B-cell non-Hodgkin Lymphoma Tumors Research Articles

Affinity Matured CD72 CAR-T Improves Efficacy Versus Low Antigen Density B-Cell Non-Hodgkin Lymphoma Models

Background: Even in the CD19 CAR-T era, many patients with relapsed/refractory B cell non-Hodgkin lymphoma (B-NHL) need additional therapeutic options. Our group has identified CD72 as a therapeutic target for high-risk B-cell malignancies and validated anti-CD72 CAR-Ts (“nanoCAR”s) for these indications in relevant preclinical models (Nix et al., Cancer Discovery 2021, Temple et al., ASH 2022). However, patient B-NHL tumors may express a wide distribution of surface CD72, and our current CAR-T designs may be less efficacious for tumors with low antigen density. We previously described the development of affinity-matured binders extending from our initial published anti-CD72 nanobody clone (NbD4), with ~20-60x higher target affinity (Temple et al.). Here, we tested the hypothesis that these higher affinity binders would better eliminate low antigen density B-NHL models, both in vitro and in vivo, and impact other properties including avidity, and antigen binding epitopes. Methods: Anti-CD72 affinity-matured binders were developed by random mutagenesis of CDR regions of the nanobody clone NbD4 (described in Temple et al.). CAR-T cells were generated by lentiviral transduction. CAR-T cell efficacy was tested by in vitro cytotoxicity assays and Incucyte live-cell imaging against lymphoma cell lines. For in vivo studies, JeKo-1 tumor cells were implanted at 1e6 per mouse and then mice were treated with 3.5e6 CAR+ T-cells at day 7. Tumors from mice bearing JeKo-1 that relapsed on CAR-T treatment were isolated and used in Incucyte assays. Results: We analyzed publicly available datasets of patient cohorts and found that CD72 is widely expressed, though with a broad distribution, across diffuse large B cell lymphoma (DLBCL), follicular lymphoma, and Burkitt lymphoma patient samples. In both DLBCL and chronic lymphocytic leukemia (CLL), high expression of CD72 correlated with significantly shorter overall survival (DLBCL: GSE10846, n=233, p = 0.0382; CLL: GSE22762, n=107, p = 0.00124, log-rank test). We confirmed expression of CD72 on 8 lymphoma cell lines by flow cytometry. In vitro against a subset of these lines, affinity-matured clone NbD4.13 showed improved cytotoxicity compared to parental NbD4 and similar cytotoxicity to a humanized variant (H24 clone). We developed a JeKo-1 model of low CD72 antigen density (4.7x MFI reduction) by CRISPR/Cas9 knockout of endogenous CD72 and re-expression of a recombinant construct. In this “CD72 low” model, affinity matured clone NbD4.13 led to improved cytotoxicity vs. H24 ( p = 5.1e-4 by t-test). We previously showed that tumor isolated from JeKo-1 mice treated with H24 CAR appear to have reduced antigen expression at relapse (Temple et al.). We cocultured these post-relapse JeKo-1 tumors with various CD72 nanoCARs and found that NbD4.13 eliminates tumor significantly faster at 24 hours than H24 by live-cell imaging (p = 0.0054 by t-test). In vivo in a JeKo-1 model, NbD4.13 also out-performed the H24 clone with respect to survival benefit (p = 0.011 by log-rank for survival) ( Fig. 1). To assess if additional differences beyond affinity may determine the efficacy of NbD4.13, we performed structural modeling using AlphaFold and HADDOCK. However, this analysis predicts NbD4, NbD4.13 and H24 bind a similar non-linear epitope on the CD72 monomer. Furthermore, acoustic force microscopy did not reveal a significant difference in binding avidity for any of the compared CD72 nanoCAR constructs. Finally, we evaluated whether it could be possible to pharmacologically increase CD72, as co-treatment strategy to enhance efficacy of CD72 nanoCARs. We found that the Protein Kinase C inhibitor Bryostatin-1, previously demonstrated to increase CD22 antigen density (Ramakrishna et al., Clin Cancer Res 2019), could also significantly increase surface CD72 in vitro in both B-ALL and B-NHL models ( Fig. 2). Conclusion: NbD4.13-based anti-CD72 CAR is a promising candidate for further preclinical development for treatment of relapsed/refractory B-cell non-Hodgkin lymphoma. Given anticipated heterogeneity in CD72 expression in various lymphomas, pharmacologic co-treatment strategies may be considered to modulate tumor antigen density.

Subtype-specific and co-occurring genetic alterations in B-cell non-Hodgkin lymphoma.

B-cell non-Hodgkin lymphoma (B-NHL) encompasses multiple clinically and phenotypically distinct subtypes of malignancy with unique molecular etiologies. Common subtypes of B-NHL, such as diffuse large B-cell lymphoma, have been comprehensively interrogated at the genomic level, but rarer subtypes, such as mantle cell lymphoma, remain less extensively characterized. Furthermore, multiple B-NHL subtypes have thus far not been comprehensively compared using the same methodology to identify conserved or subtype-specific patterns of genomic alterations. Here, we employed a large targeted hybrid-capture sequencing approach encompassing 380 genes to interrogate the genomic landscapes of 685 B-NHL tumors at high depth, including diffuse large B-cell lymphoma, mantle cell lymphoma, follicular lymphoma, and Burkitt lymphoma. We identified conserved hallmarks of B-NHL that were deregulated in the majority of tumors from each subtype, including frequent genetic deregulation of the ubiquitin proteasome system. In addition, we identified subtype-specific patterns of genetic alterations, including clusters of co-occurring mutations and DNA copy number alterations. The cumulative burden of mutations within a single cluster were more discriminatory of B-NHL subtypes than individual mutations, implicating likely patterns of genetic cooperation that contribute to disease etiology. We therefore provide the first cross-sectional analysis of mutations and DNA copy number alterations across major B-NHL subtypes and a framework of co-occurring genetic alterations that deregulate genetic hallmarks and likely cooperate in lymphomagenesis.

Genetically re-engineered K562cells significantly expand and functionally activate cord blood natural killer cells: Potential for adoptive cellular immunotherapy.

Natural killer (NK) cells play a significant role in reducing relapse in patients with hematological malignancies after allogeneic stem cell transplantation, but NK cell number and naturally occurring inhibitory signals limit their capability. Interleukin-15 (IL-15) and 4-1BBL are important modulators of NK expansion and functional activation. To overcome these limitations, cord blood mononuclear cells (CB MNCs) were exvivo expanded for 7days with genetically modified K562-mbIL15-41BBL (MODK562) or wild-type K562 (WTK562). NK cell expansion; expression of lysosome-associated membrane protein-1 (LAMP-1), granzyme B, and perforin; and invitro and invivo cytotoxicity against B-cell non-Hodgkin lymphoma (B-NHL) were evaluated. Invivo tumor growth in B-NHL-xenografted nonobese diabetic severe combined immune deficient (NOD-scid) gamma (NSG) mice was monitored by tumor volume, cell number, and survival. CB MNCs cultured with MODK562 compared with WTK562 demonstrated significantly increased NK expansion (thirty-fivefold, p<0.05); LAMP-1 (p<0.05), granzyme B, and perforin expression (p<0.001); and invitro cytotoxicity against B-NHL (p<0.01). Xenografted mice treated with MODK562 CB experienced significantly decreased B-NHL tumor volume (p=0.0086) and B-NHL cell numbers (p<0.01) at 5weeks and significantly increased survival (p<0.001) at 10weeks compared with WTK562. In summary, MODK562 significantly enhanced CB NK expansion and cytotoxicity, enhanced survival in a human Burkitt's lymphoma xenograft NSG model, and could be used in the future as adoptive cellular immunotherapy after umbilical CB transplantation. Future directions include expanding anti-CD20 chimeric receptor-modified CB NK cells to enhance B-NHL targeting invitro and invivo.

Rb silencing mediated by the down-regulation of MeCP2 is involved in cell transformation induced by long-term exposure to hydroquinone.

Hydroquinone (HQ), a metabolite of benzene, is a well-known human carcinogen; however, its molecular mechanisms of action remain unclear. MeCP2 has been traditionally described as a transcriptional repressor, though growing evidence indicates that it also activates gene expression. Here, we investigated whether some epigenetic machinery genes are aberrantly expressed as target tumor suppressor genes in HQ-transformed TK6 lymphoblastoid cells. Our results showed that treatment with 5-Aza-2'-deoxycytidine or trichostatin A enhanced the expression of Rb, resulting in cell arrest in G1-phase, and subsequently, an increase in apoptosis and a decrease in cell growth. Moreover, we hypothesised that Rb was silenced by the down-regulation of MeCP2 in HQ-transformed cells, resulting in the dynamic expression of Rb and epigenetic machinery proteins in HQ-transformed cells at different time points. The expression of Rb and MeCP2 in patients with B-cell non-Hodgkin's lymphoma (B-NHL) showed that positive staining for MeCP2 or Rb was significantly lower in B-NHL tumor tissues, and these changes were significantly and negatively correlated with the grade of B-NHL. The restoration of MeCP2 in HQ-transformed cells enhanced the expression of Rb, promoted cell apoptosis, and inhibited cell growth. The changes in the expression patterns of MeCP2 and Rb were inversely correlated with the degree of DNA methylation. A ChiP assay revealed that MeCP2 proteins were recruited to the Rb promoter with lower 5'-methylcytosine levels. In conclusion, we demonstrated that the down-regulation of MeCP2 silences Rb, a process involved in cell transformation resulting from long-term exposure to HQ. © 2016 Wiley Periodicals, Inc.

Clinical significance of mast cells and IL-9 in B-NHL

To investigate the role of mast cells and interleukin-9 (IL-9) in B-cell non-Hodgkin lymphoma (B-NHL) development and its clinical significance. The expression level of CD117 in tumor tissues of 32 B-NHL patients was determined by Western blot. The infiltration of CD117⁺ mast cells (MCs) in human B-NHL tumor tissues was observed by immunohistochemistry staining. To evaluate the correlations between the data from CD117⁺ MCs and biological markers of human B-NHL, a Spearman correlation coefficient (rs) was calculated. IL-9 levels in sera of B-NHL patients were measured by ELISA. Effects of IL-9 on expressions of functional genes of mouse bone marrow-derived mast cells (BMMCs) were detected by real-time quantitative RT-PCR. The expression of CD117 was upregulated significantly in human B-cell NHL involved tissues when compared with that of controls (0.0551±0.0064 vs 0.0192±0.0072, P<0.01). Infiltration of more CD117⁺ MCs was found in tissues from B-cell NHL subjects compared with that of controls. IL-9 level in serum samples from patients with B-cell NHL was higher than that from healthy controls. Addition of rIL-9 to the culture gave rise to increase in the purity of mouse BMMCs in the first three weeks. In vitro culture experiments showed that the addition of IL-9 could induce the differentiation of mouse BMMC and the expressions of MC-related genes, including CD117, Fcer1α, Mcpt1 and Mcpt5. Our study showed that IL-9 promoted immune response mediated by MCs, and probably played important roles in B-NHL growth. Pharmacological or targeted inhibition of mast cells or IL-9 activity may provide new strategy for B-cell NHL therapy.

Sepantronium Bromide (YM155) Enhances Response of Human B-Cell Non-Hodgkin Lymphoma to Rituximab

In the treatment of B-cell non-Hodgkin lymphoma (B-NHL) rituximab improves long-term survival in combination with conventional chemotherapy. However, because the majority of patients with B-NHL eventually relapse, the development of more effective therapies is needed. Here, we evaluated the antitumor effects of a combination treatment involving sepantronium bromide (YM155), a first-in-class survivin suppressant, and rituximab in B-NHL xenograft mice models. To determine the efficacy of the combination treatment, YM155- and rituximab-treated B-NHL cell xenografted mice were monitored for tumor size and survival and subjected to 2'-deoxy-2'-(18)F-fluoro-D-glucose ((18)F-FDG) and 3'-(18)F-fluoro-3'-deoxythymidine ((18)F-FLT) positron emission tomography (PET) imaging. In addition, the cell proliferation status of excised tumors was examined by Ki-67 immunohistochemistry. In DB, WSU-DLCL-2, and Mino xenograft-bearing mice, the combination treatment of YM155 and rituximab induced significant tumor growth inhibition and tumor regression compared with either single agent. On day 3 after the initiation of treatment a significant decrease in both (18)F-FDG and (18)F-FLT tumor uptake from pretreatment levels was observed in combination treatment groups. The Ki-67 proliferation index was significantly decreased on day 3 in the xenograft models treated with combination treatment, suggesting that the combination of YM155 and rituximab reduced cell proliferation and glucose metabolism. Furthermore, compared with monotherapy, combination treatment prolonged survival times of severe combined immunodeficient mice with disseminated WSU-FSCCL and Jeko B-NHL tumors. Our findings demonstrate that YM155 and rituximab combination treatment enhances antitumor activity in B-NHL xenografts, and (18)F-FLT and (18)F-FDG PET imaging may allow the early functional evaluation of treatment responses in patients with B-NHL.

Malignant B Cell-Derived TGF-β Controls the Generation of TH1, TH17 and Treg Cells in the Tumor Microenvironment of B-Cell Non-Hodgkin Lymphoma (NHL).

Background: Our previous work has shown that malignant B cells induce the development of intratumoral Treg cells that inhibit the host anti-tumor response. In contrast to an increase in Treg cells, we found that the number of effector T helper cells (TH1, TH2 and TH17) was low in B-cell NHL tumors, suggesting an imbalance between Treg and TH cells in the tumor microenvironment. Understanding the mechanism(s) of this imbalance is important to the development of treatments to enhance host immunity and in previous work we have shown that signaling through CD70, CD80 and CD86 plays a role. Since soluble factors, particularly TGF-β, have an important role in directing T-cell differentiation, we evaluated in this study the role of TGF-β in the lymphoma microenvironment.Goal: To determine the effect of TGF-β on the generation of intratumoral TH1, TH17 and Treg cells in human B-cell NHL.Results: Human B-cell NHL specimens were obtained from consenting patients and were used for all experiments. Using an ELISA assay, we found that malignant B cells variably secrete TGF-β - median 100 pg/ml per million cells (range: undetectable −229 pg/ml, n=7). Using flow cytometry, we showed that addition of exogenous TGF-β enhanced the expression of Foxp3+ in activated CD4+ or CD4+CD45RA+ or CD4+CD45RO+ nodal T cells, suggesting that TGF-β promotes the generation of Treg cells in tumor microenvironment. In contrast, TGF-β suppressed expression of IFN-g in activated CD4+ T cells and inhibited the up-regulation of IL-12 and IL-23-induced IFN-γ expression in CD4+ cells, indicating that TGF-β suppresses the generation of TH1 cells. TGF-β alone slightly inhibited IL-17 expression in CD4+ T cells; however, TGF-β, in the presence of IL-6 and IL-23, upregulated IL-17 expression in CD4+ T cells, suggesting proinflammatory cytokines are able to reverse the suppression induced by TGF-β. These results indicate that TGF-β plays an important role in the regulation of intratumoral TH17 cell generation. In additional experiments, TGF-β was found to exert a suppressive effect on the proliferation of both CD4+ and CD8+ intratumoral T cells. However, treatment with TGF-β enhanced IL-2 production by intratumoral CD4+ T cells detected by intracellular staining of flow cytometry. Interruption of IL-2 signaling by anti-IL-2 Ab abolished the upregulation of TGF-β-mediated Foxp3 expression and enhanced the production of IL-17 in CD4+ T cells. Furthermore, treatment with anti-IL-2 Ab reversed the inhibition of NHL B cell-mediated TH17 cell generation.Conclusion: These results suggest that TGF-β controls the generation of TH1, TH17 and Treg cells contributing to the imbalance of effector TH cells and inhibitory Treg cells in the tumor microenvironment of B-cell NHL through IL-2. Since malignant B-cells produce TGF-β, these results further support the important role of malignant B cells in the regulation of intratumoral T cell generation and the host immune response.

Rituximab blocks binding of radiolabeled anti-CD20 antibodies (Ab) but not radiolabeled anti-CD45 Ab

AbstractRituximab therapy is associated with a long in vivo persistence, yet little is known about the effect of circulating rituximab on B-cell non-Hodgkin lymphoma (B-NHL) targeting by the other available anti-CD20 monoclonal antibodies (MoAbs) 131iodine-tositumomab and 90yttrium-ibritumomab tiuxetan. Therefore we assessed the impact of preexisting rituximab on the binding and efficacy of second anti-CD20 MoAbs to B-NHL and determined whether targeting an alternative lymphoma-associated antigen, CD45, could circumvent this effect. We demonstrated that rituximab concentrations as low as 5 μg/mL nearly completely blocked the binding of a second anti-CD20 MoAbs (P < .001), but had no impact on CD45 targeting (P = .89). Serum from patients with distant exposures to rituximab also blocked binding of anti-CD20 MoAbs to patient-derived rituximab-naive B-NHL at concentrations at low as 7 μg/mL, but did not affect CD45 ligation. A mouse xenograft model (Granta, FL-18, Ramos cell lines) showed that rituximab pretreatment significantly reduced B-NHL targeting and tumor control by CD20-directed radioimmunotherapy (RIT), but had no impact on targeting CD45. These findings suggest that circulating rituximab impairs the clinical efficacy of CD20-directed RIT, imply that novel anti-CD20 MoAbs could also face this same limitation, and indicate that CD45 may represent an alternative target for RIT in B-NHL.

An apparent interlocus gene conversion-like event at a putative tumor suppressor gene locus on human chromosome 6q27 in a Burkitt's lymphoma cell line.

A region of minimal deletion in B-cell non-Hodgkin's lymphoma (B-NHL) has recently been defined between D6S186 and D6S227 spanning 5-9 Mb at 6q26-q27, predicting the presence of at least one tumor suppressor gene (TSG) at this locus. During the construction of a deletion map in the B-NHL tumor panel, we report the identification of a Burkitt's lymphoma cell line, BL74, having an apparent homozygous deletion at the D6S347 locus, internal to the critical region. Since this case may facilitate the localization of the target TSG, a detailed structural molecular characterization and search for candidate genes were undertaken at this locus. While BL74 underwent a loss of heterozygosity at 6q26-q27, D6S347 was also likely subjected to a somatic interlocus gene conversion-like event between two homologous but distinct loci, resulting in the homozygous replacement of a 1860- to 2067-bp segment of one locus with the corresponding segment copied from the other locus. Two genes at this locus were identified, but their lack of expression in B-cell lineages tentatively excludes them as candidate TSGs. Another still unidentified gene at this locus may be disrupted by the gene conversion-like event, which would represent a novel mechanism of TSG inactivation.

Cloning and Mapping of Human Chromosome 6q26–q27 Deleted in B-Cell Non-Hodgkin Lymphoma and Multiple Tumor Types

Frequent deletions of the distal region on the long arm of chromosome 6 have been reported in multiple human tumors including B-cell non-Hodgkin lymphoma (B-NHL), suggesting the presence of one or more tumor suppressor genes (TSGs) at this locus. Previously, we identified a region of minimal molecular deletion at 6q25–q27 (RMD-1) in B-NHL cases. To facilitate positional cloning efforts to identify the RMD-1 TSG(s), a yeast artificial chromosome (YAC) contig consisting of 110 clones was constructed across 6q26–q27 by sequence-tagged site/probe contentmapping. The contig integrates 79 ordered markers including restriction fragment length polymorphisms, minisatellites, microsatellites, YAC-insert termini, expressed sequence tags, and known genes. It spans 34 cM and has a minimal tiling path of approximately 12 clones, covering an estimated 9–14 Mb with nearly every marker on the map showing at least double linkage to its adjacent markers. Dual-color fluorescencein situhybridization of selected marker pairs on normal pachytene chromosome 6 further confirmed the YAC-based mappings. Utilizing a loss of constitutional heterozygosity assay in the B-NHL tumor panel, 24 additional 6q26–q27 polymorphic markers (21 mapping to the contig) further defined RMD-1 between markers D6S186 proximally and D6S227 distally. The minimal tiling path of the B-NHL RMD-1 consists of approximately 8 YAC clones, providing a size estimate of 5–9 Mb. This interval contains, in their entirety, several smaller candidate TSG critical regions previously delimited in other tumor systems. The AF-6 gene, mapping within RMD-1, revealed no mutations in a small subset of B-NHL. The deletion and physical maps presented herein provide a framework for the identification of the gene(s) involved in B-NHL as well as other malignancies and diseases mapped to this region and provide the initial reagents for large-scale genomic sequencing.

The B-Lymphocyte Maturation Promoting Transcription Factor BLIMP1/PRDI-BF1 Maps to D6S447 on Human Chromosome 6q21–q22.1 and the Syntenic Region of Mouse Chromosome 10

The human PRDI-BF1 or BLIMP1 gene and its mouse homolog Blimp1 are members of the recently realized PR domain family that includes the retinoblastoma interacting zinc finger gene RIZ and the MDS1-EVI1 leukemia cancer gene. The specific high-level expression of Blimp1 in late B and plasma cells, its induction during B-cell differentiation, and its ability to drive B-cell maturation suggest that this gene may play a role in the differentiation and pathogenesis of B cells. We have now mapped the physical location of BLIMP1 near the marker D6S447 on human chromosome 6q21–q22.1; we have also mapped Blimp1 to mouse Chromosome 10 at 14 cM distal to the Myb locus and to a region homologous to the location of BLIMP1. Deletions of the 6q21–q22 region are common in several human malignancies, particularly in B-cell non-Hodgkin lymphoma (B-NHL). The data led us to suggest that BLIMP1 may be a candidate B-NHL tumor suppressor gene.