Off-the-shelf Vδ1 gamma delta T cells engineered with glypican-3 (GPC-3)-specific chimeric antigen receptor (CAR) and soluble IL-15 display robust antitumor efficacy against hepatocellular carcinoma.

BACKGROUND: Glypican-3 (GPC-3) is an oncofetal protein that is highly expressed in various solid tumors, but rarely expressed in healthy adult tissues and represents a rational target of particular relevance in hepatocellular carcinoma (HCC). Autologous chimeric antigen receptor (CAR) αß T cell therapies have established significant clinical benefit in hematologic malignancies, although efficacy in solid tumors has been limited due to several challenges including T cell homing, target antigen heterogeneity, and immunosuppressive tumor microenvironments. Gamma delta (γδ) T cells are highly cytolytic effectors that can recognize and kill tumor cells through major histocompatibility complex (MHC)-independent antigens upregulated under stress. The Vδ1 subset is preferentially localized in peripheral tissue and engineering with CARs to further enhance intrinsic antitumor activity represents an attractive approach to overcome challenges for conventional T cell therapies in solid tumors. Allogeneic Vδ1 CAR T cell therapy may also overcome other hurdles faced by allogeneic αß T cell therapy, including graft-versus-host disease (GvHD). METHODS: We developed the first example of allogeneic CAR Vδ1 T cells that have been expanded from peripheral blood mononuclear cells (PBMCs) and genetically modified to express a 4-1BB/CD3z CAR against GPC-3. The CAR construct (GPC-3.CAR/secreted interleukin-15 (sIL)-15) additionally encodes a constitutively-secreted form of IL-15, which we hypothesized could sustain proliferation and antitumor activity of intratumoral Vδ1 T cells expressing GPC-3.CAR. RESULTS: GPC-3.CAR/sIL-15 Vδ1 T cells expanded from PBMCs on average 20,000-fold and routinely reached >80% purity. Expanded Vδ1 T cells showed a primarily naïve-like memory phenotype with limited exhaustion marker expression and displayed robust in vitro proliferation, cytokine production, and cytotoxic activity against HCC cell lines expressing low (PLC/PRF/5) and high (HepG2) GPC-3 levels. In a subcutaneous HepG2 mouse model in immunodeficient NSG mice, GPC-3.CAR/sIL-15 Vδ1 T cells primarily accumulated and proliferated in the tumor, and a single dose efficiently controlled tumor growth without evidence of xenogeneic GvHD. Importantly, compared with GPC-3.CAR Vδ1 T cells lacking sIL-15, GPC-3.CAR/sIL-15 Vδ1 T cells displayed greater proliferation and resulted in enhanced therapeutic activity. CONCLUSIONS: Expanded Vδ1 T cells engineered with a GPC-3 CAR and sIL-15 represent a promising platform warranting further clinical evaluation as an off-the-shelf treatment of HCC and potentially other GPC-3-expressing solid tumors.

Binding Rate Screen - a high-throughput assay in soluble lysate for prioritizing protein expression constructs.

Identification of constructs suitable for the recombinant protein production pipeline is a bottleneck for structural genomics efforts, as most methods require purified proteins and/or are labor-intensive. Here, we present a novel high-throughput approach, Binding Rate Screen, that can alleviate this bottleneck by screening expression constructs in crude soluble lysate. This functional screen utilizes the frequently employed hexahistidine (His(6)) tag as a reporter, and measures its binding rate to an affinity matrix as a metric to reflect aggregation, concentration, and purifiability of the target protein. The constructs with the highest binding rates also exhibit high expression of soluble monomeric protein as judged by analytical size-exclusion chromatography. Constructs expressing variations of the target protein can be prioritized on a time scale of minutes, which is at least 10-100 times faster than any other technologies currently available.

Immunotherapy using purified protein derivative in the treatment of warts: An open uncontrolled trial.

BACKGROUND: Warts are known to clear spontaneously with the development of cell-mediated immunity (CMI) to the virus. Purified protein derivative (PPD) of tuberculin bacilli has been used as a non-specific stimulant of CMI to achieve this outcome. AIM: To study the effect of PPD in the treatment of warts. METHODS: Patients with difficult-to-treat warts were selected for immunotherapy. Each patient received 2.5 TU of PPD intralesionally in a few warts. A total of four sessions were given at 2 weekly intervals and patients were followed up for 6 months after the last dose. RESULTS: Sixty-one patients were recruited of which 55 completed 6 months follow up and were available for analysis. Of these, 25 had verruca vulgaris, 18 had verruca plana and 12 had plantar warts. Forty two (76%) patients showed complete clearance after four sessions while the remaining 13 (24%) patients were non-responders. One patient developed a recurrence after total clearance during the follow-up period. Adverse effects were erythema, edema and pain at the site of injections. LIMITATIONS: As this was an uncontrolled trial, there is no comparison with a non-intervention group. Also, a Mantoux test was not done due to practical difficulties. CONCLUSION: Immunotherapy with PPD is helpful in the treatment of cutaneous warts.

Coexistence of phosphotyrosine-dependent and -independent interactions between Cbl and Bcr-Abl.

Cbl is one of the major tyrosine-phosphorylated proteins in Bcr-Abl-expressing cells. A direct association between the SH2 domain of Bcr-Abl and tyrosine-phosphorylated Cbl has been demonstrated. The purpose of this study was to determine if and how unphosphorylated Cbl and Bcr-Abl may associate. Interactions between Cbl and Bcr-Abl were investigated in yeast two- and three-hybrid systems, gel overlay assays, and immunoprecipitates from mammalian cells expressing wild-type and the Y177F mutant of Bcr-Abl. No direct interaction between Bcr-Abl and unphosphorylated Cbl was observed. Bcr-Abl did, however, associate with Grb2, an adaptor protein that binds tyrosine 177 of Bcr-Abl. Additionally, Grb2 interacted with Cbl. In a yeast three-hybrid assay, Grb2 mediated an interaction between Cbl and Bcr-Abl that was dependent on a functional Grb2 binding site. This interaction was confirmed in vitro using purified proteins. In cells expressing Bcr-Abl with a mutation in the Grb2 binding site, binding of Cbl to Bcr-Abl was significantly reduced, but Cbl tyrosine phosphorylation was maintained. Imatinib treatment of these cells further reduced but did not abrogate Cbl binding, reflecting residual kinase activity. Multiple phosphotyrosine-dependent and -independent interactions stabilize the interaction between Cbl and Abl. Grb2 or another, yet unidentified, protein may mediate an initial interaction between Cbl and Bcr-Abl that is independent of Cbl tyrosine phosphorylation. Following this initial interaction, Cbl can then become tyrosine phosphorylated and interact with the SH2 domain of Bcr-Abl, further stabilizing the complex.