FS118 reported. No dose-limiting toxicities (DLT) were observed, and an MTD was not reached. The recommended phase 2 dose of FS118 was established as 10 mg/kg weekly. The terminal half-life was 3.9 days. Immunogenicity was transient. Pharmacodynamic activity was prolonged throughout dosing as demonstrated by sustained elevation of soluble LAG-3 and increased peripheral effector cells. The overall disease control rate (DCR) was 46.5%; this disease control was observed as stable disease, except for one late partial response. Disease control of 54.8% was observed in patients receiving 1 mg/kg or greater who had acquired resistance to PD-(L)1-targeted therapy. CONCLUSIONS: FS118 was well tolerated with no DLTs observed up to and including 20 mg/kg QW. Further studies are warranted to determine clinical benefit in patients who have become refractory to anti-PD-(L)1 therapy. See related commentary by Karapetyan and Luke, p. 835.
Subject(s)
Antibodies, Bispecific , Antineoplastic Agents , Neoplasms , Humans , Interferons , B7-H1 Antigen , Neoplasms/pathology , Antineoplastic Agents/adverse effects , Antibodies, Bispecific/adverse effects , Immunotherapy , BiologyABSTRACT
Therapeutic antibodies have broad indications across diverse disease states, such as oncology, autoimmune diseases, and infectious diseases. New research continues to identify antibodies with therapeutic potential as well as methods to improve upon endogenous antibodies and to design antibodies de novo. On April 27-30, 2022, experts in antibody research across academia and industry met for the Keystone symposium "Antibodies as Drugs" to present the state-of-the-art in antibody therapeutics, repertoires and deep learning, bispecific antibodies, and engineering.
Subject(s)
Antibodies, Bispecific , Humans , Antibodies, Bispecific/therapeutic use , ImmunotherapyABSTRACT
Following the success of immune checkpoint blockade therapy against cancer, agonistic antibodies targeting T-cell costimulatory pathways are in clinical trials. The TNF superfamily of receptors (TNFRSF) members CD137 and OX40 are costimulatory receptors that stimulate T-cell proliferation and activation upon interaction with their cognate ligands. Activating CD137 and OX40 with agonistic mAbs stimulates the immune system due to their broad expression on CD4+ and CD8+ T cells and natural killer cells and has antitumor effects in preclinical models. Most TNFRSF agonist antibodies require crosslinking via Fcγ receptors (FcγR), which can limit their clinical activity. FS120 mAb2, a dual agonist bispecific antibody targeting CD137 and OX40, activated both CD4+ and CD8+ T cells in an FcγR-independent mechanism, dependent on concurrent binding. A mouse surrogate version of the bispecific antibody displayed antitumor activity in syngeneic tumor models, independent of T regulatory cell depletion and of FcγR interaction, but associated with peripheral T-cell activation and proliferation. When compared with a crosslink-independent CD137 agonist mAb, the FS120 surrogate induced lower liver T-cell infiltration. These data support initiation of clinical development of FS120, a first-in-class dual agonist bispecific antibody for the treatment of human cancer.
Subject(s)
Antibodies, Bispecific/pharmacology , Colonic Neoplasms/therapy , Killer Cells, Natural/immunology , Lymphocyte Activation/immunology , Receptors, OX40/immunology , T-Lymphocytes, Regulatory/immunology , Tumor Necrosis Factor Receptor Superfamily, Member 9/immunology , Animals , Apoptosis , Cell Proliferation , Colonic Neoplasms/immunology , Colonic Neoplasms/metabolism , Colonic Neoplasms/pathology , Female , Humans , Immunotherapy , Mice , Mice, Inbred BALB C , Tumor Cells, Cultured , Xenograft Model Antitumor AssaysABSTRACT
PURPOSE: Although programmed death-ligand 1 (PD-L1) antibody-based therapy has improved the outcome of patients with cancer, acquired resistance to these treatments limits their clinical efficacy. FS118 is a novel bispecific, tetravalent antibody (mAb2) against human lymphocyte activation gene-3 (LAG-3) and PD-L1 with the potential to reinvigorate exhausted immune cells and overcome resistance mechanisms to PD-L1 blockade. Here, using FS118 and a murine surrogate, we characterized the activity and report a novel mechanism of action of this bispecific antibody. EXPERIMENTAL DESIGN: This study characterizes the binding activity and immune function of FS118 in cell lines and human peripheral blood mononuclear cells and further investigates its antitumor activity and mechanism of action using a surrogate murine bispecific antibody (mLAG-3/PD-L1 mAb2). RESULTS: FS118 demonstrated simultaneous binding to LAG-3 and PD-L1 with high affinity and comparable or better activity than the combination of the single component parts of the mAb2 in blocking LAG-3- and PD-L1-mediated immune suppression and enhancing T-cell activity. In syngeneic tumor mouse models, mLAG-3/PD-L1 mAb2 significantly suppressed tumor growth. Mechanistic studies revealed decreased LAG-3 expression on T cells following treatment with the mouse surrogate mLAG-3/PD-L1 mAb2, whereas LAG-3 expression increased upon treatment with the combination of mAbs targeting LAG-3 and PD-L1. Moreover, following binding of mLAG-3/PD-L1 mAb2 to target-expressing cells, mouse LAG-3 is rapidly shed into the blood. CONCLUSIONS: This study demonstrates a novel benefit of the bispecific approach over a combination of mAbs and supports the further development of FS118 for the treatment of patients with cancer.
Subject(s)
Antibodies, Bispecific/pharmacology , Antigens, CD/metabolism , Antineoplastic Agents, Immunological/pharmacology , B7-H1 Antigen/antagonists & inhibitors , Lymphocyte Activation/drug effects , T-Lymphocytes/drug effects , T-Lymphocytes/metabolism , Animals , Antibody Affinity , Biomarkers, Tumor , Cell Line, Tumor , Disease Models, Animal , Humans , Immunophenotyping , Lymphocyte Activation/immunology , Mice , Protein Binding , T-Lymphocytes/immunology , Xenograft Model Antitumor Assays , Lymphocyte Activation Gene 3 ProteinABSTRACT
PURPOSE: With the increased prevalence in checkpoint therapy resistance, there remains a significant unmet need for additional therapies for patients with relapsing or refractory cancer. We have developed FS222, a bispecific tetravalent antibody targeting CD137 and PD-L1, to induce T-cell activation to eradicate tumors without the current toxicity and efficacy limitations seen in the clinic. EXPERIMENTAL DESIGN: A bispecific antibody (FS222) was developed by engineering CD137 antigen-binding sites into the Fc region of a PD-L1 IgG1 mAb. T-cell activation by FS222 was investigated using multiple in vitro assays. The antitumor efficacy, survival benefit, pharmacodynamics, and liver pharmacology of a murine surrogate molecule were assessed in syngeneic mouse tumor models. Toxicology and the pharmacokinetic/pharmacodynamic profile of FS222 were investigated in a non-human primate dose-range finding study. RESULTS: We demonstrated simultaneous binding of CD137 and PD-L1 and showed potent T-cell activation across CD8+ T-cell activation assays in a PD-L1-dependent manner with a CD137/PD-L1 bispecific antibody, FS222. FS222 also activated T cells in a human primary mixed lymphocyte reaction assay, with greater potency than the monospecific mAb combination. FS222 showed no signs of liver toxicity up to 30 mg/kg in a non-human primate dose-range finding study. A surrogate molecule caused significant tumor growth inhibition and survival benefit, concomitant with CD8+ T-cell activation, in CT26 and MC38 syngeneic mouse tumor models. CONCLUSIONS: By targeting CD137 agonism to areas of PD-L1 expression, predominantly found in the tumor microenvironment, FS222 has the potential to leverage a focused, potent, and safe immune response augmenting the PD-(L)1 axis blockade.
Subject(s)
Antibodies, Bispecific/physiology , Antineoplastic Agents, Immunological/pharmacology , B7-H1 Antigen/antagonists & inhibitors , Colorectal Neoplasms/drug therapy , Tumor Necrosis Factor Receptor Superfamily, Member 9/antagonists & inhibitors , Animals , Antibodies, Bispecific/therapeutic use , Antineoplastic Agents, Immunological/therapeutic use , Cell Line, Tumor/transplantation , Colorectal Neoplasms/immunology , Colorectal Neoplasms/pathology , Disease Models, Animal , Drug Screening Assays, Antitumor , Female , HEK293 Cells , Humans , Leukocytes, Mononuclear , Macaca fascicularis , Mice , Primary Cell Culture , Tumor Microenvironment/drug effects , Tumor Microenvironment/immunologyABSTRACT
We have used phage display to isolate a range of human domain antibodies (dAbs) that bind to mouse, rat and/or human serum albumin (SA) and can be expressed at very high levels in bacterial, yeast or mammalian cell culture. In contrast to non-SA-binding dAbs, which have terminal half-lives of less than 45 min, the half-lives of these 12 kDa 'AlbudAbs' can match the half-life of SA itself. To demonstrate the use of AlbudAbs for extending the half-lives of therapeutic drugs, we created a fusion of the interleukin-1 receptor antagonist (IL-1ra) with an AlbudAb. Soluble IL-1ra is potent inhibitor of IL-1 signalling that is approved for the treatment of rheumatoid arthritis but has a relatively short in vivo half-life. Here we show that although the AlbudAb/IL-1ra fusion has a similar in vitro potency, its in vivo efficacy can be dramatically improved due to its extended serum half-life. AlbudAbs could potentially be used to generate a range of long half-life versions of many different drugs in order to improve their dosing regimen and/or clinical effect.
Subject(s)
Albumins/chemistry , Chemistry, Pharmaceutical/methods , Drug Industry/methods , Protein Engineering/methods , Amino Acid Sequence , Animals , Collagen/chemistry , Dose-Response Relationship, Drug , Drug Design , Humans , Interleukin 1 Receptor Antagonist Protein/chemistry , Mice , Molecular Sequence Data , Peptide Library , Rats , Sequence Homology, Amino AcidABSTRACT
Antibody variable domains (domain antibodies [DAbs]) are genetically engineered antibody fragments that include individual heavy-chain (VH) or kappa-chain (Vkappa) variable domains and lack the Fc region. Human DAbs against the 65-kDa mannoprotein (MP65) or the secretory aspartyl proteinase (SAP)-2 of Candida albicans (monospecific DAbs) or against both fungal antigens (heterodimeric, bispecific DAbs) were generated from phage expression libraries. Both monospecific and bispecific DAbs inhibited fungus adherence to the epithelial cells of rat vagina and accelerated the clearance of vaginal infection with the fungus. When heterodimeric DAbs were used, the clearance of infection was at least equivalent to treatment with fluconazole. The in vivo protective effects of DAbs were demonstrated by both pre- and postchallenge schedules of DAb administration and with both fluconazole-susceptible and fluconazole-resistant strains of C. albicans. This is the first demonstration that human DAbs lacking the Fc constituent can efficiently control an infection and can act largely by inhibiting adherence.
Subject(s)
Antibodies, Fungal/immunology , Candida albicans/physiology , Candidiasis, Vulvovaginal/prevention & control , Epithelium/microbiology , Immunoglobulin Subunits/metabolism , Vagina/immunology , Animals , Aspartic Acid Endopeptidases/immunology , Candida albicans/enzymology , Candida albicans/pathogenicity , Candidiasis, Vulvovaginal/metabolism , Candidiasis, Vulvovaginal/pathology , Epithelium/pathology , Female , Fungal Proteins/immunology , Humans , Membrane Glycoproteins/immunology , Rats , Vagina/pathology , VirulenceABSTRACT
VP22, a protein of the herpes simplex virus tegument, can form complexes with fluorescein-labeled oligonucleotides. These particles, termed "Vectosomes," are efficiently taken up by cells and remain stable in the cell cytoplasm without any particular activity. Interestingly, these Vectosomes can be disrupted by light, which releases the antisense activity. Here we show that anti-c-raf1 Vectosomes are efficiently activated by light in vivo after injection into subcutaneous A549 (non-small-cell lung cancer) tumors implanted in nude mice. Moreover, two injections per week of anti-c-raf1 Vectosomes followed by illumination result in a stronger inhibition of tumor growth than injections of the antisense alone or of the different control Vectosomes. This effect correlates with a strong inhibition of the c-Raf1 protein expression. As a consequence of c-Raf1 loss, apoptosis was also detected in these tumors. Vectosomes thus represent a new powerful tool to improve the delivery of oligonucleotides in vitro and in vivo.