Long-term outcomes following CAR T cell therapy: what we know so far.

Chimeric antigen receptors (CAR) are engineered fusion proteins designed to target T cells to antigens expressed on cancer cells. CAR T cells are now an established treatment for patients with relapsed and/or refractory B cell lymphomas, B cell acute lymphoblastic leukaemia and multiple myeloma. At the time of this writing, over a decade of follow-up data are available from the initial patients who received CD19-targeted CAR T cells for B cell malignancies. Data on the outcomes of patients who received B cell maturation antigen (BCMA)-targeted CAR T cells for multiple myeloma are more limited owing to the more recent development of these constructs. In this Review, we summarize long-term follow-up data on efficacy and toxicities from patients treated with CAR T cells targeting CD19 or BCMA. Overall, the data demonstrate that CD19-targeted CAR T cells can induce prolonged remissions in patients with B cell malignancies, often with minimal long-term toxicities, and are probably curative for a subset of patients. By contrast, remissions induced by BCMA-targeted CAR T cells are typically more short-lived but also generally have only limited long-term toxicities. We discuss factors associated with long-term remissions, including the depth of initial response, malignancy characteristics predictive of response, peak circulating CAR levels and the role of lymphodepleting chemotherapy. We also discuss ongoing investigational strategies designed to improve the length of remission following CAR T cell therapy.

A comparison of chimeric antigen receptors containing CD28 versus 4-1BB costimulatory domains.

Chimeric antigen receptors (CARs) are engineered proteins designed to target T cells to cancer cells. To effectively activate the T cells in which they are expressed, CARs must contain a costimulatory domain. The CAR T cell products approved for the treatment of B cell lymphomas and/or acute lymphoblastic leukaemia or multiple myeloma incorporate either a CD28-derived or a 4-1BB-derived costimulatory domain. Almost all other clinically tested CARs also use costimulatory domains from CD28 or 4-1BB. In preclinical experiments, cytokine release is usually greater with CARs containing CD28 versus 4-1BB costimulatory domains; however, constructs with either domain confer similar anticancer activity in mouse models. T cell products expressing CARs with either CD28 or 4-1BB costimulatory domains have been highly efficacious in patients with relapsed haematological malignancies, with anti-CD19 products having similar activity regardless of the source of the costimulatory domain. In large-cohort clinical trials, the rates of neurological toxicities have been higher with CD28-costimulated CARs, although this finding is probably the result of a combination of factors rather than due to CD28 signalling alone. Future preclinical and clinical research should aim to compare different costimulatory domains while controlling for confounding variables. Herein, we provide an overview of T cell costimulation by CD28 and 4-1BB and, using the available preclinical and clinical data, compare the efficacy and toxicity profiles associated with CARs containing either costimulatory domain.

Long-Term Follow-Up of Anti-CD19 Chimeric Antigen Receptor T-Cell Therapy.

PURPOSE: Anti-CD19 chimeric antigen receptors (CARs) are artificial fusion proteins that cause CD19-specific T-cell activation. Durability of remissions and incidence of long-term adverse events are critical factors determining the utility of anti-CD19 CAR T-cell therapy, but long-term follow-up of patients treated with anti-CD19 CAR T cells is limited. This work provides the longest follow-up of patients in remission after anti-CD19 CAR T-cell therapy. METHODS: Between 2009 and 2015, we administered 46 CAR T-cell treatments to 43 patients (ClinicalTrials.gov identifier: NCT00924326). Patients had relapsed B-cell malignancies of the following types: diffuse large B-cell lymphoma or primary mediastinal B-cell lymphoma (DLBCL/PMBCL; n = 28), low-grade B-cell lymphoma (n = 8), or chronic lymphocytic leukemia (CLL; n = 7). This report focuses on long-term outcomes of these patients. The CAR used was FMC63-28Z; axicabtagene ciloleucel uses the same CAR. Cyclophosphamide plus fludarabine conditioning chemotherapy was administered before CAR T cells. RESULTS: The percentages of CAR T-cell treatments resulting in a > 3-year duration of response (DOR) were 51% (95% CI, 35% to 67%) for all evaluable treatments, 48% (95% CI, 28% to 69%) for DLBCL/PMBCL, 63% (95% CI, 25% to 92%) for low-grade lymphoma, and 50% (95% CI, 16% to 84%) for CLL. The median event-free survival of all 45 evaluable treatments was 55 months. Long-term adverse effects were rare, except for B-cell depletion and hypogammaglobulinemia. Median peak blood CAR-positive cell levels were higher among patients with a DOR of > 3 years (98/µL; range, 9-1,217/µL) than among patients with a DOR of < 3 years (18/µL; range, 0-308/µL, P = .0051). CONCLUSION: Complete remissions of a variety of B-cell malignancies lasting ≥ 3 years occurred after 51% of evaluable anti-CD19 CAR T-cell treatments. Remissions of up to 9 years are ongoing. Late adverse events were rare.

Mechanisms promoting escape from mitotic stress-induced tumor cell death.

Non-small cell lung cancer (NSCLC) is notorious for its paltry responses to first-line therapeutic regimens. In contrast to acquired chemoresistance, little is known about the molecular underpinnings of the intrinsic resistance of chemo-naïve NSCLC. Here we report that intrinsic resistance to paclitaxel in NSCLC occurs at a cell-autonomous level because of the uncoupling of mitotic defects from apoptosis. To identify components that permit escape from mitotic stress-induced death, we used a genome-wide RNAi-based strategy, which combines a high-throughput toxicity screen with a live-cell imaging platform to measure mitotic fate. This strategy revealed that prolonging mitotic arrest with a small molecule inhibitor of the APC/cyclosome could sensitize otherwise paclitaxel-resistant NSCLC. We also defined novel roles for CASC1 and TRIM69 in supporting resistance to spindle poisons. CASC1, which is frequently co-amplified with KRAS in lung tumors, is essential for microtubule polymerization and satisfaction of the spindle assembly checkpoint. TRIM69, which associates with spindle poles and promotes centrosomal clustering, is essential for formation of a bipolar spindle. Notably, RNAi-mediated attenuation of CASC1 or TRIM69 was sufficient to inhibit tumor growth in vivo. On the basis of our results, we hypothesize that tumor evolution selects for a permissive mitotic checkpoint, which may promote survival despite chromosome segregation errors. Attacking this adaptation may restore the apoptotic consequences of mitotic damage to permit the therapeutic eradication of drug-resistant cancer cells.

The 3M complex maintains microtubule and genome integrity.

CUL7, OBSL1, and CCDC8 genes are mutated in a mutually exclusive manner in 3M and other growth retardation syndromes. The mechanism underlying the function of the three 3M genes in development is not known. We found that OBSL1 and CCDC8 form a complex with CUL7 and regulate the level and centrosomal localization of CUL7, respectively. CUL7 depletion results in altered microtubule dynamics, prometaphase arrest, tetraploidy, and mitotic cell death. These defects are recaptured in CUL7 mutated 3M cells and can be rescued by wild-type, but not by 3M patient-derived CUL7 mutants. Depletion of either OBSL1 or CCDC8 results in defects and sensitizes cells to microtubule damage similarly to loss of CUL7 function. Microtubule damage reduces the level of CCDC8 that is required for the centrosomal localization of CUL7. We propose that CUL7, OBSL1, and CCDC8 proteins form a 3M complex that functions in maintaining microtubule and genome integrity and normal development.

CUL9 mediates the functions of the 3M complex and ubiquitylates survivin to maintain genome integrity.

The Cullin 9 (CUL9) gene encodes a putative E3 ligase that localizes in the cytoplasm. Cul9 null mice develop spontaneous tumors in multiple organs; however, both the cellular and the molecular mechanisms of CUL9 in tumor suppression are currently unknown. We show here that deletion of Cul9 leads to abnormal nuclear morphology, increased DNA damage, and aneuploidy. CUL9 knockdown rescues the microtubule and mitosis defects in cells depleted for CUL7 or OBSL1, two genes that are mutated in a mutually exclusive manner in 3M growth retardation syndrome and function in microtubule dynamics. CUL9 promotes the ubiquitylation and degradation of survivin and is inhibited by CUL7. Depletion of CUL7 decreases survivin level, and overexpression of survivin rescues the defects caused by CUL7 depletion. We propose a 3M-CUL9-survivin pathway in maintaining microtubule and genome integrity, normal development, and tumor suppression.

Multiple cancer testis antigens function to support tumor cell mitotic fidelity.

While the expression of genes that are normally involved in spermatogenesis is frequently detected in tumors, the extent to which these gene products are required for neoplastic behaviors is unclear. To begin to address their functional relevance to tumorigenesis, we identified a cohort of proteins which display synthetic lethality with paclitaxel in non-small-cell lung cancer and whose expression is biased toward testes and tumors. Remarkably, these testis proteins, FMR1NB, NXF2, MAGEA5, FSIP1, and STARD6, are required for accurate chromosome segregation in tumor cells. Their individual depletion enhances the generation of multipolar spindles, increases mitotic transit time, and induces micronucleation in response to an otherwise innocuous dose of paclitaxel. The underlying basis for abnormal mitosis is an alteration in microtubule function, as their depletion increases microtubule cytaster formation and disrupts microtubule stability. Given these observations, we hypothesize that reactivated testis proteins may represent unique tumor cell vulnerabilities which, if targeted, could enhance responsiveness to antimitotic therapy. Indeed, we demonstrate that combining paclitaxel with a small-molecule inhibitor of the gametogenic and tumor cell mitotic protein TACC3 leads to enhanced centrosomal abnormalities, activation of death programs, and loss of anchorage-independent growth.

Symplekin specifies mitotic fidelity by supporting microtubule dynamics.

Using a pangenomic loss-of-function screening strategy, we have previously identified 76 potent modulators of paclitaxel responsiveness in non-small-cell lung cancer. The top hit isolated from this screen, symplekin, is a well-established component of the mRNA polyadenylation machinery. Here, we performed a high-resolution phenotypic analysis to reveal the mechanistic underpinnings by which symplekin depletion collaborates with paclitaxel. We find that symplekin supports faithful mitosis by contributing to the formation of a bipolar spindle apparatus. Depletion of symplekin attenuates microtubule polymerization activity as well as expression of the critical microtubule polymerization protein CKAP5 (TOGp). Depletion of additional members of the polyadenylation complex induces similar phenotypes, suggesting that polyadenylation machinery is intimately coupled to microtubule function and thus mitotic spindle formation. Importantly, tumor cells depleted of symplekin display reduced fecundity, but the mitotic defects that we observe are not evident in immortalized cells. These results demonstrate a critical connection between the polyadenylation machinery and mitosis and suggest that tumor cells have an enhanced dependency on these components for spindle assembly.

Tumor antigen acrosin binding protein normalizes mitotic spindle function to promote cancer cell proliferation.

Cancer cells manage to divide in the context of gross chromosomal abnormalities. These abnormalities can promote bypass of normal restraints on cell proliferation but at a cost of mitotic vulnerabilities that can be attacked by chemotherapy. Determining how cancer cells balance these issues may permit chemotherapeutic sensitivity to be leveraged more efficiently. From a pan-genomic small interfering RNA screen for modifiers of chemoresponsiveness, we identified the tumor antigen acrosin binding protein (ACRBP)/OY-TES-1 as a specifier of paclitaxel resistance. ACRBP expression is normally restricted to the testes but is detected in a wide variety of cancers, including most ovarian cancers. We found that ACRBP is both necessary and sufficient for paclitaxel resistance in ovarian cancer cell lines and ovarian tumor explants. Moreover, high ACRBP expression correlated with reduced survival time and faster relapse among ovarian cancer patients. We identified the mitotic spindle protein NuMA as an ACRBP-interacting protein that could account for the effects of ACRBP on paclitaxel sensitivity. In cancer cells, ACRBP restricted a NuMA-dependent abrogation of a mitotic spindle assembly that is otherwise pathologic. As a consequence, ACRBP depletion resulted in mitotic errors and reduced proliferative fitness that could be rescued by NuMA codepletion. We propose that the codependent relationship of ACRBP and NuMA in cancer cells reflects their passage through a selection bottleneck during tumor evolution, one which requires the acquisition of traits that normalize mitotic perturbations that originally drove the plasticity of a preneoplastic genome. The molecular definition of such traits as defined by the ACRBP-NuMA complex may represent conceptually ideal intervention targets based on the wide therapeutic windows they may offer.