Generation of a neutralizing antibody against RD114-pseudotyped viral vectors

The feline endogenous RD114 glycoprotein has proved to be an attractive envelope to pseudotype both retroviral and lentiviral vectors. As a surface protein, its detection on packaging cells as well as viral particles would be useful in different fields of its use. To address this, we generated a monoclonal antibody against RD114 by immunization of rats, termed 22F10. Once seroconversion was confirmed, purified 22F10 was cloned into murine Fc and characterized with a binding affinity of 10nM. The antibody was used to detect RD114 and its variant envelopes on different stable viral packaging cell lines (FLYRD18 and WinPac-RD). 22F10 was also shown to prevent the infections of different strains of RD-pseudotyped vectors but not related envelope glycoproteins by blocking cell surface receptor binding. We are the first to report the neutralization of viral particles by a monoclonal αRD114 antibody

Combining phage display with SMRTbell next-generation sequencing for the rapid discovery of functional scFv fragments

Phage display technology in combination with next-generation sequencing (NGS) currently is a state-of-the-art method for the enrichment and isolation of monoclonal antibodies from diverse libraries. However, the current NGS methods employed for sequencing phage display libraries are limited by the short contiguous read lengths associated with second-generation sequencing platforms. Consequently, the identification of antibody sequences has conventionally been restricted to individual antibody domains or to the analysis of single domain binding moieties such as camelid VHH or cartilaginous fish IgNAR antibodies. In this study, we report the application of third-generation sequencing to address this limitation. We used single molecule real time (SMRT) sequencing coupled with hairpin adaptor loop ligation to facilitate the accurate interrogation of full-length single-chain Fv (scFv) libraries. Our method facilitated the rapid isolation and testing of scFv antibodies enriched from phage display libraries within days following panning. Two libraries against CD160 and CD123 were panned and monitored by NGS. Analysis of NGS antibody data sets led to the isolation of several functional scFv antibodies that were not identified by conventional panning and screening strategies. Our approach, which combines phage display selection of immune libraries with the full-length interrogation of scFv fragments, is an easy method to discover functional antibodies, with a range of affinities and biophysical characteristics

A primer set for the rapid isolation of scFv fragments against cell surface antigens from immunised rats

Antibody phage display is a powerful platform for discovery of clinically applicable high affinity monoclonal antibodies against a broad range of targets. Libraries generated from immunized animals offer the advantage of in vivo affinity-maturation of V regions prior to library generation. Despite advantages, few studies have described isolation of antibodies from rats using immune phage display. In our study, we describe a novel primer set, covering the full rat heavy chain variable and kappa light chain variable regions repertoire for the generation of an unbiased immune libraries. Since the immune repertoire of rats is poorly understood, we first performed a deep sequencing analysis of the V(D)J regions of VH and VLK genes, demonstrating the high abundance of IGVH2 and IGVH5 families for VH and IGVLK12 and IGVLK22 for VLK. The comparison of gene’s family usage in naïve rats have been used to validate the frequency’s distribution of the primer set, confirming the absence of PCR-based biases. The primers were used to generate and assemble a phage display library from human CD160-vaccinated rats. CD160 represents a valid therapeutic target as it has been shown to be expressed on chronic lymphocytic leukaemia cells and on the surface of newly formed vessels. We utilised a novel phage display panning strategy to isolate a high affinity pool (KD range: 0.399–233 nM) of CD160 targeting monoclonal antibodies. Subsequently, identified binders were tested for function as third generation Chimeric Antigen Receptors (CAR) T cells demonstrating specific cytolytic activity. Our novel primer set coupled with a streamlined strategy for phage display panning enable the rapid isolation and identification of high affinity antibodies from immunised rats. The therapeutic utility of these antibodies was demonstrated in CAR format

Intratumoral IL-12 delivery empowers CAR-T cell immunotherapy in a pre-clinical model of glioblastoma

Glioblastoma multiforme (GBM) is the most common and aggressive form of primary brain cancer, for which effective therapies are urgently needed. Chimeric antigen receptor (CAR)-based immunotherapy represents a promising therapeutic approach, but it is often impeded by highly immunosuppressive tumor microenvironments (TME). Here, in an immunocompetent, orthotopic GBM mouse model, we show that CAR-T cells targeting tumor-specific epidermal growth factor receptor variant III (EGFRvIII) alone fail to control fully established tumors but, when combined with a single, locally delivered dose of IL-12, achieve durable anti-tumor responses. IL-12 not only boosts cytotoxicity of CAR-T cells, but also reshapes the TME, driving increased infiltration of proinflammatory CD4+ T cells, decreased numbers of regulatory T cells (Treg), and activation of the myeloid compartment. Importantly, the immunotherapy-enabling benefits of IL-12 are achieved with minimal systemic effects. Our findings thus show that local delivery of IL-12 may be an effective adjuvant for CAR-T cell therapy for GBM

Durable Responses and Low Toxicity After Fast Off-Rate CD19 Chimeric Antigen Receptor-T Therapy in Adults With Relapsed or Refractory B-Cell Acute Lymphoblastic Leukemia

PURPOSE Prognosis for adult B-cell acute lymphoblastic leukemia (B-ALL) is poor, and there are currently no licensed CD19 chimeric antigen receptor (CAR) therapeutics. We developed a novel second-generation CD19-CAR (CAT19-41BB-Z) with a fast off rate, designed for more physiologic T-cell activation to reduce toxicity and improve engraftment. We describe the multicenter phase I ALLCAR19 (NCT02935257) study of autologous CAT19-41BB-Z CAR T cells (AUTO1) in relapsed or refractory (r/r) adult B-ALL. METHODS Patients age ≥ 16 years with r/r B-ALL were eligible. Primary outcomes were toxicity and manufacturing feasibility. Secondary outcomes were depth of response at 1 and 3 months, persistence of CAR-T, incidence and duration of hypogammaglobulinemia and B-cell aplasia, and event-free survival and overall survival at 1 and 2 years. RESULTS Twenty-five patients were leukapheresed, 24 products were manufactured, and 20 patients were infused with AUTO1. The median age was 41.5 years; 25% had prior blinatumomab, 50% prior inotuzumab ozogamicin, and 65% prior allogeneic stem-cell transplantation. At the time of preconditioning, 45% had ≥ 50% bone marrow blasts. No patients experienced ≥ grade 3 cytokine release syndrome; 3 of 20 (15%) experienced grade 3 neurotoxicity that resolved to ≤ grade 1 within 72 hours with steroids. Seventeen of 20 (85%) achieved minimal residual disease–negative complete response at month 1, and 3 of 17 underwent allogeneic stem-cell transplantation while in remission. The event-free survival at 6 and 12 months was 68.3% (42.4-84.4) and 48.3% (23.1%-69.7%), respectively. High-level expansion (Cmax 127,152 copies/µg genomic DNA) and durable CAR-T persistence were observed with B-cell aplasia ongoing in 15 of 20 patients at last follow-up. CONCLUSION AUTO1 demonstrates a tolerable safety profile, high remission rates, and excellent persistence in r/r adult B-ALL. Preliminary data support further development of AUTO1 as a stand-alone treatment for r/r adult B-ALL

Targeting T-cell receptor β-constant region for immunotherapy of T-cell malignancies

Mature T cell cancers are typically aggressive, treatment resistant and associated with poor prognosis. Clinical application of immunotherapeutic approaches has been limited by a lack of target antigens that discriminate malignant from healthy (normal) T cells. Unlike B cell depletion, pan–T cell aplasia is prohibitively toxic. We report a new targeting strategy based on the mutually exclusive expression of T cell receptor β-chain constant domains 1 and 2 (TRBC1 and TRBC2). We identify an antibody with unique TRBC1 specificity and use it to demonstrate that normal and virus-specific T cell populations contain both TRBC1+ and TRBC2+ compartments, whereas malignancies are restricted to only one. As proof of concept for anti-TRBC immunotherapy, we developed anti-TRBC1 chimeric antigen receptor (CAR) T cells, which recognized and killed normal and malignant TRBC1+, but not TRBC2+, T cells in vitro and in a disseminated mouse model of leukemia. Unlike nonselective approaches targeting the entire T cell population, TRBC-targeted immunotherapy could eradicate a T cell malignancy while preserving sufficient normal T cells to maintain cellular immunity

Dual targeting of CD19 and CD22 against B-ALL using a novel high-sensitivity aCD22 CAR

CAR T cells recognizing CD19 effectively treat relapsed and refractory B-ALL and DLBCL. However, CD19 loss is a frequent cause of relapse. Simultaneously targeting a second antigen, CD22, may decrease antigen escape, but is challenging: its density is approximately 10-fold less than CD19, and its large structure may hamper immune synapse formation. The characteristics of the optimal CD22 CAR are underexplored. We generated 12 distinct CD22 antibodies and tested CARs derived from them to identify a CAR based on the novel 9A8 antibody, which was sensitive to low CD22 density and lacked tonic signaling. We found no correlation between affinity or membrane proximity of recognition epitope within Ig domains 3–6 of CD22 with CART function. The optimal strategy for CD19/CD22 CART co-targeting is undetermined. Co-administration of CD19 and CD22 CARs is costly; single CARs targeting CD19 and CD22 are challenging to construct. The co-expression of two CARs has previously been achieved using bicistronic vectors. Here, we generated a dual CART product by co-transduction with 9A8-41BBζ and CAT-41BBζ (obe-cel), the previously described CD19 CAR. CAT/9A8 CART eliminated single- and double-positive target cells in vitro and eliminated CD19- tumors in vivo. CAT/9A8 CART is being tested in a phase I clinical study (NCT02443831)