Lisocabtagene maraleucel in chronic lymphocytic leukaemia and small lymphocytic lymphoma (TRANSCEND CLL 004): a multicentre, open-label, single-arm, phase 1-2 study.

BACKGROUND: Patients with relapsed or refractory chronic lymphocytic leukaemia or small lymphocytic lymphoma for whom treatment has failed with both Bruton tyrosine kinase (BTK) inhibitor and venetoclax have few treatment options and poor outcomes. We aimed to evaluate the efficacy and safety of lisocabtagene maraleucel (liso-cel) at the recommended phase 2 dose in patients with relapsed or refractory chronic lymphocytic leukaemia or small lymphocytic lymphoma. METHODS: We report the primary analysis of TRANSCEND CLL 004, an open-label, single-arm, phase 1-2 study conducted in the USA. Patients aged 18 years or older with relapsed or refractory chronic lymphocytic leukaemia or small lymphocytic lymphoma and at least two previous lines of therapy, including a BTK inhibitor, received an intravenous infusion of liso-cel at one of two target dose levels: 50 × 106 (dose level 1) or 100 × 106 (dose level 2, DL2) chimeric antigen receptor-positive T cells. The primary endpoint was complete response or remission (including with incomplete marrow recovery), assessed by independent review according to the 2018 International Workshop on Chronic Lymphocytic Leukemia criteria, in efficacy-evaluable patients with previous BTK inhibitor progression and venetoclax failure (the primary efficacy analysis set) at DL2 (null hypothesis of ≤5%). This trial is registered with ClinicalTrials.gov, NCT03331198. FINDINGS: Between Jan 2, 2018, and June 16, 2022, 137 enrolled patients underwent leukapheresis at 27 sites in the USA. 117 patients received liso-cel (median age 65 years [IQR 59-70]; 37 [32%] female and 80 [68%] male; 99 [85%] White, five [4%] Black or African American, two [2%] other races, and 11 [9%] unknown race; median of five previous lines of therapy [IQR 3-7]); all 117 participants had received and had treatment failure on a previous BTK inhibitor. A subset of patients had also experienced venetoclax failure (n=70). In the primary efficacy analysis set at DL2 (n=49), the rate of complete response or remission (including with incomplete marrow recovery) was statistically significant at 18% (n=9; 95% CI 9-32; p=0·0006). In patients treated with liso-cel, grade 3 cytokine release syndrome was reported in ten (9%) of 117 (with no grade 4 or 5 events) and grade 3 neurological events were reported in 21 (18%; one [1%] grade 4, no grade 5 events). Among 51 deaths on the study, 43 occurred after liso-cel infusion, of which five were due to treatment-emergent adverse events (within 90 days of liso-cel infusion). One death was related to liso-cel (macrophage activation syndrome-haemophagocytic lymphohistiocytosis). INTERPRETATION: A single infusion of liso-cel was shown to induce complete response or remission (including with incomplete marrow recovery) in patients with relapsed or refractory chronic lymphocytic leukaemia or small lymphocytic lymphoma, including patients who had experienced disease progression on a previous BTK inhibitor and venetoclax failure. The safety profile was manageable. FUNDING: Juno Therapeutics, a Bristol-Myers Squibb Company.

Early transduction produces highly functional chimeric antigen receptor-modified virus-specific T-cells with central memory markers: a Production Assistant for Cell Therapy (PACT) translational application.

BACKGROUND: Virus-specific T-cells (VSTs) proliferate exponentially after adoptive transfer into hematopoietic stem cell transplant (HSCT) recipients, eliminate virus infections, then persist and provide long-term protection from viral disease. If VSTs behaved similarly when modified with tumor-specific chimeric antigen receptors (CARs), they should have potent anti-tumor activity. This theory was evaluated by Cruz et al. in a previous clinical trial with CD19.CAR-modified VSTs, but there was little apparent expansion of these cells in patients. In that study, VSTs were gene-modified on day 19 of culture and we hypothesized that by this time, sufficient T-cell differentiation may have occurred to limit the subsequent proliferative capacity of the transduced T-cells. To facilitate the clinical testing of this hypothesis in a project supported by the NHLBI-PACT mechanism, we developed and optimized a good manufacturing practices (GMP) compliant method for the early transduction of VSTs directed to Epstein-Barr virus (EBV), Adenovirus (AdV) and cytomegalovirus (CMV) using a CAR directed to the tumor-associated antigen disialoganglioside (GD2). RESULTS: Ad-CMVpp65-transduced EBV-LCLs effectively stimulated VSTs directed to all three viruses (triVSTs). Transduction efficiency on day three was increased in the presence of cytokines and high-speed centrifugation of retroviral supernatant onto retronectin-coated plates, so that under optimal conditions up to 88% of tetramer-positive VSTs expressed the GD2.CAR. The average transduction efficiency of early-and late transduced VSTs was 55 ± 4% and 22 ± 5% respectively, and early-transduced VSTs maintained higher frequencies of T cells with central memory or intermediate memory phenotypes. Early-transduced VSTs also had higher proliferative capacity and produced higher levels of TH1 cytokines IL-2, TNF-α, IFN-γ, MIP-1α, MIP-1ß and other cytokines in vitro. CONCLUSIONS: We developed a rapid and GMP compliant method for the early transduction of multivirus-specific T-cells that allowed stable expression of high levels of a tumor directed CAR. Since a proportion of early-transduced CAR-VSTs had a central memory phenotype, they should expand and persist in vivo, simultaneously protecting against infection and targeting residual malignancy. This manufacturing strategy is currently under clinical investigation in patients receiving allogeneic HSCT for relapsed neuroblastoma and B-cell malignancies (NCT01460901 using a GD2.CAR and NCT00840853 using a CD19.CAR).

Genetic modification of cytotoxic T lymphocytes to express cytokine receptors.

Adoptive transfer of tumor-infiltrating lymphocytes (TIL) or antigen-specific cytotoxic T lymphocytes (CTL) is safe and can be effective in cancer patients. Achievement of clinical responses in these patients is associated with the in vivo expansion and persistence of the transferred T lymphocytes. For this reason, recombinant human interleukin-2 (IL-2) is frequently used to support the in vivo survival of T lymphocytes infused into patients. However, IL-2 also causes important side effects. Thus, alternative strategies are highly demanded to limit cytokine-related off-target effects and to redirect the responsiveness of specific T-cell subsets to selected cytokines. Interleukin-7 (IL-7) is a promising alternative cytokine as it possesses the above mentioned properties. However, because its receptor is downregulated in ex vivo-expanded T cells, methods are required to restore their responsiveness to this homeostatic cytokine. In this chapter, we describe the methodology to obtain the ectopic expression of IL-7 receptor alpha (IL-7Rα) in antigen-specific CTL, using Epstein-Barr virus-specific CTL (EBV-CTL), as a model.

Interleukin-7 mediates selective expansion of tumor-redirected cytotoxic T lymphocytes (CTLs) without enhancement of regulatory T-cell inhibition.

PURPOSE: The antitumor activity of chimeric antigen receptor (CAR)-redirected CTLs should be enhanced if it were possible to increase their proliferation and function after adoptive transfer without concomitantly increasing the proliferation and function of regulatory T cells (Treg). Here, we explored whether the lack of IL-7Rα in Treg can be exploited by the targeted manipulation of the interleukin-7 (IL-7) cytokine-cytokine receptor axis in CAR-engrafted Epstein-Barr Virus-specific CTLs (EBV-CTLs) to selectively augment their growth and antitumor activity even in the presence of Treg. EXPERIMENTAL DESIGN: We generated a bicistronic retroviral vector encoding a GD2-specific CAR and the IL-7Rα subunit, expressed the genes in EBV-CTLs, and assessed their capacity to control tumor growth in the presence of Treg in vitro and in vivo when exposed to either interleukin-2 (IL-2) or IL-7 in a neuroblastoma xenograft. RESULTS: We found that IL-7, in sharp contrast with IL-2, supports the proliferation and antitumor activity of IL-7Rα.CAR-GD2(+) EBV-CTLs both in vitro and in vivo even in the presence of fully functional Treg. CONCLUSIONS: IL-7 selectively favors the survival, proliferation, and effector function of IL-7Rα-transgenic/CAR-redirected EBV-CTLs in the presence of Treg both in vitro and in vivo. Thus, IL-7 can have a significant impact in sustaining expansion and persistence of adoptively CAR-redirected CTLs.

Elimination of metastatic melanoma using gold nanoshell-enabled photothermal therapy and adoptive T cell transfer.

Ablative treatments such as photothermal therapy (PTT) are attractive anticancer strategies because they debulk accessible tumor sites while simultaneously priming antitumor immune responses. However, the immune response following thermal ablation is often insufficient to treat metastatic disease. Here we demonstrate that PTT induces the expression of proinflammatory cytokines and chemokines and promotes the maturation of dendritic cells within tumor-draining lymph nodes, thereby priming antitumor T cell responses. Unexpectedly, however, these immunomodulatory effects were not beneficial to overall antitumor immunity. We found that PTT promoted the infiltration of secondary tumor sites by CD11b(+)Ly-6G/C(+) myeloid-derived suppressor cells, consequently failing to slow the growth of poorly immunogenic B16-F10 tumors and enhancing the growth of distant lung metastases. To exploit the beneficial effects of PTT activity against local tumors and on antitumor immunity whilst avoiding the adverse consequences, we adoptively transferred gp100-specific pmel T cells following PTT. The combination of local control by PTT and systemic antitumor immune reactivity provided by adoptively transferred T cells prevented primary tumor recurrence post-ablation, inhibited tumor growth at distant sites, and abrogated the outgrowth of lung metastases. Hence, the combination of PTT and systemic immunotherapy prevented the adverse effects of PTT on metastatic tumor growth and optimized overall tumor control.

Robust and cost effective expansion of human regulatory T cells highly functional in a xenograft model of graft-versus-host disease.

The low frequency of naturally occurring regulatory T cells (nTregs) in peripheral blood and the suboptimal protocols available for their ex vivo expansion limit the development of clinical trials based on the adoptive transfer of these cells. We have, therefore, generated a simplified, robust and cost-effective platform for the large-scale expansion of nTregs using a gas permeable static culture flask (G-Rex) in compliance with Good Manufacturing Practice. More than 10(9) putative Tregs co-expressing CD25 and CD4 molecules (92 ± 5%) and FoxP3 (69 ± 19%) were obtained within 21 days of culture. Expanded Tregs showed potent regulatory activity in vitro (80 ± 13% inhibition of CD8(+) cell division) and in vivo (suppression or delay of graft-versus-host disease in a xenograft mouse model) indicating that the cost-effective and simplified production of nTregs we propose will facilitate the implementation of clinical trials based on their adoptive transfer.

Interleukin 15 provides relief to CTLs from regulatory T cell-mediated inhibition: implications for adoptive T cell-based therapies for lymphoma.

PURPOSE: Systemic administration of recombinant interleukin (IL)-2 is used to support the expansion and persistence of adoptively transferred antigen-specific CTLs in patients with cancer. However, IL-2 also expands regulatory T cells (Treg) that in turn impair the antitumor activity of CTLs. As recombinant IL-15 is approaching clinical applications, we assessed the effects of this cytokine on the proliferation and antitumor activity of CTLs in the presence of Tregs. We used the model of adoptive transfer of Epstein-Barr virus (EBV)-CTLs, as these cells induce responses in patients with EBV-associated Hodgkin lymphoma, and Tregs are frequently abundant in these patients. EXPERIMENTAL DESIGN: Tregs were isolated from the peripheral blood of healthy donors and patients with Hodgkin lymphoma or from Hodgkin lymphoma tumors and assessed for their ability to inhibit the proliferation and antitumor activity of EBV-CTLs in the presence of IL-15 or IL-2. Specific molecular pathways activated by IL-15 were also explored. RESULTS: We found that in the presence of Tregs, IL-15, but not IL-2, promoted the proliferation, effector function, and resistance to apoptosis of effectors T cells and EBV-CTLs. IL-15 did not reverse or block Tregs but instead preferentially supported the proliferation of CTLs and effector T cells as compared with Tregs. CONCLUSIONS: IL-15 selectively favors the survival, proliferation, and effector function of antigen-specific CTLs in the presence of Tregs, and thus IL-15, unlike IL-2, would have a significant impact in sustaining expansion and persistence of adoptively transferred CTLs in patients with cancer, including those infused with EBV-CTLs for treatment of EBV-associated malignancies.

Rapidly generated multivirus-specific cytotoxic T lymphocytes for the prophylaxis and treatment of viral infections.

Severe and fatal viral infections remain common after hematopoietic stem cell transplantation. Adoptive transfer of cytotoxic T lymphocytes (CTLs) specific for Epstein-Barr virus (EBV), cytomegalovirus (CMV), and adenoviral antigens can treat infections that are impervious to conventional therapies, but broader implementation and extension to additional viruses is limited by competition between virus-derived antigens and time-consuming and laborious manufacturing procedures. We now describe a system that rapidly generates a single preparation of polyclonal (CD4(+) and CD8(+)) CTLs that is consistently specific for 15 immunodominant and subdominant antigens derived from 7 viruses (EBV, CMV, Adenovirus (Adv), BK, human herpes virus (HHV)-6, respiratory syncytial virus (RSV), and Influenza) that commonly cause post-transplant morbidity and mortality. CTLs can be rapidly produced (10 days) by a single stimulation of donor peripheral blood mononuclear cells (PBMCs) with a peptide mixture spanning the target antigens in the presence of the potent prosurvival cytokines interleukin-4 (IL4) and IL7. This approach reduces the impact of antigenic competition with a consequent increase in the antigenic repertoire and frequency of virus-specific T cells. Our approach can be readily introduced into clinical practice and should be a cost-effective alternative to common antiviral prophylactic agents for allogeneic hematopoietic stem cell transplant (HSCT) recipients.

Infusion of suicide-gene-engineered donor lymphocytes after family haploidentical haemopoietic stem-cell transplantation for leukaemia (the TK007 trial): a non-randomised phase I-II study.

BACKGROUND: Procedures to prevent severe graft-versus-host disease (GVHD) delay immune reconstitution secondary to transplants of haploidentical haemopoietic stem cells for the treatment of leukaemia, leading to high rates of late infectious mortality. We aimed to systematically add back genetically engineered donor lymphocytes to facilitate immune reconstitution and prevent late mortality. METHODS: In a phase I-II, multicentre, non-randomised trial of haploidentical stem-cell transplantation, we infused donor lymphocytes expressing herpes-simplex thymidine kinase suicide gene (TK-cells) after transplantation. The primary study endpoint was immune reconstitution defined as circulating CD3+ count of 100 cells per muL or more for two consecutive observations. Analysis was by intention to treat. This trial is registered with ClinicalTrials.gov, number NCT00423124. FINDINGS: From Aug 13, 2002, to March 26, 2008, 50 patients (median age 51 years, range 17-66) received haploidentical stem-cell transplants for high-risk leukaemia. Immune reconstitution was not recorded before infusion of TK-cells. 28 patients received TK-cells starting 28 days after transplantation; 22 patients obtained immune reconstitution at median 75 days (range 34-127) from transplantation and 23 days (13-42) from infusion. Ten patients developed acute GVHD (grade I-IV) and one developed chronic GVHD, which were controlled by induction of the suicide gene. Overall survival at 3 years was 49% (95% CI 25-73) for 19 patients who were in remission from primary leukaemia at the time of stem-cell transplantation. After TK-cell infusion, the last death due to infection was at 166 days, this was the only infectious death at more than 100 days. No acute or chronic adverse events were related to the gene-transfer procedure. INTERPRETATION: Infusion of TK-cells might be effective in accelerating immune reconstitution, while controlling GVHD and protecting patients from late mortality in those who are candidates for haploidentical stem-cell transplantation. FUNDING: MolMed SpA, Italian Association for Cancer Research.

Transcriptional enhancers induce insertional gene deregulation independently from the vector type and design.

The integration characteristics of retroviral (RV) vectors increase the probability of interfering with the regulation of cellular genes, and account for a tangible risk of insertional mutagenesis in treated patients. To assess the potential genotoxic risk of conventional or self-inactivating (SIN) gamma-RV and lentiviral (LV) vectors independently from the biological consequences of the insertion event, we developed a quantitative assay based on real-time reverse transcriptase--PCR on low-density arrays to evaluate alterations of gene expression in individual primary T-cell clones. We show that the Moloney leukemia virus long terminal repeat (LTR) enhancer has the strongest activity in both a gamma-RV and a LV vector context, while an internal cellular promoter induces deregulation of gene expression less frequently, at a shorter range and to a lower extent in both vector types. Downregulation of gene expression was observed only in the context of LV vectors. This study indicates that insertional gene activation is determined by the characteristics of the transcriptional regulatory elements carried by the vector, and is largely independent from the vector type or design.

Temporal, quantitative, and functional characteristics of single-KIR-positive alloreactive natural killer cell recovery account for impaired graft-versus-leukemia activity after haploidentical hematopoietic stem cell transplantation.

In this study, we have characterized reconstitution of the natural killer (NK) cell repertoire after haploidentical CD34(+) selected hematopoietic stem cell transplantation (HSCT) for high-risk hematologic malignancies. Analysis focused on alloreactive single-KIR(+) NK cells, which reportedly are potent antileukemic effectors. One month after HSCT, CD56(bright)/CD56(dim) NK-cell subsets showed inverted ratio and phenotypic features. CD25 and CD117 down-regulation on CD56(bright), and NKG2A and CD62L up-regulation on CD56(dim), suggest sequential CD56(bright)-to-CD56(dim) NK-cell maturation in vivo. Consistently, the functional potential of these maturation intermediates against leukemic blasts was impaired. Mature receptor repertoire reconstitution took at least 3 months. Importantly, at this time point, supposedly alloreactive, single-KIR(+) NK cells were not yet fully functional. Frequency of these cells was highly variable, independently from predicted NK alloreactivity, and below 1% of NK cells in 3 of 6 alloreactive patients studied. In line with these observations, no clinical benefit of predicted NK alloreactivity was observed in the total cohort of 56 patients. Our findings unravel the kinetics, and limits, of NK-cell differentiation from purified haploidentical hematopoietic stem cells in vivo, and suggest that NK-cell antileukemic potential could be best exploited by infusion of mature single-KIR(+) NK cells selected from an alloreactive donor.