Sortase-Mediated Site-Specific Conjugation to Prepare Fluorine-18-Labeled Nanobodies.

Single-domain antibodies, or nanobodies (Nbs), are promising biomolecules for use in molecular imaging due to their excellent affinity, specificity, and fast clearance from the blood. Given their short blood half-life, pairing Nbs with short-lived imaging radioisotopes is desirable. Because fluorine-18 (18F) is routinely used for clinical imaging, it is an attractive radioisotope for Nbs. We report a novel sortase-based, site-specific 18F-labeling method applied to three nanobodies. Labeled nanobodies were synthesized either by a two-step indirect radiolabeling method in one pot or by a one-step direct labeling method using a sortase-mediated conjugation of either the radiolabeled chelator (H-GGGK((±)-Al[18F]FH3RESCA)-NH2) or the unlabeled chelator (H-GGGK((±)-H3RESCA)-NH2) followed by labeling with Al[18F]F, respectively. The overall radiochemical yields were 15-43% (n = 22, decay-corrected) in 70 min (indirect labeling) and 23-58% (n = 12, decay-corrected) in 50 min (direct labeling). The radiochemical purities of the labeled nanobodies prepared by both methods were >98% with a specific activity of 400-600 Ci/mmol (n = 22) for each of the three Nbs tested and exhibited excellent stability profiles under physiological conditions. This simple, site-specific, reproducible, and generalizable 18F-labeling method to prepare nanobodies (Nb-Al[18F]F-RESCA) or other low molecular weight biomolecules can easily be adopted in various settings for preclinical and clinical studies.

rhIL-7-hyFc, a long-acting interleukin-7, improves efficacy of CAR-T cell therapy in solid tumors.

BACKGROUND: Chimeric antigen receptor T-cell (CAR-T) therapy has achieved remarkable remission in patients with B-cell malignancies. However, its efficacy in treating solid tumors remains limited. Here, we investigated a combination therapy approach using an engineered long-acting interleukin (IL)-7 (rhIL-7-hyFc or NT-I7) and CAR-T cells targeting three antigens, glypican-2 (GPC2), glypican-3 (GPC3), and mesothelin (MSLN), against multiple solid tumor types including liver cancer, neuroblastoma, ovarian cancer, and pancreatic cancer in mice. METHODS: CAR-T cells targeting GPC2, GPC3, and MSLN were used in combination with NT-I7 to assess the anticancer activity. Xenograft tumor models, including the liver cancer orthotopic model, were established using NOD scid gamma mice engrafted with cell lines derived from hepatocellular carcinoma, neuroblastoma, ovarian cancer, and pancreatic cancer. The mice were monitored by bioluminescence in vivo tumor imaging and tumor volume measurement using a caliper. Immunophenotyping of CAR-T cells on NT-I7 stimulation was evaluated for memory markers, exhaust markers, and T-cell signaling molecules by flow cytometry and western blotting. RESULTS: Compared with the IL-2 combination, preclinical evaluation of NT-I7 exhibited regression of solid tumors via enhanced occupancy of CD4+ CAR-T, improved T-cell expansion, reduced exhaustion markers (programmed cell death protein 1 or PD-1 and lymphocyte-activation gene 3 or LAG-3) expression, and increased generation of stem cell-like memory CAR-T cells. The STAT5 pathway was demonstrated to be downstream of NT-I7 signaling, mediated by increased expression of the IL-7 receptor expression in CAR-T cells. Furthermore, CAR-T cells improved efficacy against tumors with low antigen density when combined with NT-I7 in mice, presenting an avenue for patients with heterogeneous antigenic profiles. CONCLUSION: This study provides a rationale for NT-I7 plus CAR-T cell combination therapy for solid tumors in humans.

Cancer therapy with antibodies.

The greatest challenge in cancer therapy is to eradicate cancer cells with minimal damage to normal cells. Targeted therapy has been developed to meet that challenge, showing a substantially increased therapeutic index compared with conventional cancer therapies. Antibodies are important members of the family of targeted therapeutic agents because of their extraordinarily high specificity to the target antigens. Therapeutic antibodies use a range of mechanisms that directly or indirectly kill the cancer cells. Early antibodies were developed to directly antagonize targets on cancer cells. This was followed by advancements in linker technologies that allowed the production of antibody-drug conjugates (ADCs) that guide cytotoxic payloads to the cancer cells. Improvement in our understanding of the biology of T cells led to the production of immune checkpoint-inhibiting antibodies that indirectly kill the cancer cells through activation of the T cells. Even more recently, bispecific antibodies were synthetically designed to redirect the T cells of a patient to kill the cancer cells. In this Review, we summarize the different approaches used by therapeutic antibodies to target cancer cells. We discuss their mechanisms of action, the structural basis for target specificity, clinical applications and the ongoing research to improve efficacy and reduce toxicity.

Cleavage-intermediate Lassa virus trimer elicits neutralizing responses, identifies neutralizing nanobodies, and reveals an apex-situated site-of-vulnerability.

Lassa virus (LASV) infection is expanding outside its traditionally endemic areas in West Africa, posing a pandemic biothreat. LASV-neutralizing antibodies, moreover, have proven difficult to elicit. To gain insight into LASV neutralization, here we develop a prefusion-stabilized LASV glycoprotein trimer (GPC), pan it against phage libraries comprising single-domain antibodies (nanobodies) from shark and camel, and identify one, D5, which neutralizes LASV. Cryo-EM analyses reveal D5 to recognize a cleavage-dependent site-of-vulnerability at the trimer apex. The recognized site appears specific to GPC intermediates, with protomers lacking full cleavage between GP1 and GP2 subunits. Guinea pig immunizations with the prefusion-stabilized cleavage-intermediate LASV GPC, first as trimer and then as a nanoparticle, induce neutralizing responses, targeting multiple epitopes including that of D5; we identify a neutralizing antibody (GP23) from the immunized guinea pigs. Collectively, our findings define a prefusion-stabilized GPC trimer, reveal an apex-situated site-of-vulnerability, and demonstrate elicitation of LASV-neutralizing responses by a cleavage-intermediate LASV trimer.

Cooperative Armoring of CAR and TCR T Cells by T Cell-Restricted IL15 and IL21 Universally Enhances Solid Tumor Efficacy.

PURPOSE: Chimeric antigen receptor (CAR) and T-cell receptor (TCR) T-cell therapies are effective in a subset of patients with solid tumors, but new approaches are needed to universally improve patient outcomes. Here, we developed a technology to leverage the cooperative effects of IL15 and IL21, two common cytokine-receptor gamma chain family members with distinct, pleiotropic effects on T cells and other lymphocytes, to enhance the efficacy of adoptive T cells. EXPERIMENTAL DESIGN: We designed vectors that induce the constitutive expression of either membrane-tethered IL15, IL21, or IL15/IL21. We used clinically relevant preclinical models of transgenic CARs and TCRs against pediatric and adult solid tumors to determine the effect of the membrane-tethered cytokines on engineered T cells for human administration. RESULTS: We found that self-delivery of these cytokines by CAR or TCR T cells prevents functional exhaustion by repeated stimulation and limits the emergence of dysfunctional natural killer (NK)-like T cells. Across different preclinical murine solid tumor models, we observed enhanced regression with each individual cytokine but the greatest antitumor efficacy when T cells were armored with both. CONCLUSIONS: The coexpression of membrane-tethered IL15 and IL21 represents a technology to enhance the resilience and function of engineered T cells against solid tumors and could be applicable to multiple therapy platforms and diseases. See related commentary by Ruffin et al., p. 1431.

Modular chimeric cytokine receptors with leucine zippers enhance the antitumour activity of CAR T cells via JAK/STAT signalling.

The limited availability of cytokines in solid tumours hinders maintenance of the antitumour activity of chimeric antigen receptor (CAR) T cells. Cytokine receptor signalling pathways in CAR T cells can be activated by transgenic expression or injection of cytokines in the tumour, or by engineering the activation of cognate cytokine receptors. However, these strategies are constrained by toxicity arising from the activation of bystander cells, by the suboptimal biodistribution of the cytokines and by downregulation of the cognate receptor. Here we show that replacement of the extracellular domains of heterodimeric cytokine receptors in T cells with two leucine zipper motifs provides optimal Janus kinase/signal transducer and activator of transcription signalling. Such chimeric cytokine receptors, which can be generated for common γ-chain receptors, interleukin-10 and -12 receptors, enabled T cells to survive cytokine starvation without induction of autonomous cell growth, and augmented the effector function of CAR T cells in vitro in the setting of chronic antigen exposure and in human tumour xenografts in mice. As a modular design, leucine zippers can be used to generate constitutively active cytokine receptors in effector immune cells.

Camel nanobody-based B7-H3 CAR-T cells show high efficacy against large solid tumours.

Rational design of chimeric antigen receptor T (CAR-T) cells based on the recognition of antigenic epitopes capable of evoking the most potent CAR activation is an important objective in optimizing immune therapy. In solid tumors, the B7-H3 transmembrane protein is an emerging target that harbours two distinct epitope motifs, IgC and IgV, in its ectodomain. Here, we generate dromedary camel nanobodies targeting B7-H3 and demonstrate that CAR-T cells, based on the nanobodies recognizing the IgC but not IgV domain, had potent antitumour activity against large tumors in female mice. These CAR-T cells are characterized by highly activated T cell signaling and significant tumor infiltration. Single-cell transcriptome RNA sequencing coupled with functional T-cell proteomics analysis uncovers the top-upregulated genes that might be critical for the persistence of polyfunctional CAR-T cells in mice. Our results highlight the importance of the specific target antigen epitope in governing optimal CAR-T activity and provide a nanobody-based B7-H3 CAR-T product for use in solid tumor therapy.

Identification of nurse shark VNAR single-domain antibodies targeting the spike S2 subunit of SARS-CoV-2.

SARS-CoV-2 is the etiological agent of the COVID-19 pandemic. Antibody-based therapeutics targeting the spike protein, specifically the S1 subunit or the receptor binding domain (RBD) of SARS-CoV-2, have gained attention due to their clinical efficacy in treating patients diagnosed with COVID-19. An alternative to conventional antibody therapeutics is the use of shark new antigen variable receptor domain (VNAR ) antibodies. VNAR s are small (<15 kDa) and can reach deep into the pockets or grooves of the target antigen. Here, we have isolated 53 VNAR s that bind to the S2 subunit by phage panning from a naïve nurse shark VNAR phage display library constructed in our laboratory. Among those binders, S2A9 showed the best neutralization activity against the original pseudotyped SARS-CoV-2 virus. Several binders, including S2A9, showed cross-reactivity against S2 subunits from other ß coronaviruses. Furthermore, S2A9 showed neutralization activity against all variants of concern (VOCs) from alpha to omicron (including BA1, BA2, BA4, and BA5) in both pseudovirus and live virus neutralization assays. Our findings suggest that S2A9 could be a promising lead molecule for the development of broadly neutralizing antibodies against SARS-CoV-2 and emerging variants. The nurse shark VNAR phage library offers a novel platform that can be used to rapidly isolate single-domain antibodies against emerging viral pathogens.

The IgG4 hinge with CD28 transmembrane domain improves VHH-based CAR T cells targeting a membrane-distal epitope of GPC1 in pancreatic cancer.

Heterogeneous antigen expression is a key barrier influencing the activity of chimeric antigen receptor (CAR) T cells in solid tumors. Here, we develop CAR T cells targeting glypican-1 (GPC1), an oncofetal antigen expressed in pancreatic cancer. We report the generation of dromedary camel VHH nanobody (D4)-based CAR T cells targeting GPC1 and the optimization of the hinge (H) and transmembrane domain (TM) to improve activity. We find that a structurally rigid IgG4H and CD28TM domain brings the two D4 fragments in proximity, driving CAR dimerization and leading to enhanced T-cell signaling and tumor regression in pancreatic cancer models with low antigen density in female mice. Furthermore, single-cell-based proteomic and transcriptomic analysis of D4-IgG4H-CD28TM CAR T cells reveals specific genes (e.g., HMGB1) associated with high T-cell polyfunctionality. This study demonstrates the potential of VHH-based CAR T for pancreatic cancer therapy and provides an engineering strategy for developing potent CAR T cells targeting membrane-distal epitopes.

Site-Specifically Conjugated Single-Domain Antibody Successfully Identifies Glypican-3-Expressing Liver Cancer by Immuno-PET.

Primary liver cancer is the third leading cause of cancer-related deaths, and its incidence and mortality are increasing worldwide. Hepatocellular carcinoma (HCC) accounts for 80% of primary liver cancer cases. Glypican-3 (GPC3) is a heparan sulfate proteoglycan that histopathologically defines HCC and represents an attractive tumor-selective marker for radiopharmaceutical imaging and therapy for this disease. Single-domain antibodies are a promising scaffold for imaging because of their favorable pharmacokinetic properties, good tumor penetration, and renal clearance. Although conventional lysine-directed bioconjugation can be used to yield conjugates for radiolabeling full-length antibodies, this stochastic approach risks negatively affecting target binding of the smaller single-domain antibodies. To address this challenge, site-specific approaches have been explored. Here, we used conventional and sortase-based site-specific conjugation methods to engineer GPC3-specific human single-domain antibody (HN3) PET probes. Methods: Bifunctional deferoxamine (DFO) isothiocyanate was used to synthesize native HN3 (nHN3)-DFO. Site-specifically modified HN3 (ssHN3)-DFO was engineered using sortase-mediated conjugation of triglycine-DFO chelator and HN3 containing an LPETG C-terminal tag. Both conjugates were radiolabeled with 89Zr, and their binding affinity in vitro and target engagement of GPC3-positive (GPC3+) tumors in vivo were determined. Results: Both 89Zr-ssHN3 and 89Zr-nHN3 displayed nanomolar affinity for GPC3 in vitro. Biodistribution and PET/CT image analysis in mice bearing isogenic A431 and A431-GPC3+ xenografts, as well as in HepG2 liver cancer xenografts, showed that both conjugates specifically identify GPC3+ tumors. 89Zr-ssHN3 exhibited more favorable biodistribution and pharmacokinetic properties, including higher tumor uptake and lower liver accumulation. Comparative PET/CT studies on mice imaged with both 18F-FDG and 89Zr-ssHN3 showed more consistent tumor accumulation for the single-domain antibody conjugate, further establishing its potential for PET imaging. Conclusion: 89Zr-ssHN3 showed clear advantages in tumor uptake and tumor-to-liver signal ratio over the conventionally modified 89Zr-nHN3 in xenograft models. Our results establish the potential of HN3-based single-domain antibody probes for GPC3-directed PET imaging of liver cancers.

Soluble programmed death ligand-1-induced immunosuppressive effects on chimeric antigen receptor-natural killer cells targeting Glypican-3 in hepatocellular carcinoma.

Although the pre-clinical study of chimeric antigen receptor (CAR)-natural killer (NK) cell was effective against various tumours, immunosuppression mediated by tumour microenvironment hampers their application and several efforts have been explored to improve their effect in combating solid tumours. Glypican 3 (GPC3) is a promising target for hepatocellular carcinoma (HCC), and CAR-T cells targeting GPC3 have been tested in clinical trials. Based on an affinity-enhanced antibody (hYP7) targeting GPC3, we constructed GPC3-CAR-NK cells to explore their potential function in the treatment of HCC. We found that patients with HCC secreted high levels of soluble programmed death-ligand 1 (sPD-L1), which inhibits the function of CAR-NK cells targeting GPC3. In addition, we combined high-affinity sPD-L1 variant (L3C7c-Fc) with GPC3-CAR-NK cells to solve the problem of GPC3-CAR-NK inhibition. Our studies demonstrated that L3C7c-Fc could enhance the therapeutic effect of CAR-NK cells by reversing the suppression of sPD-L1, which provides the experimental evidence for the subsequent development of HCC immunotherapy strategies.

Preclinical optimization of a GPC2-targeting CAR T-cell therapy for neuroblastoma.

BACKGROUND: Although most patients with newly diagnosed high-risk neuroblastoma (NB) achieve remission after initial therapy, more than 50% experience late relapses caused by minimal residual disease (MRD) and succumb to their cancer. Therapeutic strategies to target MRD may benefit these children. We developed a new chimeric antigen receptor (CAR) targeting glypican-2 (GPC2) and conducted iterative preclinical engineering of the CAR structure to maximize its anti-tumor efficacy before clinical translation. METHODS: We evaluated different GPC2-CAR constructs by measuring the CAR activity in vitro. NOD-SCID mice engrafted orthotopically with human NB cell lines or patient-derived xenografts and treated with human CAR T cells served as in vivo models. Mechanistic studies were performed using single-cell RNA-sequencing. RESULTS: Applying stringent in vitro assays and orthotopic in vivo NB models, we demonstrated that our single-chain variable fragment, CT3, integrated into a CAR vector with a CD28 hinge, CD28 transmembrane, and 4-1BB co-stimulatory domain (CT3.28H.BBζ) elicits the best preclinical anti-NB activity compared with other tested CAR constructs. This enhanced activity was associated with an enrichment of CD8+ effector T cells in the tumor-microenvironment and upregulation of several effector molecules such as GNLY, GZMB, ZNF683, and HMGN2. Finally, we also showed that the CT3.28H.BBζ CAR we developed was more potent than a recently clinically tested GD2-targeted CAR to control NB growth in vivo. CONCLUSION: Given the robust preclinical activity of CT3.28H.BBζ, these results form a promising basis for further clinical testing in children with NB.

Human VH-based chimeric antigen receptor T cells targeting glypican 3 eliminate tumors in preclinical models of HCC.

BACKGROUND AND AIMS: Efficacy of chimeric antigen receptor (CAR) T cells for treating solid tumors, including HCC, remains a challenge. Nanobodies are emerging building blocks of CAR T cells due to their small size and high expression. Membrane proximal sites have been shown as attractive epitopes of CAR T cells. However, current CAR formats are not tailored toward nanobodies or targeting membrane distal epitopes. APPROACH AND RESULTS: Using hYP7 Fv (membrane proximal) and HN3 VH nanobody (membrane distal) as GPC3 targeting elements, we sought to determine how hinges and transmembrane portions of varying structures and sizes affect CAR T-cell function. We generated multiple permutations of CAR T cells containing CD8, CD28, IgG4, and Fc domains. We show that engineered HN3 CAR T cells can be improved by 2 independent, synergistic changes in the hinge and transmembrane domains. The T cells expressing the HN3 CAR which contains the hinge region of IgG4 and the CD28 transmembrane domain (HN3-IgG4H-CD28TM) exhibited high cytotoxic activity and caused complete HCC tumor eradication in immunodeficient mice. HN3-IgG4H-CD28TM CAR T cells were enriched for cytotoxic-memory CD8+ T cells and NFAT signals, and reduced ß catenin levels in HCC cells. CONCLUSION: Our findings indicate that altering the hinge and transmembrane domains of a nanobody-based CAR targeting a distal GPC3 epitope, in contrast to a membrane proximal epitope, lead to robust T-cell signaling and induce swift and durable eradication of HCC tumors.

Humanization of the Shark VNAR Single Domain Antibody Using CDR Grafting.

The variable domain of the new antigen receptor (VNAR ) of shark single domain antibodies is evolutionarily distant from the variable regions (VH ) of mammalian immunoglobulins, yet it still has complementarity-determining regions (CDRs) that are involved in antigen recognition, therefore making it possible to humanize by grafting these CDRs to the framework of human VH homologs. Here, we show the VNAR CDR based on an analysis of currently available VNAR -antigen structure complexes in the global Protein Data Bank archive of 3D structure data, and describe the detailed protocol to humanize VNAR by CDR grafting, using B6 (an anti-Pseudomonas exotoxin VNAR ), the most common type (Type II) of shark VNAR s, as an example. Ongoing efforts will further optimize the protocol for moving shark VNAR s to the clinic for treating cancer and other human diseases. Published 2023. This article is a U.S. Government work and is in the public domain in the USA. Basic Protocol: Humanize shark VNAR sequence by CDR grafting Support Protocol 1: VNAR structure prediction and comparison Support Protocol 2: Measure binding kinetics of humanized VNAR using bio-layer interferometry (BLI).

The emergence of AntibodyPlus: the future trend of antibody-based therapeutics.

To date, close to 100 canonical monoclonal antibody drugs have been approved by the FDA; furthermore, a number of antibody-derived therapeutics in nontraditional formats have reached late development stages and the market, and many more are being evaluated in early-stage development. To better reflect this trend and to set up a framework for forward thinking, we herein introduce the concept of AntibodyPlus, embracing any therapeutics with an antibody component. AntibodyPlus therapeutics contain effector modules, in the form of small molecules, nucleic acids, proteins or even cells, to enhance their therapeutic activities against cancer, virus infection and other diseases. In this short review, we discuss historic perspective and current status of therapeutic antibody development, and the scope and categories of AntibodyPlus therapeutics along with their advantages, applications and challenges. We also present several examples that highlight their design principles, potentials and future trends.

Immunotherapy for hepatobiliary cancers: Emerging targets and translational advances.

Over the past several decades, primary liver cancer (PLC), mostly hepatocellular carcinoma (HCC) and intrahepatic cholangiocarcinoma (iCCA), has become the focus of rising concern mainly due to the increasing rates of incidence and high global mortality. Immunotherapy, as an emerging treatment approach, represents an effective and promising option against PLC. However, the selection of immunotherapeutic targets while considering tumor heterogeneity and immunosuppressive tumor microenvironment is a major challenge. The purpose of this review is to summarize and present the emerging immunotherapeutic targets for HCC and iCCA and to evaluate their translation advances in currently ongoing clinical trials. To better provide a framework for the liver cancer target selection, this chapter will highlight cell surface antigens expressed in both tumor cells and immune cells. Particular focus will be on the development, biology and function of Glypican-3 (GPC3) and Mesothelin (MSLN) in the cancer progress of HCC and iCCA, respectively. By doing so, we will explore the prospects and applications of various immunotherapeutic strategies such as vaccines, monoclonal antibodies, immunotoxins, antibody-drug conjugates (ADCs) and chimeric antigen receptors (CARs) T cells that have been developed targeting GPC3 and MSLN.

An optimized bicistronic chimeric antigen receptor against GPC2 or CD276 overcomes heterogeneous expression in neuroblastoma.

Chimeric antigen receptor (CAR) T cell therapies targeting single antigens have performed poorly in clinical trials for solid tumors due to heterogenous expression of tumor-associated antigens (TAAs), limited T cell persistence, and T cell exhaustion. Here, we aimed to identify optimal CARs against glypican 2 (GPC2) or CD276 (B7-H3), which were highly but heterogeneously expressed in neuroblastoma (NB), a lethal extracranial solid tumor of childhood. First, we examined CAR T cell expansion in the presence of targets by digital droplet PCR. Next, using pooled competitive optimization of CAR by cellular indexing of transcriptomes and epitopes by sequencing (CITE-Seq), termed P-COCC, we simultaneously analyzed protein and transcriptome expression of CAR T cells to identify high-activity CARs. Finally, we performed cytotoxicity assays to identify the most effective CAR against each target and combined the CARs into a bicistronic "OR" CAR (BiCisCAR). BiCisCAR T cells effectively eliminated tumor cells expressing GPC2 or CD276. Furthermore, the BiCisCAR T cells demonstrated prolonged persistence and resistance to exhaustion when compared with CARs targeting a single antigen. This study illustrated that targeting multiple TAAs with BiCisCAR may overcome heterogenous expression of target antigens in solid tumors and identified a potent, clinically relevant CAR against NB. Moreover, our multimodal approach integrating competitive expansion, P-COCC, and cytotoxicity assays is an effective strategy to identify potent CARs among a pool of candidates.

Production and Purification of Shark and Camel Single-Domain Antibodies from Bacterial and Mammalian Cell Expression Systems.

Single-domain antibodies, including the antigen-binding variable domains of the shark immunoglobulin new antigen receptor and the camelid variable region of the heavy chain, are the smallest antigen recognition domains (∼15 kDa) and have unique characteristics compared to conventional antibodies. They are capable of binding epitopes that are hard to access for classical antibodies and can also be used for therapeutics or diagnostics or as modular building blocks for multi-domain constructs, antibody-drug conjugates, immunotoxins, or chimeric antigen receptor therapy. This article contains detailed procedures for the purification and validation of two single-domain antibodies (one shark and one camel), which bind to the S2 subunit of the SARS-CoV-2 spike protein, using both bacterial and mammalian cell expression systems. It provides a comprehensive reference for the production of single-domain antibodies with high yield, good quality, and purity. © Published 2022. This article is a U.S. Government work and is in the public domain in the USA. Basic Protocol: Production of single-domain antibodies from Escherichia coli Alternate Protocol: Production of single-domain antibodies using the mammalian cell line Expi293F Support Protocol 1: Production and purification of single-domain antibodies on a small scale with the polymyxin B method Support Protocol 2: Validation of single-domain antibodies by ELISA.

Highly active CAR T cells that bind to a juxtamembrane region of mesothelin and are not blocked by shed mesothelin.

SignificanceMesothelin (MSLN) is a cell-surface protein that is a popular target for antibody-based therapies. We have identified shed MSLN as a major obstacle to successful antibody therapies and prepared a monoclonal antibody that inhibits shedding and makes very active CAR T cells whose activity is not blocked by shed MSLN and merits further preclinical development.

Development of Highly Effective Anti-Mesothelin hYP218 Chimeric Antigen Receptor T Cells With Increased Tumor Infiltration and Persistence for Treating Solid Tumors.

Mesothelin targeting CAR T cells have limited activity in patients. In this study, we sought to determine if efficacy of anti-mesothelin CAR T cells is dependent on the mesothelin epitopes that are recognized by them. To do so, we developed hYP218 (against membrane-proximal epitope) and SS1 (against membrane-distal epitope) CAR T cells. Their efficacy was assessed in vitro using mesothelin-positive tumor cell lines and in vivo in NSG mice with mesothelin-expressing ovarian cancer (OVCAR-8), pancreatic cancer (KLM-1), and mesothelioma patient-derived (NCI-Meso63) tumor xenografts. Persistence and tumor infiltration of CAR T cells was determined using flow cytometry. hYP218 CAR T cells killed cancer cells more efficiently than SS1 CAR T cells, with a two- to fourfold lower ET50 value (effector-to-target ratio for 50% killing of tumor cells). In mice with established tumors, single intravenous administration of hYP218 CAR T cells lead to improved tumor response and survival compared with SS1 CAR T cells, with complete regression of OVCAR-8 and NCI-Meso63 tumors. Compared with SS1 CAR T cells, there was increased peripheral blood expansion, persistence, and tumor infiltration of hYP218 CAR T cells in the KLM-1 tumor model. Persistence of hYP218 CAR T cells in treated mice led to antitumor immunity when rechallenged with KLM-1 tumor cells. Our results show that hYP218 CAR T cells, targeting mesothelin epitope close to cell membrane, are very effective against mesothelin-positive tumors and are associated with increased persistence and tumor infiltration. These results support its clinical development to treat patients with mesothelin-expressing cancers.