16th GCC Closed Forum: ICH M10 implementation; NGS, qPCR/dPCR, flow cytometry validation; tissue biomarkers; IS response; immunogenicity harmonization; bioanalytical industry status.

The 16th GCC Closed Forum was held in Orlando, FL, USA, on 23 June 2023. Representatives from international bioanalytical Contract Research Organizations were in attendance in order to discuss scientific and regulatory issues specific to bioanalysis. The issues discussed at the meeting included: IS response, flow cytometry, changes to the bioanalytical industry, NGS assays, biomarker assay for tissues, dPCR validation, immunogenicity harmonization and ICH M10 implementation. Conclusions and consensus from discussions of these topics are included in this article.

Modulation of Morphine Analgesia, Antinociceptive Tolerance, and Mu-Opioid Receptor Binding by the Cannabinoid CB2 Receptor Agonist O-1966.

Acutely, non-selective cannabinoid (CB) agonists have been shown to increase morphine antinociceptive effects, and we and others have also demonstrated that non-selective CB agonists attenuate morphine antinociceptive tolerance. Activation of cannabinoid CB2 receptors reverses allodynia and hyperalgesia in models of chronic pain, and co-administration of morphine with CB2 receptor selective agonists has been shown to be synergistic. CB2 receptor activation has also been shown to reduce morphine-induced hyperalgesia in rodents, an effect attributed to CB2 receptor modulation of inflammation. In the present set of experiments, we tested both the acute and chronic interactions between morphine and the CB2 receptor selective agonist O-1966 treatments on antinociception and antinociceptive tolerance in C57Bl6 mice. Co-administration of morphine and O-1966 was tested under three dosing regimens: simultaneous administration, morphine pre-treated with O-1966, and O-1966 pre-treated with morphine. The effects of O-1966 on mu-opioid receptor binding were determined using [3H]DAMGO and [35S]GTPγS binding assays, and these interactions were further examined by FRET analysis linked to flow cytometry. Results yielded surprising evidence of interactions between the CB2 receptor selective agonist O-1966 and morphine that were dependent upon the order of administration. When O-1966 was administered prior to or simultaneous with morphine, morphine antinociception was attenuated and antinociceptive tolerance was exacerbated. When O-1966 was administered following morphine, morphine antinociception was not affected and antinociceptive tolerance was attenuated. The [35S]GTPγS results suggest that O-1966 interrupts functional activity of morphine at the mu-opioid receptor, leading to decreased potency of morphine to produce acute thermal antinociceptive effects and potentiation of morphine antinociceptive tolerance. However, O-1966 administered after morphine blocked morphine hyperalgesia and led to an attenuation of morphine tolerance, perhaps due to well-documented anti-inflammatory effects of CB2 receptor agonism.

Decade-long leukaemia remissions with persistence of CD4+ CAR T cells.

The adoptive transfer of T lymphocytes reprogrammed to target tumour cells has demonstrated potential for treatment of various cancers1-7. However, little is known about the long-term potential and clonal stability of the infused cells. Here we studied long-lasting CD19-redirected chimeric antigen receptor (CAR) T cells in two patients with chronic lymphocytic leukaemia1-4 who achieved a complete remission in 2010. CAR T cells remained detectable more than ten years after infusion, with sustained remission in both patients. Notably, a highly activated CD4+ population emerged in both patients, dominating the CAR T cell population at the later time points. This transition was reflected in the stabilization of the clonal make-up of CAR T cells with a repertoire dominated by a small number of clones. Single-cell profiling demonstrated that these long-persisting CD4+ CAR T cells exhibited cytotoxic characteristics along with ongoing functional activation and proliferation. In addition, longitudinal profiling revealed a population of gamma delta CAR T cells that prominently expanded in one patient concomitant with CD8+ CAR T cells during the initial response phase. Our identification and characterization of these unexpected CAR T cell populations provide novel insight into the CAR T cell characteristics associated with anti-cancer response and long-term remission in leukaemia.

Tobacco smoke and morphine alter peripheral and CNS inflammation following HIV infection in a humanized mouse model.

Tobacco smoking is common in HIV-infected patients, and is prevalent among intravenous opiate abusers. Conversely, intravenous opiate abusers are more likely HIV-infected, and opiate abuse is associated with more severe neuroinflammation. Given the coincident use of tobacco smoking among HIV-infected intravenous drug users (IVDUs), we set out to study the effects of smoke exposure, chronic morphine administration, and HIV infection using the NSG humanized mouse model. Our results show that smoke, morphine, and the combination promotes the decline in CD4+ T cells in HIV-infected mice. Further, chronic morphine administration increases the numbers of circulating CD8+ T cells which express the inhibitory receptor PD-1, as well as the cytolytic proteins perforin and granzyme B in the infected mice. We also found that the combination of smoke and morphine inhibited the expression of IL-1α, IL-4 and IL-17A. Finally, the combination of smoke and morphine exposure induces microglial activation following infection, as well as in the absence of HIV infection. To our knowledge, this is the first report to assess the combined effects of smoke and chronic morphine exposure on the inflammation associated with HIV infection, and demonstrate that these two insults exert significant neuroinflammatory activity.

Identification, recovery, and impact of ghost fishing gear in the Mullica River-Great Bay Estuary (New Jersey, USA): Stakeholder-driven restoration for smaller-scale systems.

The impact of ghost fishing in large coastal ecosystems has generated considerable interest. In smaller, understudied systems with fewer stakeholders, derelict fishing gear (DFGs) may have impacts similar to these larger systems at the same relative scale. Four years of side scan sonar surveys in the Mullica River-Great Bay Estuary (New Jersey, USA) supported the recovery of 1776 DFGs off-season by commercial partners. Locations with high densities of recovered DFGs (>200 DFGs/km2) occupied intersections of recreational vessel traffic and commercial crabbing activity. Condition and depth-in-sediment of recovered DFGs was used to evaluate true bycatch (terrapins, whelks, blue crabs) versus species utilizing degraded gear as habitat (juvenile tautog, oyster toadfish). Critically, gear recovered in-season with low cost sonars (an additional 225 DFGs) prevented the accumulation of new DFGs which likely generate the highest percentages of bycatch. Removal of DFGs in this system led to significant ecological (reduced bycatch), economic (>$61,000 in direct pay, reused gear), and anticipated future benefits (increased harvest).

Induction of resistance to chimeric antigen receptor T cell therapy by transduction of a single leukemic B cell.

We report a patient relapsing 9 months after CD19-targeted CAR T cell (CTL019) infusion with CD19- leukemia that aberrantly expressed the anti-CD19 CAR. The CAR gene was unintentionally introduced into a single leukemic B cell during T cell manufacturing, and its product bound in cis to the CD19 epitope on the surface of leukemic cells, masking it from recognition by and conferring resistance to CTL019.

Disruption of TET2 promotes the therapeutic efficacy of CD19-targeted T cells.

Cancer immunotherapy based on genetically redirecting T cells has been used successfully to treat B cell malignancies1-3. In this strategy, the T cell genome is modified by integration of viral vectors or transposons encoding chimaeric antigen receptors (CARs) that direct tumour cell killing. However, this approach is often limited by the extent of expansion and persistence of CAR T cells4,5. Here we report mechanistic insights from studies of a patient with chronic lymphocytic leukaemia treated with CAR T cells targeting the CD19 protein. Following infusion of CAR T cells, anti-tumour activity was evident in the peripheral blood, lymph nodes and bone marrow; this activity was accompanied by complete remission. Unexpectedly, at the peak of the response, 94% of CAR T cells originated from a single clone in which lentiviral vector-mediated insertion of the CAR transgene disrupted the methylcytosine dioxygenase TET2 gene. Further analysis revealed a hypomorphic mutation in this patient's second TET2 allele. TET2-disrupted CAR T cells exhibited an epigenetic profile consistent with altered T cell differentiation and, at the peak of expansion, displayed a central memory phenotype. Experimental knockdown of TET2 recapitulated the potency-enhancing effect of TET2 dysfunction in this patient's CAR T cells. These findings suggest that the progeny of a single CAR T cell induced leukaemia remission and that TET2 modification may be useful for improving immunotherapies.

Determinants of response and resistance to CD19 chimeric antigen receptor (CAR) T cell therapy of chronic lymphocytic leukemia.

Tolerance to self-antigens prevents the elimination of cancer by the immune system1,2. We used synthetic chimeric antigen receptors (CARs) to overcome immunological tolerance and mediate tumor rejection in patients with chronic lymphocytic leukemia (CLL). Remission was induced in a subset of subjects, but most did not respond. Comprehensive assessment of patient-derived CAR T cells to identify mechanisms of therapeutic success and failure has not been explored. We performed genomic, phenotypic and functional evaluations to identify determinants of response. Transcriptomic profiling revealed that CAR T cells from complete-responding patients with CLL were enriched in memory-related genes, including IL-6/STAT3 signatures, whereas T cells from nonresponders upregulated programs involved in effector differentiation, glycolysis, exhaustion and apoptosis. Sustained remission was associated with an elevated frequency of CD27+CD45RO-CD8+ T cells before CAR T cell generation, and these lymphocytes possessed memory-like characteristics. Highly functional CAR T cells from patients produced STAT3-related cytokines, and serum IL-6 correlated with CAR T cell expansion. IL-6/STAT3 blockade diminished CAR T cell proliferation. Furthermore, a mechanistically relevant population of CD27+PD-1-CD8+ CAR T cells expressing high levels of the IL-6 receptor predicts therapeutic response and is responsible for tumor control. These findings uncover new features of CAR T cell biology and underscore the potential of using pretreatment biomarkers of response to advance immunotherapies.

Chimeric antigen receptor T cells persist and induce sustained remissions in relapsed refractory chronic lymphocytic leukemia.

Patients with multiply relapsed or refractory chronic lymphocytic leukemia (CLL) have a poor prognosis. Chimeric antigen receptor (CAR)-modified T cells targeting CD19 have the potential to improve on the low complete response rates with conventional therapies by inducing sustained remissions in patients with refractory B cell malignancies. We previously reported preliminary results on three patients with refractory CLL. We report the mature results from our initial trial using CAR-modified T cells to treat 14 patients with relapsed and refractory CLL. Autologous T cells transduced with a CD19-directed CAR (CTL019) lentiviral vector were infused into patients with relapsed/refractory CLL at doses of 0.14 × 10(8) to 11 × 10(8) CTL019 cells (median, 1.6 × 10(8) cells). Patients were monitored for toxicity, response, expansion, and persistence of circulating CTL019 T cells. The overall response rate in these heavily pretreated CLL patients was 8 of 14 (57%), with 4 complete remissions (CR) and 4 partial remissions (PR). The in vivo expansion of the CAR T cells correlated with clinical responses, and the CAR T cells persisted and remained functional beyond 4 years in the first two patients achieving CR. No patient in CR has relapsed. All responding patients developed B cell aplasia and experienced cytokine release syndrome, coincident with T cell proliferation. Minimal residual disease was not detectable in patients who achieved CR, suggesting that disease eradication may be possible in some patients with advanced CLL.

Computation of symmetric, time-periodic solutions of the vortex sheet with surface tension.

A numerical method is introduced for the computation of time-periodic vortex sheets with surface tension separating two immiscible, irrotational, two-dimensional ideal fluids of equal density. The approach is based on minimizing a nonlinear functional of the initial conditions and supposed period that is positive unless the solution is periodic, in which case it is zero. An adjoint-based optimal control technique is used to efficiently compute the gradient of this functional. Special care is required to handle singular integrals in the adjoint formulation. Starting with a solution of the linearized problem about the flat rest state, a family of smooth, symmetric breathers is found that, at quarter-period time intervals, alternately pass through a flat state of maximal kinetic energy, and a rest state in which all the energy is stored as potential energy in the interface. In some cases, the interface overturns before returning to the initial, flat configuration. It is found that the bifurcation diagram describing these solutions contains several disjoint curves separated by near-bifurcation events.

Regularization of the Kelvin-Helmholtz instability by surface tension.

The Kelvin-Helmholtz instability is present in the motion of a vortex sheet without surface tension. This can be seen from the linearization of the equations of motion, and there have also been proofs of ill-posedness for the full nonlinear equations. In the presence of surface tension, the linearized equations no longer exhibit an instability, and it has been believed that the full equations should then be well-posed. In this paper, I sketch a proof that the vortex sheet with surface tension is well-posed in the case of both two- and three-dimensional fluids. The proof in the case of three-dimensional fluids is the joint work with Nader Masmoudi. The method is to first reformulate the problem using suitable variables and parametrizations, and then to perform energy estimates. The choice of variables and parametrizations in the two-dimensional case is the same as that of Hou et al. in a prior numerical work.