Allogeneic CD20-targeted γδ T cells exhibit innate and adaptive antitumor activities in preclinical B-cell lymphoma models.

OBJECTIVES: Autologous chimeric antigen receptor (CAR) αß T-cell therapies have demonstrated remarkable antitumor efficacy in patients with haematological malignancies; however, not all eligible cancer patients receive clinical benefit. Emerging strategies to improve patient access and clinical responses include using premanufactured products from healthy donors and alternative cytotoxic effectors possessing intrinsic tumoricidal activity as sources of CAR cell therapies. γδ T cells, which combine innate and adaptive mechanisms to recognise and kill malignant cells, are an attractive candidate platform for allogeneic CAR T-cell therapy. Here, we evaluated the manufacturability and functionality of allogeneic peripheral blood-derived CAR+ Vδ1 γδ T cells expressing a second-generation CAR targeting the B-cell-restricted CD20 antigen. METHODS: Donor-derived Vδ1 γδ T cells from peripheral blood were ex vivo-activated, expanded and engineered to express a novel anti-CD20 CAR. In vitro and in vivo assays were used to evaluate CAR-dependent and CAR-independent antitumor activities of CD20 CAR+ Vδ1 γδ T cells against B-cell tumors. RESULTS: Anti-CD20 CAR+ Vδ1 γδ T cells exhibited innate and adaptive antitumor activities, such as in vitro tumor cell killing and proinflammatory cytokine production, in addition to in vivo tumor growth inhibition of B-cell lymphoma xenografts in immunodeficient mice. Furthermore, CD20 CAR+ Vδ1 γδ T cells did not induce xenogeneic graft-versus-host disease in immunodeficient mice. CONCLUSION: These preclinical data support the clinical evaluation of ADI-001, an allogeneic CD20 CAR+ Vδ1 γδ T cell, and a phase 1 study has been initiated in patients with B-cell malignancies (NCT04735471).

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.

The PET-Tracer 89Zr-Df-IAB22M2C Enables Monitoring of Intratumoral CD8 T-cell Infiltrates in Tumor-Bearing Humanized Mice after T-cell Bispecific Antibody Treatment.

CD8-expressing T cells are the main effector cells in cancer immunotherapy. Treatment-induced changes in intratumoral CD8+ T cells may represent a biomarker to identify patients responding to cancer immunotherapy. Here, we have used a 89Zr-radiolabeled human CD8-specific minibody (89Zr-Df-IAB22M2C) to monitor CD8+ T-cell tumor infiltrates by PET. The ability of this tracer to quantify CD8+ T-cell tumor infiltrates was evaluated in preclinical studies following single-agent treatment with FOLR1-T-cell bispecific (TCB) antibody and combination therapy of CEA-TCB (RG7802) and CEA-targeted 4-1BB agonist CEA-4-1BBL. In vitro cytotoxicity assays with peripheral blood mononuclear cells and CEA-expressing MKN-45 gastric or FOLR1-expressing HeLa cervical cancer cells confirmed noninterference of the anti-CD8-PET-tracer with the mode of action of CEA-TCB/CEA-4-1BBL and FOLR1-TCB at relevant doses. In vivo, the extent of tumor regression induced by combination treatment with CEA-TCB/CEA-4-1BBL in MKN-45 tumor-bearing humanized mice correlated with intratumoral CD8+ T-cell infiltration. This was detectable by 89Zr-IAB22M2C-PET and γ-counting. Similarly, single-agent treatment with FOLR1-TCB induced strong CD8+ T-cell infiltration in HeLa tumors, where 89Zr-Df-IAB22M2C again was able to detect CD8 tumor infiltrates. CD8-IHC confirmed the PET imaging results. Taken together, the anti-CD8-minibody 89Zr-Df-IAB22M2C revealed a high sensitivity for the detection of intratumoral CD8+ T-cell infiltrates upon either single or combination treatment with TCB antibody-based fusion proteins. These results provide further evidence that the anti-CD8 tracer, which is currently in clinical phase II, is a promising monitoring tool for intratumoral CD8+ T cells in patients treated with cancer immunotherapy. SIGNIFICANCE: Monitoring the pharmacodynamic activity of cancer immunotherapy with novel molecular imaging tools such as 89Zr-Df-IAB22M2C for PET imaging is of prime importance to identify patients responding early to cancer immunotherapy.

Enfortumab Vedotin Antibody-Drug Conjugate Targeting Nectin-4 Is a Highly Potent Therapeutic Agent in Multiple Preclinical Cancer Models.

The identification of optimal target antigens on tumor cells is central to the advancement of new antibody-based cancer therapies. We performed suppression subtractive hybridization and identified nectin-4 (PVRL4), a type I transmembrane protein and member of a family of related immunoglobulin-like adhesion molecules, as a potential target in epithelial cancers. We conducted immunohistochemical analysis of 2,394 patient specimens from bladder, breast, lung, pancreatic, ovarian, head/neck, and esophageal tumors and found that 69% of all specimens stained positive for nectin-4. Moderate to strong staining was especially observed in 60% of bladder and 53% of breast tumor specimens, whereas the expression of nectin-4 in normal tissue was more limited. We generated a novel antibody-drug conjugate (ADC) enfortumab vedotin comprising the human anti-nectin-4 antibody conjugated to the highly potent microtubule-disrupting agent MMAE. Hybridoma (AGS-22M6E) and CHO (ASG-22CE) versions of enfortumab vedotin (also known as ASG-22ME) ADC were able to bind to cell surface-expressed nectin-4 with high affinity and induced cell death in vitro in a dose-dependent manner. Treatment of mouse xenograft models of human breast, bladder, pancreatic, and lung cancers with enfortumab vedotin significantly inhibited the growth of all four tumor types and resulted in tumor regression of breast and bladder xenografts. Overall, these findings validate nectin-4 as an attractive therapeutic target in multiple solid tumors and support further clinical development, investigation, and application of nectin-4-targeting ADCs. Cancer Res; 76(10); 3003-13. ©2016 AACR.

AGS16F Is a Novel Antibody Drug Conjugate Directed against ENPP3 for the Treatment of Renal Cell Carcinoma.

PURPOSE: New cancer-specific antigens are required for the design of novel antibody-drug conjugates (ADC) that deliver tumor-specific and highly potent cytotoxic therapy. EXPERIMENTAL DESIGN: Suppression subtractive hybridization identified ectonucleotide pyrophosphatase/phosphodiesterase 3 (ENPP3 or CD203c) as a potential human cancer-specific antigen. Antibodies targeting the extracellular domain of human ENPP3 were produced and selected for specific binding to ENPP3. Expression of ENPP3 in normal and cancer tissue specimens was evaluated by immunohistochemistry (IHC). ADCs comprising anti-ENPP3 Ab conjugated with maleimidocaproyl monomethyl auristatin F via a noncleavable linker (mcMMAF) were selected for therapeutic potential using binding and internalization assays, cytotoxicity assays, and tumor growth inhibition in mouse xenograft models. Pharmacodynamic markers were evaluated by IHC in tissues and ELISA in blood. RESULTS: ENPP3 was highly expressed in clear cell renal cell carcinoma: 92.3% of samples were positive and 83.9% showed high expression. By contrast, expression was negligible in normal tissues examined, with the exception of the kidney. High expression was less frequent in papillary renal cell carcinoma and hepatocellular carcinoma samples. AGS16F, an anti-ENPP3 antibody-mcMMAF conjugate, inhibited tumor growth in three different renal cell carcinoma (RCC) xenograft models. AGS16F localized to tumors, formed the active metabolite Cys-mcMMAF, induced cell-cycle arrest and apoptosis, and increased blood levels of caspase-cleaved cytokeratin-18, a marker of epithelial cell death. CONCLUSIONS: AGS16F is a promising new therapeutic option for patients with RCC and is currently being evaluated in a phase I clinical trial.

Discoidin domain receptor 1 contributes to tumorigenesis through modulation of TGFBI expression.

Discoidin domain receptor 1 (DDR1) is a member of the receptor tyrosine kinase family. The receptor is activated upon binding to its ligand, collagen, and plays a crucial role in many fundamental processes such as cell differentiation, adhesion, migration and invasion. Although DDR1 is expressed in many normal tissues, upregulated expression of DDR1 in a variety of human cancers such as lung, colon and brain cancers is known to be associated with poor prognosis. Using shRNA silencing, we assessed the oncogenic potential of DDR1. DDR1 knockdown impaired tumor cell proliferation and migration in vitro and tumor growth in vivo. Microarray analysis of tumor cells demonstrated upregulation of TGFBI expression upon DDR1 knockdown, which was subsequently confirmed at the protein level. TGFBI is a TGFß-induced extracellular matrix protein secreted by the tumor cells and is known to act either as a tumor promoter or tumor suppressor, depending on the tumor environment. Here, we show that exogenous addition of recombinant TGFBI to BXPC3 tumor cells inhibited clonogenic growth and migration, thus recapitulating the phenotypic effect observed from DDR1 silencing. BXPC3 tumor xenografts demonstrated reduced growth with DDR1 knockdown, and the same xenograft tumors exhibited an increase in TGFBI expression level. Together, these data suggest that DDR1 expression level influences tumor growth in part via modulation of TGFBI expression. The reciprocal expression of DDR1 and TGFBI may help to elucidate the contribution of DDR1 in tumorigenesis and TGFBI may also be used as a biomarker for the therapeutic development of DDR1 specific inhibitors.