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Purpose: Metastatic melanoma is a tumor amenable to immunotherapy in part due to the presence of antigen-specific tumor-infiltrating lymphocytes (TIL). These T cells can be activated and expanded for adoptive cell transfer (ACT), which has resulted in relatively high rates of clinical responses. Similarly, immune checkpoint inhibitors, specifically programmed cell death protein 1 (PD-1) blocking antibodies, augment antitumor immunity and increase the influx of T cells into tumors. Thus, we hypothesized that addition of PD-1 inhibition may improve the outcomes for patients undergoing ACT with TILs. Patients and Methods: Patients with stage III/IV metastatic melanoma with unresectable disease who were anti–PD-1 treatment-naïve were enrolled. TILs were generated in the presence of anti–4-1BB antibody in vitro and expanded for ACT. Patients in cohort 1 received TIL infusion followed by nivolumab. Patients in cohort 2 also received nivolumab prior to surgical harvest and during TIL production. Results: A total of 11 patients were enrolled, all of whom were evaluated for response, and nine completed ACT. Predominantly CD8+ TILs were successfully expanded from all ACT-treated patients and were tumor reactive in vitro. The trial met its safety endpoint, as there were no protocol-defined dose-limiting toxicity events. The objective response rate was 36%, and median progression-free survival was 5 months. Two nonresponders who developed new metastatic lesions were analyzed to determine potential mechanisms of therapeutic resistance, which included clonal divergence and intrinsic TIL dysfunction. Conclusions: Combination therapy with TILs and nivolumab was safe and feasible for patients with metastatic melanoma and provides important insights for future therapeutic developments in ACT with TILs.

Purpose:Metastatic melanoma is a tumor amenable to immunotherapy in part due to the presence of antigen-specific tumor-infiltrating lymphocytes (TIL). These T cells can be activated and expanded for adoptive cell transfer (ACT), which has resulted in relatively high rates of clinical responses. Similarly, immune checkpoint inhibitors, specifically programmed cell death protein 1 (PD-1) blocking antibodies, augment antitumor immunity and increase the influx of T cells into tumors. Thus, we hypothesized that addition of PD-1 inhibition may improve the outcomes for patients undergoing ACT with TILs.Patients and Methods:Patients with stage III/IV metastatic melanoma with unresectable disease who were anti–PD-1 treatment-naïve were enrolled. TILs were generated in the presence of anti–4-1BB antibody in vitro and expanded for ACT. Patients in cohort 1 received TIL infusion followed by nivolumab. Patients in cohort 2 also received nivolumab prior to surgical harvest and during TIL production.Results:A total of 11 patients were enrolled, all of whom were evaluated for response, and nine completed ACT. Predominantly CD8+ TILs were successfully expanded from all ACT-treated patients and were tumor reactive in vitro. The trial met its safety endpoint, as there were no protocol-defined dose-limiting toxicity events. The objective response rate was 36%, and median progression-free survival was 5 months. Two nonresponders who developed new metastatic lesions were analyzed to determine potential mechanisms of therapeutic resistance, which included clonal divergence and intrinsic TIL dysfunction.Conclusions:Combination therapy with TILs and nivolumab was safe and feasible for patients with metastatic melanoma and provides important insights for future therapeutic developments in ACT with TILs.

The Supplementary Methods Table contains the antibodies and panels in support of the Flow Cytometry section of the Materials and Methods.

Supplementary Figure 1 shows that central memory T cells were increase in the CD8+ TIL compartment of responders.

Supplementary Table 2 contains the Representativeness Table which demonstrates how this study fits into the larger patient population for metastatic melanoma.

Supplementary Table 1 shows the distribution of Grade 3 and 4 non-hematologic adverse events reported in this study.

Data not shown in reference to related statements in manuscript.

Supplementary Data from Therapeutic Potential of Adult Bone Marrow–Derived Mesenchymal Stem Cells in Prostate Cancer Bone Metastasis

Purpose: Current evidence indicates that an osteoblast lesion in prostate cancer is preceded by osteolysis. Thus, prevention of osteolysis would reduce complications of bone metastasis. Bone marrow–derived mesenchymal stem cells have the ability to differentiate into osteoblast and produce osteoprotegerin, a decoy receptor for the receptor activator for nuclear factor κB ligand, naturally. The present study examined the potential of unmodified mesenchymal stem cells to prevent osteolytic bone lesions in a preclinical mouse model of prostate cancer.Experimental Design: The human prostate cancer cell line PC3 was implanted in tibiae of severe combined immunodeficient mice. After establishment of the tumor, either unmodified or genetically engineered mesenchymal stem cells overexpressing osteoprotegerin was injected at the site of tumor growth. The effects of therapy were monitored by bioluminescence imaging, micro–computed tomography, immunohistochemistry, and histomorphometry.Results: Data indicated significant (P < 0.001) inhibition of tumor growth and restoration of bone in mice treated with unmodified and modified mesenchymal stem cells. Detailed analysis suggested that the donor mesenchymal stem cell inhibited tumor progression by producing woven bone around the growing tumor cells in the tibiae and by preventing osteoclastogenesis.Conclusions: Overcoming the limitation of the number of mesenchymal stem cells available in the bone can provide significant amelioration for osteolytic damage without further modification. (Clin Cancer Res 2009;15(23):7175–85)

In this study, we evaluated the feasibility of expanding tumor infiltrating lymphocytes (TIL) from surgically resected renal cell carcinoma (RCC) tumors. Tumors were collected from 43 patients undergoing surgery to remove primary kidney tumors. Tumor types included clear cell RCC (86.0%), papillary RCC (11.6%) as well as chromophobe RCC (2.3%). Tumors were minced into fragments, placed in individual wells of a 24-well plate containing media with high dose IL-2 (6000 IU/mL), and cultured for four weeks. Successful expansion was considered when at least one fragment expanded to a minimum of 2 wells. Successfully expanded TIL were then evaluated for T-cell phenotypes as well as reactivity to the autologous tumor. Of all RCC tumors collected, TIL were successfully grown for 79% of the samples (34/43). The T-cell phenotype of these TIL was highly variable across samples, although there was a higher percentage of CD4+ T-cells relative to CD8+ T-cells. The reactivity of expanded TIL was then assessed by IFNγ release. We found that TIL secreted IFNγ in response to autologous tumor in 71% of the samples. Together, these results demonstrated the feasibility of expanding functional tumor reactive TIL from RCC. Given that hypoxia plays an important role in the development of RCC tumors and is associated with poor prognosis, we then evaluated the ability to expand TIL under hypoxic conditions. To this end, primary TIL were cultured at various O2 levels (20% O2, 5% O2 or 1% O2) and the cell yield as well as the T-cell memory phenotypes were evaluated. We found that the yield of TIL grown in hypoxic conditions was lower when compared to normoxic conditions (20% O2), suggesting a reduction of TIL proliferation in hypoxia. TIL grown in hypoxic conditions displayed increased percentage of central memory T-cells compared to TIL cultured at 20% O2, as evaluated by CD45RA and CCR7 expression (CD45RA-CCR7+). Primary TIL were then expanded to larger numbers using a rapid expansion protocol (REP) by stimulation with anti-CD3 antibody in the presence of allogenic feeder cells. REP of TIL was carried out at either atmospheric O2 (20% O2) or at a hypoxic O2 level (5% O2), and the T-cell phenotype as well as reactivity to the autologous tumor were assessed. Unlike pre-REP TIL, TIL that underwent REP were able to expand in hypoxic conditions (5% O2) and displayed increased percentage of tissue resident memory T-cells (CD69+CD103+; 19%) when compared to the starting TIL population (0.11%) or TIL expanded at atmospheric O2 (0.17%). When co-cultured with the autologous tumor, hypoxic TILs displayed increased IFNγ, TNFα as well as increased Granzyme B release (p Citation Format: Mohammed Alkhouli, Veronica Martinez-Brockhus, Matthew S. Beatty, Robert J. Gillies, Shari Pilon-Thomas, Jad Chahoud. Ex vivo expansion of tumor infiltrating lymphocytes (TIL) from human renal cell carcinoma (RCC) tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 575.

Immunotherapy, including adoptive cell transfer (ACT) with tumor-infiltrating lymphocytes (TIL) predominantly targets improvement in MHC Class I-mediated anti-tumor immune responses. The primary objective of this study is to better understand the role of CD4+ TIL in ACT as a complementary avenue for therapeutic efficacy. Briefly, CD4+ TIL were isolated by negative selection from metastatic melanoma patients who received TIL therapy at Moffitt Cancer Center on IRB approved protocols. Individual T cell clones were tracked by TCRbeta sequencing to quantify clonal persistence in patients. CD4+ TIL clones were found to be decidedly persistent in a candidate patient who achieved a complete response (CR) after infusion of 88% CD4+ T cells. CD4+ TIL from additional patients were stimulated with anti-CD3/CD28 in vitro and those who were clinical responders demonstrated a pleiotropic cytokine profile marked by an elevated ratio of Th1:Th2 cytokines (p=0.07, n=13). When cultured with APCs loaded with autologous tumor (AT), CD4+ TIL produced high levels of IFN-gamma in an MHC Class II-dependent manner. Induction of MHC Class II on melanoma cell lines and AT determined that CD4+ TIL secreted IFNg and TNFa directly in response to AT. ACT of tumor-reactive CD4+ TIL in immunodeficient (NSG) mice provided significant control of AT growth when compared to non-reactive CD4+ TIL. Preliminary data in syngeneic mouse models also suggests that antigen-specific CD4+ T cells aid in initial tumor rejection, memory formation and epitope spreading, resulting in an overall increased therapeutic efficacy during ACT. This data supports the conclusion that CD4+ TIL are tumor-reactive and instrumental to an effective anti-tumor immune response in cancer patients.

Immunomodulatory drugs, such as lenalidomide, pomalidomide, and CC122 are glutarimide derivatives that alter T cell effector functions, reverse tolerance, and increase cytokine production. We hypothesized that cereblon (CRBN), the molecular target of these drugs, is a negative regulator of T cell function. Therefore, germline Crbn knockout mice were used to investigate its role in T cell regulation. Similar to human T cells treated with immunomodulatory drugs, stimulated Crbn−/− T cells exhibit increased proliferation and cytokine production and maintain this phenotype in the absence of CD28 co-stimulation. Using Gene Set Enrichment Analysis (GSEA) to identify transcriptional drivers of differentially expressed genes, we found that Myc and its family member Max may drive the observed activated phenotype in Crbn−/− T cell. Crbn−/− T cells have Myc-related phenotypic changes, such as increased cell size, CD98 expression, glucose uptake, glutamine and arginine uptake, increased intracellular polyamine biosynthesis, and oxygen consumption. Similar increases in Myc-related processes were observed in activated human T cells treated with immunomodulatory drugs. While c-myc mRNA is similar, Crbn−/− T cells and drug-treated human T cells have prolonged c-Myc protein expression. CRBN, a substrate receptor for the DDB1/Cul4A/Rbx1 E3 ubiquitin ligase complex, has only one known endogenous substrate which is glutamine synthetase (GS). GS interacts with CRBN through acetylated lysine residues yielding a protein that is succeptable to polyubiquitinlyation. Collectively, this suggests that CRBN tunes c-Myc-regulated pathways in activated T cells and that this process is blocked by immunomodulatory drugs.

Druggable targets that reinvigorate T cells may improve the efficacy of immunotherapy in melanoma or other cancers. T cells exist in a hostile tumor microenvironment associated with hypoxia, high lactic acid, and poor nutrient availability which limits their anti-tumor activity. We found that the genetic deficiency of Crbn, a novel E3 ubiquitin ligase substrate receptor, improves effector T cell function. Growth of subcutaneous B16 tumors was slower in Crbn−/− mice and the adoptive transfer of Crbn−/− T cells into mice with established B16 tumors lead to a significant growth reduction. While similar total tumor infiltrating lymphocytes (TIL) numbers and normal splenic populations were present, a significant increase in the proportions of CD44+ and PD1+ CD8+ TIL but not KLRG1+CD8+ TIL were present in Crbn−/− mice. These results suggest that CRBN represses antigen-dependent anti-tumor T cell immunity and represents a novel regulator of T cells in the tumor microenvironment. In melanoma, hypoxia is a mode of T cell suppression. Activated Crbn−/− T cells demonstrated higher extracellular acidification rate, oxygen consumption and glucose uptake compared to Crbn+/+ T cells. Under hypoxic conditions (2% O2), effector cell cytotoxicity is reduced at high densities of B16 melanoma compared to normal atmospheric oxygen (~21% O2) and proliferation is suppressed due to reduction of OxPhos. Using anti-CD3ɛ/anti-CD28 stimulation in hypoxic conditions, Crbn−/− T cells retained their proliferative capacity compared to Crbn+/+ T cells indicating that deletion of Crbn restores metabolic activity of T cells in a hostile tumor microenvironment and may serve as a novel drug target in immunotherapy.

Protein degraders have been developed that can aide in understanding gene function. These chemicals contain complementary bifunctional components that 1) interact with specific proteins, and 2) engage an E3 ligase which leads to polyubiquitination and intracellular degradation by the proteasome. Thalidomide, infamously known for its teratogenic effects, is central to this new technology since it binds cereblon (CRBN) which promiscuously recruits the DDB1/Cul4A/Rbx1 complex to trigger target destruction. Thalidomide, and especially its immunomodulatory derivatives lenalidomide and pomalidomide, induce NK and T-cell activation by degrading IKZF1 which is a transcription factor that suppresses IL-2. Currently, it is debated whether mouse cereblon has conserved function due to a single non-conserved amino acid in mouse CRBN, Ile390 (equivalent to Val388 in human CRBN). First, our work confirms that lenalidomide induces IL-2 and the ubiquitin-dependent degradation of IKZF1 only in human and not mouse T-cells treated with lenalidomide. To further test for species-related effects, a series of theoretical and physical binding assays using mutant CRBN proteins show that the binding of IMiD compounds is not impaired by amino acid differences in mouse CRBN. Using dBET1, a novel thalidomide-JQ1 protein degrader, we show for the first time that mouse CRBN can trigger CRBN-dependent degradation of BRD4 and confirm that the CRBN/DDB1/Cul4A/Rbx1 complex is functional in mouse T-cells. Collectively, our results suggest that IKZF1 has divergent regulation in mouse cells rather than the IMiD complex and provide information relevant to the development of chemical conjugates that induce targeted intracellular protein degradation.

Thalidomide, and its immunomodulatory drug analogs (IMiD) including lenalidomide and pomalidomide, activate T-cells and reverse T-cell anergy in cancer patients. Previously we have shown that cereblon, the molecular target of IMiD compounds, is a negative regulator of T-cell metabolism and signaling following suboptimal antigen stimulation. To explore this mechanistically, differentially-regulated genes were subjected to gene set enrichment analysis (GSEA) for transcriptional regulators and global metabolomic profiling was conducted by mass spectrometry in activated Crbn+/+ versus Crbn−/− T-cells. With results confirmed by 1H-NMR spectra analysis, we show that Crbn deficiency leads to an increase in intermediates of the “Arginine/Proline” pathway and enhanced polyamine (putrescine, spermidine, and spermine) biosynthesis. Wang et al showed previously that c-MYC controls polyamine synthesis downstream of mTORC1 during T-cell activation. In activated Crbn−/− CD8+ T-cells, the kinetics and magnitude of c-Myc mRNA were similar to Crbn+/+ T-cells. However, c-MYC is largely regulated through post-transcriptional mechanisms including ubiquitin proteasome-mediated degradation and regulation of c-Myc mRNA translation. Interestingly, polyamines directly bind U- and AU-rich elements (AREs) in the 3′-untranslated region (3′-UTR) of c-Myc mRNA and spermidine is the substrate for a unique post-translational modification required for the efficient translation of c-MYC. Western blot analysis revealed more rapid and sustained expression of c-MYC in activated Crbn−/− T-cells compared to Crbn+/+ T-cells suggesting that CRBN is a regulator of the MYC-polyamine circuit controlling T-cell metabolic fate.

Cancer gene therapy approaches have benefited greatly from the utilization of molecular-based therapeutics. Of these, adenovirus-based interventions hold much promise as a platform for targeted therapeutic delivery to tumors. However, a barrier to this progression is the lack of native adenovirus receptor expression on a variety of cancer types. As such, any adenovirus-based cancer therapy must take into consideration retargeting the vector to nonnative cellular surface receptors. Predicated upon the knowledge gained in native adenovirus biology, several strategies to transductionally retarget adenovirus have emerged. Herein, we describe the biological hurdles as well as strategies utilized in adenovirus transductional targeting, covering the progress of both adapter-based and genetic manipulation-based targeting. Additionally, we discuss recent translation of these targeting strategies into a clinical setting.