Intratumoral pan-ErbB targeted CAR-T for head and neck squamous cell carcinoma: interim analysis of the T4 immunotherapy study.

BACKGROUND: Locally advanced/recurrent head and neck squamous cell carcinoma (HNSCC) is associated with significant morbidity and mortality. To target upregulated ErbB dimer expression in this cancer, we developed an autologous CD28-based chimeric antigen receptor T-cell (CAR-T) approach named T4 immunotherapy. Patient-derived T-cells are engineered by retroviral transduction to coexpress a panErbB-specific CAR called T1E28ζ and an IL-4-responsive chimeric cytokine receptor, 4αß, which allows IL-4-mediated enrichment of transduced cells during manufacture. These cells elicit preclinical antitumor activity against HNSCC and other carcinomas. In this trial, we used intratumoral delivery to mitigate significant clinical risk of on-target off-tumor toxicity owing to low-level ErbB expression in healthy tissues. METHODS: We undertook a phase 1 dose-escalation 3+3 trial of intratumoral T4 immunotherapy in HNSCC (NCT01818323). CAR T-cell batches were manufactured from 40 to 130 mL of whole blood using a 2-week semiclosed process. A single CAR T-cell treatment, formulated as a fresh product in 1-4 mL of medium, was injected into one or more target lesions. Dose of CAR T-cells was escalated in 5 cohorts from 1×107-1×109 T4+ T-cells, administered without prior lymphodepletion. RESULTS: Despite baseline lymphopenia in most enrolled subjects, the target cell dose was successfully manufactured in all cases, yielding up to 7.5 billion T-cells (67.5±11.8% transduced), without any batch failures. Treatment-related adverse events were all grade 2 or less, with no dose-limiting toxicities (Common Terminology Criteria for Adverse Events V.4.0). Frequent treatment-related adverse events were tumor swelling, pain, pyrexias, chills, and fatigue. There was no evidence of leakage of T4+ T-cells into the circulation following intratumoral delivery, and injection of radiolabeled cells demonstrated intratumoral persistence. Despite rapid progression at trial entry, stabilization of disease (Response Evaluation Criteria in Solid Tumors V.1.1) was observed in 9 of 15 subjects (60%) at 6 weeks post-CAR T-cell administration. Subsequent treatment with pembrolizumab and T-VEC oncolytic virus achieved a rapid complete clinical response in one subject, which was durable for over 3 years. Median overall survival was greater than for historical controls. Disease stabilization was associated with the administration of an immunophenotypically fitter, less exhausted, T4 CAR T-cell product. CONCLUSIONS: These data demonstrate the safe intratumoral administration of T4 immunotherapy in advanced HNSCC.

Development and Validation of a Good Manufacturing Process for IL-4-Driven Expansion of Chimeric Cytokine Receptor-Expressing CAR T-Cells.

Adoptive cancer immunotherapy using chimeric antigen receptor (CAR) engineered T-cells holds great promise, although several obstacles hinder the efficient generation of cell products under good manufacturing practice (GMP). Patients are often immune compromised, rendering it challenging to produce sufficient numbers of gene-modified cells. Manufacturing protocols are labour intensive and frequently involve one or more open processing steps, leading to increased risk of contamination. We set out to develop a simplified process to generate autologous gamma retrovirus-transduced T-cells for clinical evaluation in patients with head and neck cancer. T-cells were engineered to co-express a panErbB-specific CAR (T1E28z) and a chimeric cytokine receptor (4αß) that permits their selective expansion in response to interleukin (IL)-4. Using peripheral blood as starting material, sterile culture procedures were conducted in gas-permeable bags under static conditions. Pre-aliquoted medium and cytokines, bespoke connector devices and sterile welding/sealing were used to maximise the use of closed manufacturing steps. Reproducible IL-4-dependent expansion and enrichment of CAR-engineered T-cells under GMP was achieved, both from patients and healthy donors. We also describe the development and approach taken to validate a panel of monitoring and critical release assays, which provide objective data on cell product quality.

An overview of CAR T-cell clinical trial activity to 2021.

Immunotherapy of cancer using chimeric antigen receptor-engineered T-cells has transformed the management of selected haematological malignancies, triggering intense clinical trial activity in this arena. This article summarises trial activity that has been published to date across the spectrum of haematological and solid tumour types.

TGF-ß1 potentiates Vγ9Vδ2 T cell adoptive immunotherapy of cancer.

Despite its role in cancer surveillance, adoptive immunotherapy using γδ T cells has achieved limited efficacy. To enhance trafficking to bone marrow, circulating Vγ9Vδ2 T cells are expanded in serum-free medium containing TGF-ß1 and IL-2 (γδ[T2] cells) or medium containing IL-2 alone (γδ[2] cells, as the control). Unexpectedly, the yield and viability of γδ[T2] cells are also increased by TGF-ß1, when compared to γδ[2] controls. γδ[T2] cells are less differentiated and yet display increased cytolytic activity, cytokine release, and antitumor activity in several leukemic and solid tumor models. Efficacy is further enhanced by cancer cell sensitization using aminobisphosphonates or Ara-C. A number of contributory effects of TGF-ß are described, including prostaglandin E2 receptor downmodulation, TGF-ß insensitivity, and upregulated integrin activity. Biological relevance is supported by the identification of a favorable γδ[T2] signature in acute myeloid leukemia (AML). Given their enhanced therapeutic activity and compatibility with allogeneic use, γδ[T2] cells warrant evaluation in cancer immunotherapy.

Use of gene-modified regulatory T-cells to control autoimmune and alloimmune pathology: is now the right time?

Adoptive immunotherapy using genetically targeted T-cells has recently begun to achieve impressive clinical impact in selected tumor types. Furthermore, long-term follow-up studies indicate thus far that integrating viral vectors do not elicit clinically evident genotoxicity in T-cells, unlike hematopoietic stem cells. The optimism engendered by this clinical experience provides a platform for consideration of the extended use of this technology in other disease types. One area of particular interest entails the harnessing of regulatory T-cells (Tregs) in order to down-regulate unwanted immune responses. Increasing evidence supports the efficacy of this approach in pre-clinical models of autoimmune disease and allograft rejection. Nonetheless, questions remain about optimal host cell, transgene cargo, phenotypic stability of engineered cells in vivo and potential for toxicity. Here, we review the evidence that genetically engineered Tregs can effectively dampen pathogenic immune responses and critically evaluate the prospects for clinical development of this approach.

Antitumor immunity: easy as 1, 2, 3 with monoclonal bispecific trifunctional antibodies?

Monoclonal antibodies occupy an increasing niche in the arsenal available to treat cancer. Several developments have rendered this the fastest growing sector in the pharmaceutical industry. Traditionally, antibodies were developed to block key signaling molecules implicated in tumor progression. However, antibodies also recruit additional immune effector mechanisms against tumors, a property that may be exploited for clinical benefit. Bispecific antibodies represent one such strategy in which elements derived from two monoclonal antibodies are incorporated into a single molecular species. Commonly, the bispecific approach is used to achieve simultaneous cross-linking of CD3 and a tumor antigen such as epithelial cell adhesion molecule (EpCAM), thereby recruiting T-cell activation to the tumor cell surface. A further sophistication involves the engineering of trifunctional derivatives such as the clinically approved agent, catumaxomab. Catumaxomab has antigen-binding arms that engage CD3 and EpCAM and a constant domain that recruits Fc receptor-bearing cells, notably monocytes, dendritic cells, and natural killer cells. Owing to this triangular binding capability, catumaxomab can activate both innate and adaptive immune effector mechanisms in addition to promoting immunologic memory. Recent data indicate that this agent can also promote immunogenic cell death, particularly when used in combination with selected chemotherapeutic agents such as oxaliplatin.

Naturally occurring mutations in the thrombomodulin gene leading to impaired expression and function.

Sporadic mutations in the thrombomodulin (TM) gene occur in patients with both arterial and venous thrombosis, but the effects of these mutations on expression and function are largely unexplored. Full-length wild-type TM complementary DNA (cDNA) was incorporated into vector pcDNA6 for transfection into COS-7 cells for transient expression. Mutagenesis was performed to create 7 TM mutants with natural mutations either previously identified (Ala25Thr, Gly61Ala, Asp468Tyr, Pro477Ser, Pro483Leu) or reported here (an 11-base pair [bp] deletion, del791-801, leading to STOP306, and a missense mutation, Arg385Ser). Four mutations were found to detrimentally affect the level of expression of the TM protein. Of the missense mutations, 3 had reduced expression compared to wild-type TM (100%), Arg385Ser (50.2% +/- 5%, P <.001), Pro477Ser (76.8% +/- 1%, P <.001), Pro483Leu (82.1% +/- 8%, P <.007). No TM protein expression could be detected on the cell surface for mutation del791-801. The cofactor activity of TM in protein C activation was also evaluated. The Michaelis constant (K(m)) for wild-type thrombin-TM complex was 634 +/- 6 nmol/L. Two mutants, with Arg385Ser and Pro477Ser, had increased (P <.0001) K(m), 2967 +/- 283 nM, and 2342 +/- 219 nM, respectively, demonstrating impaired function of the thrombin-TM complex. This work presents biochemical evidence that certain (but not all) natural mutations in the TM gene reduce expression and impair function of the protein on the cell surface, and helps clarify the suggested contribution that these mutations might make to the risk of thromboembolic disease.