ABSTRACT
Antibodies that target the tumor microenvironment can be used to deliver pro-inflammatory payloads, such as cytokines. Cytokines are small proteins able to modulate the activity of the immune system, and antibody-cytokine fusion proteins have been tested in preclinical and clinical settings. In this study, we describe Tripokin, a novel multi-specific fusion protein that combines interleukin-2 and a single amino acid mutant of tumor necrosis factor. The two pro-inflammatory payloads were fused to the L19 antibody, a clinical-grade antibody against the extradomain B of fibronectin. The human payloads were used for clinical applications, while the corresponding murine cytokines were used for preclinical studies. The resulting fusion proteins were produced in mammalian cells and purified to homogeneity. The murine Tripokin product was well tolerated in tumor-bearing mice at three doses of 30 µg in a 2-day interval and promoted rapid tumor eradication in murine models, more efficiently than single-agent immunocytokines. Tripokin induced rapid tumor necrosis and stimulated a robust immune response, impacting innate and adaptive immune pathways. In addition, the combination with immune checkpoint inhibitors further boosted the therapeutic efficacy of our molecule. Tripokin represents a promising clinical candidate for the simultaneous delivery of interleukin-2 and tumor necrosis factor to neoplastic sites.
ABSTRACT
BACKGROUND: Anti-PD-1 antibodies have revolutionized cancer immunotherapy due to their ability to induce long-lasting complete remissions in a proportion of patients. Current research efforts are attempting to identify biomarkers and suitable combination partners to predict or further improve the activity of immune checkpoint inhibitors. Antibody-cytokine fusions are a class of pharmaceuticals that showed the potential to boost the anticancer properties of other immunotherapies. Extradomain A-fibronectin (EDA-FN), which is expressed in most solid and hematological tumors but is virtually undetectable in healthy adult tissues, is an attractive target for the delivery of cytokine at the site of the disease. METHODS: In this work, we describe the generation and characterization of a novel interleukin-7-based fusion protein targeting EDA-FN termed F8(scDb)-IL7. The product consists of the F8 antibody specific to the alternatively spliced EDA of FN in the single-chain diabody (scDb) format fused to human IL-7. RESULTS: F8(scDb)-IL7 efficiently stimulates human peripheral blood mononuclear cells in vitro. Moreover, the product significantly increases the expression of T Cell Factor 1 (TCF-1) on CD8+T cells compared with an IL2-fusion protein. TCF-1 has emerged as a pivotal transcription factor that influences the durability and potency of immune responses against tumors. In preclinical cancer models, F8(scDb)-IL7 demonstrates potent single-agent activity and eradicates sarcoma lesions when combined with anti-PD-1. CONCLUSIONS: Our results provide the rationale to explore the combination of F8(scDb)-IL7 with anti-PD-1 antibodies for the treatment of patients with cancer.
Subject(s)
CD8-Positive T-Lymphocytes , Fibronectins , Interleukin-7 , Humans , Fibronectins/metabolism , Fibronectins/genetics , CD8-Positive T-Lymphocytes/immunology , CD8-Positive T-Lymphocytes/metabolism , Interleukin-7/metabolism , Interleukin-7/pharmacology , Animals , Mice , Recombinant Fusion Proteins/pharmacology , Recombinant Fusion Proteins/therapeutic use , Programmed Cell Death 1 Receptor/antagonists & inhibitors , Programmed Cell Death 1 Receptor/metabolism , Neoplasms/drug therapy , Neoplasms/immunology , Hepatocyte Nuclear Factor 1-alpha/metabolism , Immune Checkpoint Inhibitors/pharmacology , Immune Checkpoint Inhibitors/therapeutic use , Up-Regulation , Female , Cell Line, TumorABSTRACT
DNA-Encoded Libraries (DELs) allow the parallel screening of millions of compounds for various applications, including de novo discovery or affinity maturation campaigns. However, library construction and HIT resynthesis can be cumbersome, especially when library members present an unknown stereochemistry. We introduce a permutational encoding strategy suitable for the construction of highly pure single-stranded single-pharmacophore DELs, designed to distinguish isomers at the sequencing level (e.g., stereoisomers, regio-isomers, and peptide sequences). This approach was validated by synthesizing a mock 921,600-member 4-amino-proline single-stranded DEL ("DEL1"). While screening DEL1 against different targets, high-throughput sequencing results showed selective enrichment of the most potent stereoisomers, with enrichment factors that outperform conventional encoding strategies. The versatility of our methodology was additionally validated by encoding 24 scaffolds derived from different permutations of the amino acid sequence of a previously described cyclic peptide targeting Fibroblast Activation Protein (FAP-2286). The resulting library ("DEL2") was interrogated against human FAP, showing selective enrichment of five cyclic peptides. We observed a direct correlation between enrichment factors and on-DNA binding affinities. The presented encoding methodology accelerates drug discovery by facilitating library synthesis and streamlining HIT resynthesis while enhancing enrichment factors at the DEL sequencing level. This facilitates the identification of HIT candidates prior to medicinal chemistry and affinity maturation campaigns.
Subject(s)
DNA, Single-Stranded , DNA, Single-Stranded/chemistry , DNA, Single-Stranded/genetics , Gene Library , Drug Discovery/methods , Stereoisomerism , Humans , Peptides, Cyclic/chemistry , Small Molecule Libraries/chemistry , Small Molecule Libraries/pharmacology , Amino Acid SequenceABSTRACT
AIMS: Pulmonary vascular and right ventricular remodelling processes are important for development and progression of pulmonary hypertension (PH). The current study analyzed the functional role of the extra domain A containing fibronectin (ED-A+ Fn) for the development of PH by comparing ED-A+ Fn knockout (KO) and wild-type (WT) mice as well as the effects of an antibody-based therapeutical approach in a model of monocrotaline (MCT)-induced PH, which will be validated in a model of Sugen 5416/Hypoxia induced PH. METHODS AND RESULTS: PH was induced using monocrotaline (MCT) (PH mice). 69 mice were divided into the following groups: sham-treated controls (WT: n=7; KO: n=7), PH mice without specific treatment (WT: n=12; KO: n=10), PH mice treated with a dual endothelin receptor antagonist (MAC; WT: n=6; KO: n=11), WT PH mice treated with the F8 antibody, specifically recognizing ED-A+ Fn, (n=8) and WT PH mice treated with an antibody of irrelevant antigen specificity (KSF, n=8). Compared to controls, WT_PH mice showed a significant elevation of the right ventricular systolic pressure (RVPsys, p=0.04) and RV functional impairment including increased basal right ventricular (RVbasal, p=0.016) diameter or tricuspid annular plane systolic excursion (TAPSE, p=0.008). In contrast, KO PH did not show such effects compared to controls (p=n.s.). In WT_PH mice treated with F8, hemodynamic and echocardiographic parameters were significantly improved compared to untreated WT_PH mice or those treated with the KSF antibody (p<0.05). On the microscopic level, KO_PH mice showed significantly less tissue damage compared to the WT_PH mice (p=0.008). Furthermore, lung tissue damage could significantly be reduced after F8 treatment (p=0.04). Additionally, these findings could be verified in the Sugen 5416/Hypoxia mouse model, in which F8 significantly improved echocardiographic, hemodynamic and histologic parameters. CONCLUSION: ED-A+ Fn is of crucial importance for PH pathogenesis representing a promising therapeutic target in PH. We here show a novel therapeutic approach using antibody-mediated functional blockade of ED-A+ Fn capable to attenuate and partially reverse PH-associated tissue remodelling.
ABSTRACT
Fibroblast activation protein (FAP) is a protein biomarker widely expressed in most solid human malignancies of epithelial origin. In recent years, a number of FAP-targeted small organic radioligands, including OncoFAP, have been utilized in the clinic for the detection and diagnosis of cancer. Despite their selective accumulation, conventional FAP ligands present a relatively short half-life in tumors, corresponding to a few hours after systemic administration. In order to maximize their efficacy, FAP-targeted radioligand therapeutics must possess prolonged tumor retention, thus irradiating tumor cells for days. In this work, we describe the development of compact OncoFAP multimers with improved FAP affinity (low picomolar IC50s), aimed at increasing tumor-residence time for therapeutic applications. An in silico analysis of the interaction of the multimers with FAP revealed a wide and deep pocket and six additional secondary binding sites. TriOncoFAP-DOTAGA emerged for its favorable in vitro profile and superior in vivo biodistribution performance in tumor-bearing mice.
Subject(s)
Endopeptidases , Animals , Humans , Mice , Endopeptidases/metabolism , Tissue Distribution , Membrane Proteins/metabolism , Cell Line, Tumor , Serine Endopeptidases/metabolism , Gelatinases/metabolism , Female , Neoplasms/drug therapyABSTRACT
The first drugs discovered using DNA-encoded chemical library (DEL) screens have entered late-stage clinical development. However, DEL technology as a whole still suffers from poor chemical purity resulting in suboptimal performance. In this work, we report a technique to overcome this issue through self-purifying release of the DEL after magnetic bead-based synthesis. Both the first and last building blocks of each assembled library member were linked to the beads by tethers that could be cleaved by mutually orthogonal chemistry. Sequential cleavage of the first and last tether, with washing in between, ensured that the final library comprises only the fully complete compounds. The outstanding purity attained by this approach enables a direct correlation of chemical display and encoding, allows for an increased chemical reaction scope, and facilitates the use of more diversity elements while achieving greatly improved signal-to-noise ratios in selections.
Subject(s)
DNA , Drug Discovery , Small Molecule Libraries , Solid-Phase Synthesis Techniques , DNA/chemistry , Drug Discovery/methods , Small Molecule Libraries/chemistry , Small Molecule Libraries/chemical synthesis , Solid-Phase Synthesis Techniques/methodsABSTRACT
Cytokines are small proteins that modulate the activity of the immune system. Because of their potent immunomodulatory properties, some recombinant cytokines have undergone clinical development and have gained marketing authorization for the therapy of certain forms of cancer. Recombinant cytokines are typically administered at ultralow doses, as many of them can cause substantial toxicity even at submilligram quantities. In an attempt to increase the therapeutic index, fusion proteins based on tumor-homing antibodies (also called "immunocytokines") have been considered, and some products in this class have reached late-stage clinical trials. While antibody-cytokine fusions, which preferentially localize in the neoplastic mass, can activate tumor-resident leukocytes and may be more efficacious than their nontargeted counterparts, such products typically conserve an intact cytokine activity, which may prevent escalation to curative doses. To further improve tolerability, several strategies have been conceived for the development of antibody-cytokine fusions with "activity-on-demand", acting on tumors but helping spare normal tissues from undesired toxicity. In this article, we have reviewed some of the most promising strategies, outlining their potential as well as possible limitations.
Subject(s)
Cytokines , Neoplasms , Humans , Neoplasms/drug therapy , Cytokines/metabolism , Animals , Recombinant Fusion Proteins/therapeutic use , Recombinant Fusion Proteins/pharmacology , Biological Products/therapeutic use , Biological Products/pharmacologyABSTRACT
Prostate-specific membrane antigen (PSMA) is a tumor-associated protein that has been successfully targeted with small organic ligands and monoclonal antibodies. Pluvicto™ is a PSMA-targeted radioligand therapeutic (RLT) recently approved by the FDA for the treatment of metastatic castration-resistant prostate cancer (2022 FDA marketing authorization). Although a large Phase III clinical trial (VISION trial) demonstrated clinical benefits in patients treated with Pluvicto™, the therapeutic window of the drug is narrowed by its undesired accumulation in healthy organs. Glutamate carboxypeptidase III (GCPIII), an enzyme sharing 70% identity with PSMA, may be responsible for the off-target accumulation of PSMA-RLTs in salivary glands and kidneys. In this work, we designed and synthesized affinity and selectivity maturation DNA-encoded chemical libraries (ASM-DELs) comprising 18'284'658 compounds that were screened in parallel against PSMA and GCPIII with the aim to identify potent and selective PSMA ligands for tumor-targeting applications. Compound A70-B104 was isolated as the most potent and selective ligand (KD of 900 pM for PSMA, KD of 40 nM for GCPIII). 177Lu-A70-B104-DOTA, a radiolabeled derivative of compound A70-B104, presented selective accumulation in PSMA-positive cancer lesions (i.e., 7.4% ID g-1, 2 hour time point) after systemic administration in tumor-bearing mice. The results of autoradiography experiments showed that 177Lu-A70-B104-DOTA selectively binds to PSMA-positive cancer tissues, while negligible binding on human salivary glands was observed.
ABSTRACT
Prostate-specific membrane antigen (PSMA)-targeted radio ligand therapeutics (RLTs), such as [177Lu]Lu-PSMA-617 (Pluvicto), have been shown to accumulate in salivary glands and kidneys, potentially leading to undesired side effects. As unwanted accumulation in normal organs may derive from the cross-reactivity of PSMA ligands to glutamate carboxypeptidase III (GCPIII), it may be convenient to block this interaction with GCPIII-selective ligands. Parallel screening of a DNA-encoded chemical library (DEL) against GCPIII and PSMA allowed the identification of GCPIII binders. Structure-activity relationship (SAR) studies resulted in the identification of nanomolar GCPIII ligands with up to 1000-fold selectivity over PSMA. We studied the ability of GCPIII ligands to counteract the binding of [177Lu]Lu-PSMA-617 to human salivary glands by autoradiography and could demonstrate a partial radioprotection.
Subject(s)
Dipeptides , Heterocyclic Compounds, 1-Ring , Lutetium , Humans , Antigens, Surface , Autoradiography , Dipeptides/chemistry , Dipeptides/metabolism , Glutamate Carboxypeptidase II , Heterocyclic Compounds, 1-Ring/chemistry , Heterocyclic Compounds, 1-Ring/metabolism , Ligands , Lutetium/chemistry , Lutetium/metabolism , Prostate-Specific Antigen , Radioisotopes/chemistry , Radioisotopes/metabolism , Radiopharmaceuticals/chemistry , Radiopharmaceuticals/metabolism , Radiopharmaceuticals/pharmacokinetics , Salivary Glands/metabolism , Structure-Activity Relationship , Tissue DistributionABSTRACT
PURPOSE: Pluvicto™ ([177Lu]Lu-PSMA-617), a radioligand therapeutic targeting prostate-specific membrane antigen (PSMA), has been recently approved for the treatment of metastatic castration-resistant prostate cancer (mCRPR). The drug suffers from salivary gland and kidney uptake that prevents its dose escalation to potentially curative doses. In this work, we sought to potentiate the in vivo anti-cancer activity of Pluvicto™ by combining it with L19-IL2, a clinical-stage investigational medicinal product based on tumor-targeted interleukin-2. METHODS: We established a new PSMA-expressing model (HT-1080.hPSMA) and validated it using a fluoresceine analogue of PSMA-617 (compound 1). The HT-1080.hPSMA model was used to study the saturation and tumor retention of Pluvicto™ (compound 2) and to run combination therapy studies with L19-IL2. To complement our understanding of the mechanism of action of this novel combination, we conducted proteomics experiments on tumor samples after therapy with Pluvicto™ alone or in combination with the immunocytokine. RESULTS: High, selective, and long-lived tumor uptake was observed for Pluvicto™ (2) in the novel HT-1080.hPSMA model. Therapy studies in HT-1080.hPSMA tumor-bearing mice revealed that the combination of Pluvicto™ (2) plus L19-IL2 mediated curative and durable responses in all animals. Potent in vivo anti-cancer activity was observed solely for the combination modality, at doses that were well tolerated by treated animals. Proteomics studies indicated that L19-IL2 boosts the activation of the immune system in animals pre-treated with Pluvicto™. CONCLUSION: The therapeutic efficacy of Pluvicto™ at low radioactive doses can be effectively enhanced by the combination with L19-IL2. Our findings warrant further clinical exploration of this novel combination modality.
Subject(s)
Interleukin-2 , Animals , Mice , Humans , Cell Line, Tumor , Male , Heterocyclic Compounds, 1-Ring/chemistry , Heterocyclic Compounds, 1-Ring/therapeutic use , Glutamate Carboxypeptidase II/metabolism , Antineoplastic Agents/pharmacology , Antineoplastic Agents/therapeutic use , Antineoplastic Agents/pharmacokinetics , Dipeptides/therapeutic use , Dipeptides/pharmacology , Dipeptides/pharmacokinetics , Lutetium/therapeutic use , Tissue Distribution , Antigens, Surface , Prostate-Specific AntigenABSTRACT
Metastatic colorectal cancer remains a leading cause of cancer-related deaths, with a 5-year survival rate of only 15%. T cell-engaging bispecific antibodies (TCBs) represent a class of biopharmaceuticals that redirect cytotoxic T cells toward tumor cells, thereby turning immunologically "cold" tumors into "hot" ones. The carcinoembryonic antigen (CEA) is an attractive tumor-associated antigen that is overexpressed in more than 98% of patients with colorectal cancer. In this study, we report the comparison of four different TCB formats employing the antibodies F4 (targeting human CEA) and 2C11 (targeting mouse CD3ε). These formats include both antibody fragment-based and IgG-based constructs, with either one or two binding specificities of the respective antibodies. The 2 + 1 arrangement, using an anti-CEA single-chain diabody fused to an anti-CD3 single-chain variable fragment, emerged as the most potent design, showing tumor killing at subnanomolar concentrations across three different CEA+ cell lines. The in vitro activity was three times greater in C57BL/6 mouse colon adenocarcinoma cells (MC38) expressing high levels of CEA compared with those expressing low levels, highlighting the impact of CEA density in this assay. The optimal TCB candidate was tested in two different immunocompetent mouse models of colorectal cancer and showed tumor growth retardation. Ex vivo analysis of tumor infiltrates showed an increase in CD4+ and CD8+ T cells upon TCB treatment. This study suggests that bivalent tumor targeting, monovalent T-cell targeting, and a short spatial separation are promising characteristics for CEA-targeting TCBs.
Subject(s)
Antibodies, Bispecific , Carcinoembryonic Antigen , Colorectal Neoplasms , Antibodies, Bispecific/pharmacology , Antibodies, Bispecific/immunology , Colorectal Neoplasms/immunology , Colorectal Neoplasms/drug therapy , Colorectal Neoplasms/pathology , Animals , Carcinoembryonic Antigen/immunology , Humans , Mice , Cell Line, Tumor , T-Lymphocytes/immunology , CD3 Complex/immunology , Xenograft Model Antitumor Assays , Disease Models, AnimalABSTRACT
Cytokine-based therapeutics have been shown to mediate objective responses in certain tumor entities but suffer from insufficient selectivity, causing limiting toxicity which prevents dose escalation to therapeutically active regimens. The antibody-based delivery of cytokines significantly increases the therapeutic index of the corresponding payload but still suffers from side effects associated with peak concentrations of the product in blood upon intravenous administration. Here we devise a general strategy (named "Intra-Cork") to mask systemic cytokine activity without impacting anti-cancer efficacy. Our technology features the use of antibody-cytokine fusions, capable of selective localization at the neoplastic site, in combination with pathway-selective inhibitors of the cytokine signaling, which rapidly clear from the body. This strategy, exemplified with a tumor-targeted IL12 in combination with a JAK2 inhibitor, allowed to abrogate cytokine-driven toxicity without affecting therapeutic activity in a preclinical model of cancer. This approach is readily applicable in clinical practice.
Subject(s)
Cytokines , Neoplasms , Humans , Neoplasms/drug therapy , ImmunotherapyABSTRACT
Small molecule-drug conjugates (SMDCs) are increasingly considered as a therapeutic alternative to antibody-drug conjugates (ADCs) for cancer therapy. OncoFAP is an ultra-high affinity ligand of Fibroblast Activation Protein (FAP), a stromal tumor-associated antigen overexpressed in a wide variety of solid human malignancies. We have recently reported the development of non-internalizing OncoFAP-based SMDCs, which are activated by FAP thanks to selective proteolytic cleavage of the -GlyPro- linker with consequent release of monomethyl auristatin E (MMAE) in the tumor microenvironment. In this article, we describe the generation and the in vivo characterization of FAP-cleavable OncoFAP-drug conjugates based on potent topoisomerase I inhibitors (DXd, SN-38, and exatecan) and an anti-tubulin payload (MMAE), which are already exploited in clinical-stage and approved ADCs. The Glycine-Proline FAP-cleavable technology was directly benchmarked against linkers found in Adcetris™, Enhertu™, and Trodelvy™ structures by means of in vivo therapeutic experiments in mice bearing tumors with cellular or stromal FAP expression. OncoFAP-GlyPro-Exatecan and OncoFAP-GlyPro-MMAE emerged as the most efficacious anti-cancer therapeutics against FAP-positive cellular models. OncoFAP-GlyPro-MMAE exhibited a potent antitumor activity also against stromal models, and was therefore selected for clinical development.
Subject(s)
Antineoplastic Agents , Immunoconjugates , Humans , Animals , Mice , Pharmaceutical Preparations , Tubulin , Tumor Microenvironment , Immunoconjugates/therapeutic use , Immunoconjugates/chemistry , Camptothecin/therapeutic use , Cell Line, TumorABSTRACT
BACKGROUND: Pathogenesis of pulmonary hypertension (PH) is a multifactorial process driven by inflammation and pulmonary vascular remodeling. To target these two aspects of PH, we recently tested a novel treatment: Interleukin-9 (IL9) fused to F8, an antibody that binds to the extra-domain A of fibronectin (EDA+ Fn). As EDA+ Fn is not found in healthy adult tissue but is expressed during PH, IL9 is delivered specifically to the tissue affected by PH. We found that F8IL9 reduced pulmonary vascular remodeling and attenuated PH compared with sham-treated mice. PURPOSE: To evaluate possible F8IL9 effects on PH-associated inflammatory processes, we analysed the expression of genes involved in pulmonary immune responses. METHODS: We applied the monocrotaline (MCT) model of PH in mice (n = 44). Animals were divided into five experimental groups: sham-induced animals without PH (control, n = 4), MCT-induced PH without treatment (PH, n = 8), dual endothelin receptor antagonist treatment (dual ERA, n = 8), F8IL9 treatment (n = 12, 2 formats with n = 6 each), or with KSFIL9 treatment (KSFIL9, n = 12, 2 formats with n = 6 each, KSF: control antibody with irrelevant antigen specificity). After 28 days, a RT-PCR gene expression analysis of inflammatory response (84 genes) was performed in the lung. RESULTS: Compared with the controls, 19 genes exhibited relevant (+2.5-fold) upregulation in the PH group without treatment. Gene expression levels in F8IL9-treated lung tissue were reduced compared to the PH group without treatment. This was the case especially for CCL20, CXCL5, C-reactive protein, pentraxin related (CRPPR), and Kininogen-1 (KNG1). CONCLUSION: In accordance with the hypothesis stated above, F8IL9 treatment diminished the upregulation of some genes associated with inflammation in a PH animal model. Therefore, we hypothesize that IL9-based immunocytokine treatment will likely modulate various inflammatory pathways.
Subject(s)
Hypertension, Pulmonary , Interleukin-9 , Animals , Mice , Antibodies , Hypertension, Pulmonary/drug therapy , Hypertension, Pulmonary/immunology , Immunoconjugates/therapeutic use , Inflammation/drug therapy , Interleukin-9/immunology , Interleukin-9/therapeutic use , Lung , Vascular Remodeling , Disease Models, AnimalABSTRACT
Immune-stimulating antibody conjugates (ISACs) equipped with imidazoquinoline (IMD) payloads can stimulate endogenous immune cells to kill cancer cells, ultimately inducing long-lasting anticancer effects. A novel ISAC was designed, featuring the IMD Resiquimod (R848), a tumor-targeting antibody specific for Carbonic Anhydrase IX (CAIX) and the protease-cleavable Val-Cit-PABC linker. In vitro stability analysis showed not only R848 release in the presence of the protease Cathepsin B but also under acidic conditions. The ex vivo mass spectrometry-based biodistribution data confirmed the low stability of the linker-drug connection while highlighting the selective accumulation of the IgG in tumors and its long circulatory half-life.
ABSTRACT
DNA-encoded chemical libraries (DELs) are powerful drug discovery tools, enabling the parallel screening of millions of DNA-barcoded compounds. We investigated how the DEL input affects the hit discovery rate in DEL screenings. Evaluation of selection fingerprints revealed that the use of approximately 105 copies of each library member is required for the confident identification of nanomolar hits, using generally applicable methodologies.
ABSTRACT
On-target off-tumour toxicity limits the anticancer applicability of chimaeric antigen receptor (CAR) T cells. Here we show that the tumour-targeting specificity and activity of T cells with a CAR consisting of an antibody with a lysine residue that catalytically forms a reversible covalent bond with a 1,3-diketone hapten can be regulated by the concentration of a small-molecule adapter. This adapter selectively binds to the hapten and to a chosen tumour antigen via a small-molecule binder identified via a DNA-encoded library. The adapter therefore controls the formation of a covalent bond between the catalytic antibody and the hapten, as well as the tethering of the CAR T cells to the tumour cells, and hence the cytotoxicity and specificity of the cytotoxic T cells, as we show in vitro and in mice with prostate cancer xenografts. Such small-molecule switches of T-cell cytotoxicity and specificity via an antigen-independent 'universal' CAR may enhance the control and safety profile of CAR-based cellular immunotherapies.
ABSTRACT
Neutralizing monoclonal antibodies have achieved great efficacy and safety for the treatment of numerous infectious diseases. However, their neutralization potency is often rapidly lost when the target antigen mutates. Instead of isolating new antibodies each time a pathogen variant arises, it can be attractive to adapt existing antibodies, making them active against the new variant. Potential benefits of this approach include reduced development time, cost, and regulatory burden. Here a methodology is described to rapidly evolve neutralizing antibodies of proven activity, improving their function against new pathogen variants without losing efficacy against previous ones. The reported procedure is based on structure-guided affinity maturation using combinatorial mutagenesis and phage display technology. Its use against the novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is demonstrated, but it is suitable for any other pathogen. As proof of concept, the method is applied to CoV-X2, a human bispecific antibody that binds with high affinity to the early SARS-CoV-2 variants but lost neutralization potency against Delta. Antibodies emerging from the affinity maturation selection exhibit significantly improved neutralization potency against Delta and no loss of efficacy against the other viral sequences tested. These results illustrate the potential application of structure-guided affinity maturation in facilitating the rapid adaptation of neutralizing antibodies to pathogen variants.