Prognostic value of MYC rearrangement in cases of B-cell lymphoma, unclassifiable, with features intermediate between diffuse large B-cell lymphoma and Burkitt lymphoma

BACKGROUND:

B‐cell lymphoma, Unclassifiable with features intermediate between diffuse large B‐cell lymphoma (DLBCL) and Burkitt lymphoma, for convenience referred to here as unclassifiable B‐cell lymphoma, is a category in the 2008 World Health Organization system used for a group of histologically aggressive neoplasms that are difficult to classify definitively. Currently, there is no established standard therapy for these neoplasms.

METHODS:

The authors assessed MYC status and correlated it with treatment response and outcome in a group of 52 patients with unclassifiable B‐cell lymphoma treated with either a standard DLBCL regimen (R‐CHOP [rituximab plus cyclophosphamide, doxorubicin, vincristine, and prednisolone‐related therapy]) or more intensive regimens, such as R‐hyper‐CVAD (rituximab plus hyperfractionated cyclophosphamide, vincristine, doxorubicin, and dexamethasone alternating with high‐dose methotrexate and cytarabine). The regimens were selected by the treating clinicians based on the overall clinical and pathological findings.

RESULTS:

Thirty (58%) unclassifiable B‐cell lymphomas had MYC abnormalities (MYC⁺) including 27 with rearrangement, 2 with amplification, and 1 with both. The MYC⁺ and MYC^? groups were similar in their age distribution and International Prognostic Index scores. Progression‐free survival of patients with MYC⁺ unclassifiable B‐cell lymphoma treated initially with R‐CHOP was significantly worse than patients treated with R‐hyper‐CVAD (P = .0358). In contrast, for the MYC^? unclassifiable B‐cell lymphoma group, some patients responded to R‐CHOP, and others were refractory to R‐hyper‐CVAD.

CONCLUSIONS:

MYC aberrations are common in unclassifiable B‐cell lymphoma. The presence of MYC aberrations identifies a patient subset that requires more aggressive therapy than R‐CHOP. In contrast, MYC^? unclassifiable B‐cell lymphoma patients responded variably to either R‐CHOP or aggressive therapy, and the latter showed no survival advantage. Cancer 2011;. © 2011 American Cancer Society.     

Chimeric antigen receptor‐engineered cytokine‐induced killer cells overcome treatment resistance of pre‐B‐cell acute lymphoblastic leukemia and enhance survival

Pre‐emptive cancer immunotherapy by donor lymphocyte infusion (DLI) using cytokine‐induced killer (CIK) cells may be beneficial to prevent relapse with a reduced risk of causing graft‐versus‐host‐disease. CIK cells are a heterogeneous effector cell population including T cells (CD3⁺ CD56^?), natural killer (NK) cells (CD3^?CD56⁺) and natural killer T (T‐NK) cells (CD3⁺ CD56⁺) that exhibit non‐major histocompatibility complex (MHC)‐restricted cytotoxicity and are generated by ex vivo expansion of peripheral blood mononuclear cells in the presence of interferon (IFN)‐γ, anti‐CD3 antibody, interleukin‐2 (IL‐2) and interleukin‐15 (IL‐15). To facilitate selective target‐cell recognition and enhance specific cytotoxicity against B‐cell acute lymphoblastic leukemia (B‐ALL), we transduced CIK cells with a lentiviral vector encoding a chimeric antigen receptor (CAR) that carries a composite CD28‐CD3ζ domain for signaling and a CD19‐specific scFv antibody fragment for cell binding (CAR 63.28.z). In vitro analysis revealed high and specific cell killing activity of CD19‐targeted CIK/63.28.z cells against otherwise CIK‐resistant cancer cell lines and primary B‐ALL blasts, which was dependent on CD19 expression and CAR signaling. In a xenograft model in immunodeficient mice, treatment with CIK/63.28.z cells in contrast to therapy with unmodified CIK cells resulted in complete and durable molecular remissions of established primary pre‐B‐ALL. Our results demonstrate potent antileukemic activity of CAR‐engineered CIK cells in vitro and in vivo, and suggest this strategy as a promising approach for adoptive immunotherapy of refractory pre‐B‐ALL.     

NY‐ESO‐1 specific antibody and cellular responses in melanoma patients primed with NY‐ESO‐1 protein in ISCOMATRIX and boosted with recombinant NY‐ESO‐1 fowlpox virus

Vaccination strategies based on repeated injections of NY‐ESO‐1 protein formulated in ISCOMATRIX particles (NY‐ESO‐1 ISCOMATRIX) have shown to elicit combined NY‐ESO‐1 specific antibody and T cell responses. However, it remains unclear whether heterologous prime‐boost strategies based on the combination with NY‐ESO‐1 ISCOMATRIX with different NY‐ESO‐1 boosting reagents could be used to increase NY‐ESO‐1 CD8⁺ or CD4⁺ T cell responses. To address this question, we carried out a randomized clinical trial in 39 high‐risk, resected melanoma patients vaccinated with NY‐ESO‐1 ISCOMATRIX, and then boosted with repeated injections of either recombinant fowlpox virus encoding full length NY‐ESO‐1 (rF‐NY‐ESO‐1) (Arm A) or NY‐ESO‐1 ISCOMATRIX alone (Arm B). We have comprehensively analyzed NY‐ESO‐1 specific T cells and B cells response in all patients before and after vaccination for a total of seven time points per patient. NY‐ESO‐1 ISCOMATRIX alone elicited a strong NY‐ESO‐1 specific CD4⁺ T cell and antibody response, which was maintained by both regiments at similar levels. However, CD8⁺ T cell responses were significantly boosted in 3 out of 18 patients in Arm A after the first rF‐NY‐ESO‐1 injection and such responses were maintained until the end of the trial, while no patients in Arm B showed similar CD8⁺ T cell responses. In addition, our results clearly identified immunodominant regions in the NY‐ESO‐1 protein: NY‐ESO‐1_79–102 and NY‐ESO‐1_115–138 for CD4+ T cells and NY‐ESO‐1_85–108 for CD8+ T cells in a large proportion of vaccinated patients. These regions of NY‐ESO‐1 protein should be considered in future clinical trials as immunodominant epitopes.     

Targeting high‐grade B cell lymphoma with CD19‐specific T cells

Adoptive T cell therapy is an important additional treatment option for malignant diseases resistant to chemotherapy. Using a murine high‐grade B cell lymphoma model, we have addressed the question whether the B cell differentiation antigen CD19 can act as rejection antigen. CD19^?/? mice inoculated with CD19⁺ B cell lymphoma cells showed higher survival rates than WT mice and were protected against additional tumor challenge. T cell depletion prior to tumor transfer completely abolished the protective response. By heterotypic vaccination of CD19^?/? mice against murine CD19, survival after tumor challenge was significantly increased. To define protective epitopes within the CD19 molecule, T cells collected from mice that had survived the tumor transfer were analyzed for IFNγ secretion in response to CD19‐derived peptides. The majority of mice exhibited a CD4⁺ T cell response to CD19 peptide 27, which was the most dominant epitope after CD19 vaccination. A peptide 27‐specific CD4⁺ T cell line protected CD19^?/? mice against challenge with CD19⁺ lymphoma and also cured a significant proportion of WT mice from recurrent disease in a model of minimal residual disease after chemotherapy. In conclusion, our data highlight CD19‐specific CD4⁺ T cells for adoptive T cell therapy of B cell lymphomas.     

IgG1 anti‐epidermal growth factor receptor antibodies induce CD8‐dependent antitumor activity

Anti‐EGFR monoclonal antibodies (mAb) like Cetuximab are commonly used for treatment of EGFR⁺ solid tumors mainly by exerting their therapeutic effect through inhibition of signal transduction. Additionally, IgG1 is a potent mediator of antibody‐dependent cytotoxicity (ADCC). In case of the IgG1, Cetuximab induction of ADCC in vivo is controversially discussed. In our study, we investigated the efficiency of Cetuximab‐mediated ADCC in a humanized mouse tumor model in vivo and analyzed the contribution of immunologic processes toward antitumor activity. Therefore, we used immunodeficient NOD/Scid mice transgenic for human MHC class I molecule HLA‐A2 and adoptively transferred human HLA‐A2⁺ PBMC after engraftment of human epidermoid cell carcinoma A431. Here, we show that high doses of anti‐EGFR mAb induced strong tumor regression independent of the immune system. However, tumor regression by low doses of anti‐EGFR mAb treatment was ADCC dependent and mediated by tumor infiltrating CD8⁺ T effector cells. This novel mechanism of ADCC conducted by CD8⁺ T effector cells was restricted to IgG1 anti‐EGFR mAb, dependent of binding to CD16 on T cells and could be inhibited after EGFR blockade on tumor cells. Furthermore, CD8⁺ T effector cell‐mediated ADCC was enhanced in the presence of IL‐15 and strongly improved after glycosylation of anti‐EGFR mAb indicating the potential of glycoengineered therapeutic mAb as efficient biologicals in cancer therapy.     

Alternating R‐CHOP and R‐cytarabine is a safe and effective regimen for transplant‐ineligible patients with a newly diagnosed mantle cell lymphoma

Implementation of cytarabine into induction therapy became standard of care for younger patients with mantle cell lymphoma (MCL). On the basis of its beneficial impact, many centers incorporated cytarabine at lower doses also into first‐line treatments of elderly patients. We conducted a multicenter observational study that prospectively analyzed safety and efficacy of alternating 3 + 3 cycles of R‐CHOP and R‐cytarabine for newly diagnosed transplant‐ineligible MCL patients. A total of 73 patients were enrolled with median age 70 years. Most patients had intermediate (39.7%) and high‐risk (50.7%) disease according to MCL international prognostic index. Rituximab maintenance was initiated in 58 patients. Overall response rate reached 89% by positron emission tomography–computed tomography, including 75.3% complete remissions. Two patients (2.7%) did not complete the induction therapy because of toxicity. Three patients (4.1%) were considered nonresponders, which led to therapy change before completion of induction. Estimated progression‐free survival and overall survival were 51.3% and 68.6% at 4 years, respectively. Mantle cell lymphoma international prognostic index, bulky disease (≥ 5 cm), and achievement of positron emission tomography–negativity independently correlated with progression‐free survival. Grade 3 to 4 hematologic and nonhematologic toxicity was documented in 48% and 20.5% patients, respectively. Alternation of R‐CHOP and R‐cytarabine represents feasible and very effective regimen for elderly/comorbid MCL patients. This study was registered at GovTrial ( clinicaltrials.gov ) NCT03054883.     

Efficacy and toxicity of 2 schedules of frontline rituximab plus cyclophosphamide,doxorubicin, vincristine,and prednisone plus bortezomib in patients with B‐cell lymphoma

BACKGROUND:

Bortezomib demonstrated promising activity in lymphomas. The authors conducted a randomized phase 2 trial of frontline rituximab plus cyclophosphamide, doxorubicin, vincristine, and prednisone (R‐CHOP) with the addition of bortezomib in patients with B‐cell lymphoma.

METHODS:

Patients were randomized between 2 schedules of bortezomib, Arm A (Days 1, 4, 8, and 11) and Arm B (Days 1 and 8), combined with 6 cycles of R‐CHOP. For the first patients (Step 1), bortezomib was given at a dose of 1 mg/m² in Arm A and 1.3 mg/m² in Arm B. For the next patients (Step 2), doses were increased to 1.3 mg/m² and 1.6 mg/m² in Arms A and B, respectively. The primary endpoint was the rate of complete response (CR) and unconfirmed CR (CR/CRu) after 6 cycles.

RESULTS:

Forty‐nine patients were included in the study, and 41 patients (84%) achieved a CR/CRu, ie, 18 of 20 patients (90%) in Arm A and 23 of 29 patients (79%) in Arm B. There were 6 partial responses and 2 patients with progressive disease. Neurologic toxicity occurred in 21 patients (43%) and was grade 2 in 11 patients (7 patients in Step 2) and grade 3 in 10 patients (9 patients in Step 2). Other grade 3 and 4 toxicities included constipation (n = 1), infections (n = 3), and cardiac events (n = 2). Grade 3 and 4 thrombocytopenia and leucopenia occurred in 14% and 41% of cycles, respectively.

CONCLUSIONS:

R‐CHOP + bortezomib was an effective regimen and produced an 84% CR rate. However, the dose‐limiting neurotoxicity should be kept in mind for further trials with vinca alkaloids or other potentially neurotoxic drugs combination therapies. Cancer 2009. © 2009 American Cancer Society.     

PD‐1 blockade enhances the antitumor efficacy of GM‐CSF surface‐modified bladder cancer stem cells vaccine

Eliminating cancer stem cells (CSCs) is a key issue in eradicating tumor. The streptavidin–granulocyte‐macrophage‐colony stimulating factor (SA–GM‐CSF) surface‐modified bladder CSCs vaccine previously developed using our protein–anchor technology could effectively induce specific immune response for eliminating CSCs. However, program death receptor‐1 (PD‐1)/program death ligand 1 (PD‐L1) signaling in tumor microenvironment results in tumor‐adaptive immune resistance. Although the CSCs vaccine could increase the number of CD8⁺T cells, a part of these CD8⁺T cells expressed PD‐1. Moreover, the CSCs vaccine upregulated the PD‐L1 expression of tumor cells, resulting in immune resistance. Adding PD‐1 blockade to the CSCs vaccine therapy increased the population of CD4⁺, CD8⁺ and CD8⁺IFN‐γ⁺ but not CD4⁺ Foxp3⁺T cells and induced the highest production of IFN‐γ. PD‐1 blockade could effectively enhance the functions of tumor‐specific T lymphocytes generated by the CSCs vaccine. This combination therapy improved the cure rate among mice and effectively protected the mice against a second CSCs cell challenge, but not a RM‐1 cell challenge. These results indicate that PD‐1 blockade combined with the GM‐CSF‐modified CSCs vaccine effectively induced a strong and specific antitumor immune response against bladder cancer.     

Combination immunotherapy with interleukin‐2 surface‐modified tumor cell vaccine and programmed death receptor‐1 blockade against renal cell carcinoma

Immunotherapy may be an effective way to prevent postoperative recurrence of renal cell carcinoma. Streptavidin‐interleukin‐2 (SA‐IL‐2) surface‐modified tumor cell vaccine developed through our protein‐anchor technology could induce specific antitumor T‐cell responses, but this immunotherapy cannot completely eradicate the tumor. These effector T cells highly expressed programmed death receptor‐1 (PD‐1), and the expression of programmed death ligand‐1 (PD‐L1) in the tumor environment also was upregulated after SA‐IL‐2‐modified vaccine therapy. PD‐1/PD‐L1 interaction promotes tumor immune evasion. Adding PD‐1 blockade to SA‐IL‐2‐modified vaccine therapy increased the number of CD4⁺, CD8⁺ and CD8⁺interferon‐γ⁺ but not CD4⁺Foxp3⁺ T cells. PD‐1 blockade could rescue the activity of tumor‐specific T lymphocytes induced by the SA‐IL‐2‐modified vaccine. Combination therapy delayed tumor growth and protected mice against a second Renca cells but not melanoma cells challenge. Taken together, PD‐1 blockade could reverse immune evasion in the treatment with SA‐IL‐2‐modified vaccine, and eventually induce a stronger specific antitumor immune response against renal cell carcinoma.     

Efficacy and safety of the combination of rituximab,fludarabine, and mitoxantrone for rituximab‐naive,recurrent/refractory follicular non‐Hodgkin lymphoma with high tumor burden

BACKGROUND:

This phase 2 trial was undertaken to evaluate the efficacy and safety of rituximab combined with intravenous fludarabine and mitoxantrone (R‐FM) for patients with recurrent/refractory follicular lymphoma who had high tumor burden according to Groupe d'Etude des Lymphomes Folliculaires (GELF) criteria.

METHODS:

Fifty patients were enrolled who had received a maximum of 2 previous regimens, including 1 cyclophosphamide, doxorubicin, vincristine, and prednisone (CHOP)/CHOP‐like regimen but no previous exposure to rituximab, fludarabine, or mitoxantrone. At baseline, 58% of patients had bulky disease (lesion >7cm), 56% had high‐risk Follicular Lymphoma International Prognostic Index (FLIPI) scores (range, 3‐5), and 22% were refractory. Treatment consisted of 4 courses of R‐FM (rituximab 375 mg/m² intravenously on Day 1, fludarabine 25 mg/m² intravenously on Days 2 through 4, and mitoxantrone 10 mg/m² intravenously on Day 2, recycling at Day 28) and consolidation with 2 courses of fludarabine and mitoxantrone (the same regimen without rituximab).

RESULTS:

The best response (84% overall response rate including 68% complete response/complete response unconfirmed) was achieved after 4 courses of R‐FM. Response rates were high regardless of age, refractoriness to last previous therapy, and FLIPI score. After a median follow‐up of 4 years, the 3‐year progression‐free survival rate was 47%, the event‐free survival rate was 41%, and the 3‐year overall survival rate was 66%. Grade ≥3 neutropenia and infections were the most common toxicities and occurred in 72% and 14% of patients, respectively.

CONCLUSIONS:

Cytoreduction with 4 courses of R‐FM was safe and highly efficient in patients with recurrent/refractory follicular lymphoma who had high tumor burden; however, better consolidation than FM is needed to further improve outcome. Cancer 2010. © 2010 American Cancer Society.     

Multicentre phase II study of CyclOBEAP plus rituximab in patients with diffuse large B‐cell lymphoma

The R‐CHOP regimen has been found to improve the outcome of diffuse large B‐cell lymphoma (DLBCL). However, it does not provide a satisfactory treatment outcome in the high‐risk group. We previously administered the CyclOBEAP regimen to patients with DLBCL, and reported its safety and efficacy. The R‐CyclOBEAP regimen was administered over a total period of 12 weeks, and rituximab 375 mg/m² was given every 2 weeks. There were 101 eligible patients. CR was achieved in 96 patients (95%). The 5‐year overall survival (OS) rate was 85% and progression‐free survival (PFS) rate was 76%. When the patients were divided according to the IPI, the 5‐year OS and PFS rates did not significantly differ among the risk groups. The 5‐year PFS of the germinal centre B‐cell group was 80% and that of the non‐GCB group was 74% (NS). Univariate analysis showed that the presence of B symptoms, extranodal lesions ≧2, and sIL‐2R were significant poor prognostic factors. Grade 4 neutropenia was observed in 91 patients and thrombocytopenia in 9 patients. The addition of rituximab to CyclOBEAP therapy may enhance the effect of CyclOBEAP therapy for DLBCL. Copyright © 2010 John Wiley & Sons, Ltd.     

Immunotherapy of B‐cell non‐Hodgkin lymphoma by targeting the chemokine receptor CXCR5 in a preclinical mouse model

Bispecific antibodies are promising agents for immunotherapy. Here, we describe a quadroma‐based trifunctional bispecific antibody binding the chemokine receptor CXCR5 and the T‐cell antigen CD3 that efficiently prevents tumor growth in a mouse B‐cell lymphoma model. CXCR5 regulates the tissue homeostasis of mature B cells and is highly expressed on B‐cell non‐Hodgkin and lymphocyte‐predominant Hodgkin lymphoma, as well as on a subset of CD4⁺ T cells known as follicular T‐helper cells. In vitro, the bispecific CXCR5::CD3 antibody efficiently recruited effector T cells to CXCR5 expressing B cells and induced a co‐stimulation‐independent activation of CD8⁺ and CD4⁺ T cells as demonstrated by the de novo expression of CD25 and CD69, and secretion of the cytokines IFN‐γ, TNF‐α, IL‐6 and IL‐10 by peripheral blood mononuclear cells. Notably, at low antibody concentrations, CXCR5::CD3 displayed a significantly higher cytotoxic activity against autologous B cells than its parental antibodies or rituximab. In vivo imaging revealed that CXCR5::CD3 and its parental CXCR5 antibody efficiently prevent tumor growth in a xenograft model of B‐cell lymphoma in mice and prolong their survival. Taken together, our results identify CXCR5 as a promising target for antibody‐based therapies in the treatment of B‐cell malignancies.     

Blockade of TGF‐β enhances tumor vaccine efficacy mediated by CD8+ T cells

Though TGF‐β inhibition enhances antitumor immunity mediated by CD8⁺ T cells in several tumor models, it is not always sufficient for rejection of tumors. In this study, to maximize the antitumor effect of TGF‐β blockade, we tested the effect of anti‐TGF‐β combined with an irradiated tumor vaccine in a subcutaneous CT26 colon carcinoma tumor model. The irradiated tumor cell vaccine alone in prophylactic setting significantly delayed tumor growth, whereas anti‐TGF‐β antibodies alone did not show any antitumor effect. However, tumor growth was inhibited significantly more in vaccinated mice treated with anti‐TGF‐β antibodies compared to vaccinated mice without anti‐TGF‐β, suggesting that anti‐TGF‐β synergistically enhanced irradiated tumor vaccine efficacy. CD8⁺ T‐cell depletion completely abrogated the vaccine efficacy, and so protection required CD8⁺ T cells. Depletion of CD25⁺ T regulatory cells led to the almost complete rejection of tumors without the vaccine, whereas anti‐TGF‐β did not change the number of CD25⁺ T regulatory cells in unvaccinated and vaccinated mice. Though the abrogation of CD1d‐restricted NKT cells, which have been reported to induce TGF‐β production by MDSC through an IL‐13‐IL‐4R‐STAT6 pathway, partially enhanced antitumor immunity regardless of vaccination, abrogation of the NKT cell‐IL‐13‐IL‐4R‐STAT‐6 immunoregulatory pathway did not enhance vaccine efficacy. Taken together, these data indicated that anti‐TGF‐β enhances efficacy of a prophylactic vaccine in normal individuals despite their not having the elevated TGF‐β levels found in patients with cancer and that the effect is not dependent on TGF‐β solely from CD4⁺CD25⁺ T regulatory cells or the NKT cell‐IL‐13‐IL‐4R‐STAT‐6 immunoregulatory pathway.     

The influence of myeloid‐derived suppressor cells on angiogenesis and tumor growth after cancer surgery

While myeloid‐derived suppressor cells (MDSCs) have been reported to participate in the promotion of angiogenesis and tumor growth, little is known about their presence and function during perioperative period. Here, we demonstrated that human MDSCs expressing CD11b⁺, CD33⁺ and HLA‐DR^– significantly increased in lung cancer patients after thoracotomy. CD11b⁺ CD33⁺ HLA‐DR^– MDSCs isolated 24 hr after surgery from lung cancer patients were more efficient in promoting angiogenesis and tumor growth than MDSCs isolated before surgical operation in allograft tumor model. In addition, CD11b⁺ CD33⁺ HLA‐DR^– MDSCs produced high levels of MMP‐9. Using an experimental lung metastasis mouse model, we demonstrated that the numbers of metastases on lung surface and Gr‐1⁺ CD11b⁺ MDSCs at postoperative period were enhanced in proportion to the degree of surgical manipulation. We also examined that syngeneic bone marrow mesenchymal stem cells (BMSCs) significantly inhibited the induction and proliferation of Gr‐1⁺ CD11b⁺ MDSCs and further prevented lung metastasis formation in the mice undergoing laparotomy. Taken together, our results suggest that postoperatively induced MDSCs were qualified with potent proangiogenic and tumor‐promotive ability and this cell population should be considered as a target for preventing postoperative tumor metastasis.     

Induction of CD4+ and CD8+ anti‐tumor effector T cell responses by bacteria mediated tumor therapy

Facultative anaerobic bacteria like E. coli can colonize solid tumors often resulting in tumor growth retardation or even clearance. Little mechanistic knowledge is available for this phenomenon which is however crucial for optimization and further implementation in the clinic. Here, we show that intravenous injections with E. coli TOP10 can induce clearance of CT26 tumors in BALB/c mice. Importantly, re‐challenging mice which had cleared tumors showed that clearance was due to a specific immune reaction. Accordingly, lymphopenic mice never showed tumor clearance after infection. Depletion experiments revealed that during induction phase, CD8⁺ T cells are the sole effectors responsible for tumor clearance while in the memory phase CD8⁺ and CD4⁺ T cells were involved. This was confirmed by adoptive transfer. CD4⁺ and CD8⁺ T cells could reject newly set tumors while CD8⁺ T cells could even reject established tumors. Detailed analysis of adoptively transferred CD4⁺ T cells during tumor challenge revealed expression of granzyme B, FasL, TNF‐α and IFN‐γ in such T cells that might be involved in the anti‐tumor activity. Our findings should pave the way for further optimization steps of this promising therapy.     

Specific thermal ablation of tumor cells using single‐walled carbon nanotubes targeted by covalently‐coupled monoclonal antibodies

CD22 is broadly expressed on human B cell lymphomas. Monoclonal anti‐CD22 antibodies alone, or coupled to toxins, have been used to selectively target these tumors both in SCID mice with xenografted human lymphoma cell lines and in patients with B cell lymphomas. Single‐walled carbon nanotubes (CNTs) attached to antibodies or peptides represent another approach to targeting cancer cells. CNTs convert absorbed near‐infrared (NIR) light to heat, which can thermally ablate cells that have bound the CNTs. We have previously demonstrated that monoclonal antibodies (MAbs) noncovalently coupled to CNTs can specifically target and kill cells in vitro. Here, we describe the preparation of conjugates in which the MAbs are covalently conjugated to the CNTs. The specificity of both the binding and NIR‐mediated killing of the tumor cells by the MAb‐CNTs is demonstrated by using CD22⁺CD25^? Daudi cells, CD22^?CD25⁺ phytohemagglutinin‐activated normal human peripheral blood mononuclear cells, and CNTs covalently modified with either anti‐CD22 or anti‐CD25. We further demonstrate that the stability and specificity of the MAb‐CNT conjugates are preserved following incubation in either sodium dodecyl sulfate or mouse serum, indicating that they should be stable for in vivo use. © 2009 UICC     

Humanized anti‐CD19 chimeric antigen receptor‐T cell therapy is safe and effective in lymphoma and leukemia patients with chronic and resolved hepatitis B virus infection

Chimeric antigen receptor‐T (CAR‐T) cell therapy is a promising treatment for CD19⁺ B‐cell malignancies. However, elimination of B cells by anti‐CD19 CAR‐T cells may lead to the reactivation of hepatitis B virus (HBV) and related hepatitis in patients with HBV infection. This study aims to evaluate the safety and efficacy of humanized anti‐CD19 CAR‐T (hCAR‐T) therapy in B‐cell malignancies with HBV infection. Twenty relapsed/refractory (r/r) diffuse large B‐cell lymphoma (DLBCL) and acute lymphoblastic leukemia (ALL) patients with HBV infection were treated with hCAR‐T therapy. Among them, five hepatitis B antigen‐positive patients who received antiviral prophylaxis did not develop HBV reactivation, including two patients who received both hCAR‐T and allogeneic hematopoietic stem cell transplantation (allo‐HSCT). Among 15 patients with resolved HBV infection, two received antiviral prophylaxis, and the other 13 did not experience HBV reactivation without antiviral prophylaxis. One patient with resolved HBV infection experienced HBV reactivation 6 months after hCAR‐T therapy and sequential allo‐HSCT. Moreover, HBV infection did not affect in vivo expansion of hCAR‐T cells or increase the risk of severe cytokine release syndrome. In conclusion, hCAR‐T therapy is safe and effective in DLBCL and ALL patients with chronic and resolved HBV infection under proper antiviral prophylaxis.     

Prognostic value of arginase‐II expression and regulatory T‐cell infiltration in head and neck squamous cell carcinoma

Tumor‐infiltrating lymphocytes are present in a variety of tumors and play a central role in antitumor immune responses. Nevertheless, most cancers progress probably because tumors are only weakly immunogenic and develop multiple immunosuppressive mechanisms. In the present study, on head and neck squamous cell carcinoma, we found high intraepithelial infiltration of regulatory FOXP3⁺ T cells, and relatively high levels of BDCA2⁺ and FOXP3⁺ cells in stromal (peripheral) regions of the tumors. Tumor‐infiltrating (intraepithelial) FOXP3⁺ T cells were significantly more frequent in patients with oropharynx and oral cavity squamous cell carcinoma and in patients without lymph node metastasis. Furthermore, arginase‐II (ARG2) was expressed by 60%, inducible nitric oxide synthetase by 9%, cyclooxygenase‐2 by 43%, and B‐cell lymphoma 2 (BCL2) by 26% of tumors. Interestingly, the absence of ARG2 expression, enhanced stromal infiltration of CD11c⁺ myeloid dendritic cells, and high numbers of FOXP3⁺ T cells were each significantly associated with prolonged overall survival, and the latter two parameters were also confirmed by multivariate analysis. For disease‐free survival, multivariate analysis revealed significant negative correlations with BCL2 and ARG2 expression by tumor cells. These findings shed new light on mechanisms of cancer progression, and provide rationales for therapeutic inhibition of immunosuppressive mechanisms in head and neck squamous cell carcinoma.     

Evidence of long‐lasting anti‐CD19 activity of engrafted CD19 chimeric antigen receptor–modified T cells in a phase I study targeting pediatrics with acute lymphoblastic leukemia

Ninety percent of relapse/refractory B‐cell acute lymphatic leukemia (R/R B‐ALL) patients can achieve complete remission (CR) after CD19‐targeting chimeric antigen receptor T (CAR‐T) cell therapy. However, around 50% of them relapse in 1 year. Persistent CAR‐T cell engraftment is considered as the key to remain durable remission. Here, we initiated a phase I study to treat 10 pediatric B‐ALL patients using a CD19‐targeted second generation CAR with a 4‐1BB intracellular costimulatory domain. All patients received a standard fludarabine and cyclophosphamide (FC) preconditioning regiment, followed by a CAR‐T infusion with a median number of 0.5 (0.3‐1.58) × 10⁶ CAR+ T cells/kg. The pretreatment tumor burdens were high with a median bone marrow (BM) blasts percentage of 59.2% (7.31%‐86.2%), excluding one patient only with brain infiltration of leukemia cells (0% BM blasts). The initial CR rate was 80% (n = 8/10). Four patients (40%) experienced serious (grade > 2) cytokine release syndrome (CRS) and three patients (30%) with obvious neurotoxicity. Monthly assessments of CD19+ minimal residual disease (MRD) and CAR‐T engraftment demonstrated the anti‐CD19 activity of long‐term engrafted CAR‐T cell clones in one patient for more than 2 years.     

Irrespective of CD34 expression,lineage‐committed cell fraction reconstitutes and re‐establishes transformed Philadelphia chromosome‐positive leukemia in NOD / SCID / IL‐2Rγc−/− mice

(Cancer Sci 2010; 101: 631–638) Stem cells of acute myeloid leukemia (AML) have been identified as immunodeficient mouse‐repopulating cells with a Lin^?CD34⁺38^? phenotype similar to normal hematopoietic stem cells. To identify the leukemia‐propagating stem cell fraction of Philadelphia chromosome‐positive (Ph⁺) leukemia, we serially transplanted human leukemia cells from patients with chronic myeloid leukemia blast crisis (n = 3) or Ph⁺ acute lymphoblastic leukemia (n = 3) into NOD/SCID/IL‐2Rγc^?/? mice. Engrafted cells were almost identical to the original leukemia cells as to phenotypes, IGH rearrangements, and karyotypes. CD34⁺CD38^?CD19⁺, CD34⁺38⁺CD19⁺, and CD34^?CD38⁺CD19⁺ fractions could self‐renew and transfer the leukemia, whereas the CD34^?CD38⁺CD19⁺ fraction did not stably propagate in NOD/SCID mice. These findings suggest that leukemia‐repopulating cells in transformed Ph⁺ leukemia are included in a lineage‐committed but multilayered fraction, and that CD34⁺ leukemia cells potentially emerge from CD34^? populations.