B7-H3-Targeted CAR-Vδ1T Cells Exhibit Potent Broad-Spectrum Activity Against Solid Tumors.

Vδ1T cells, a rare subset of γδT cells, hold promise for treating solid tumors. Unlike conventional T cells, they recognize tumor antigens independently of the MHC antigen-presentation pathway, making them a potential "off-the-shelf" cell therapy product. However, isolation and activation of Vδ1T cells is challenging, which has limited their clinical investigation. Here, we developed a large-scale clinical-grade manufacturing process for Vδ1T cells and validated the therapeutic potential of B7-H3-CAR-modified Vδ1T cells in treating solid tumors. Co-expression of interleukin-2 with the B7-H3-CAR led to durable anti-tumor activity of Vδ1T cells in vitro and in vivo. In multiple subcutaneous and orthotopic mouse xenograft tumor models, a single intravenous administration of the CAR-Vδ1T cells resulted in complete tumor regression. These modified cells demonstrated significant in vivo expansion and robust homing ability to tumors, akin to natural tissue-resident immune cells. Additionally, the B7-H3-CAR-Vδ1T cells exhibited a favorable safety profile. In conclusion, B7-H3-CAR-modified Vδ1T cells represent a promising strategy for treating solid tumors.

Enhanced efficacy of CD19/CD22 bispecific CAR-T cells with EAAAK linker on B-cell malignancies.

OBJECTIVES: Despite the great success of CD19 CAR-T cell therapy, its clinical efficacy has been greatly hampered by the high relapse rate. In this study, we designed and compared four structures of CD19/CD22 bispecific CAR-T cells with different linkers and different orders of the antibody sequences. METHODS: We detected the cytotoxicity, cytokine secretion levels, sustainable killing ability, differentiation, exhaustion of these four CAR-T cells in vitro. The optimal Bis-C CAR-T cells were evaluated the efficacy using NSG mice. RESULTS: The two structures of CD19/CD22 bispecific CAR-T cells using (EAAAK)3 as linker had more significant cytotoxicity and cytokine secretion levels. In the process of continuous killing, Bis-C CAR-T cells showed better sustained killing ability, memory phenotype differentiation, and exhaustion. In the in vivo experiment mimicking CD19-negative relapse, Bis-C CAR-T was more able to control the tumor progression of mice in the CD19 low expression or no expression groups than CD19 CAR-T. CONCLUSIONS: This study has generated a novel bispecific CAR-T cell that can simultaneously target CD19 or CD22 positive tumor cells, providing a new strategy to address the limitations of single-targeted CAR-T therapy in B-cell tumors (limited response or relapse).

Preconditioning of radiotherapy enhances efficacy of B7-H3-CAR-T in treating solid tumor models.

AIMS: Limited efficacy of chimeric antigen receptor T (CAR-T) cells in treating solid tumors is largely due to the antigen heterogeneity and immunosuppressive tumor microenvironment (TME). B7-H3 is over-expressed in most kind of solid tumors, making it a promising target for cancer treatment. This study aims to explore the effect of B7-H3-CAR-T therapy combined with radiotherapy in treating solid tumor models. METHODS: Irradiated tumor cell lines were prepared and tested. A humanized B7-H3-CAR-T was constructed, and it was evaluated that B7-H3-CAR-T cytotoxicity against solid tumor models with preconditioning of radiotherapy in vitro and vivo. RESULTS: Irradiation was found to increase expression level of B7-H3 in pancreatic cancer (PANC-1), colorectal cancer (HCT-15, SW620), acute myelocytic leukemia (AML-5), epidermoid carcinoma (KB) and glioma (U87-MG) human cell lines significantly. 6Gy irradiation was also found to up-regulate tumor-infiltration molecule like intracellular adhesion molecule-1 ICAM-1 or FAS in HCT-15 cells, supporting a possible synergistic enhancement effect of radiotherapy. In vitro and in vivo experiments demonstrated that irradiation indeed significantly enhanced the ability of B7-H3-CAR-T to infiltrate and kill tumors. Interestingly in dual-tumor mouse model study, not only tumor cells on irradiation side were eradicated completely, irradiation also enhanced CAR-T tumor-killing ability on non-irradiated side, confirming the abscopal effect of irradiation existed with CAR-T therapy. CONCLUSIONS: Our results suggest that B7-H3-CAR-T therapy combined with radiotherapy may be a promising modality in treating solid tumors.

CD7 protein plays a crucial role in T cell infiltration in tumors.

CD7 protein as a target is being used to treat CD7+ lymphoma; however, the role of CD7 in the hematopoietic system remains largely unknown. Therefore, we evaluated the effects of CD7 KO in mice. The differentiation of the hematopoietic system in the bone marrow and the number of various cell types in the thymus and spleen did not differ between CD7 KO and WT mice. After subcutaneous inoculation of B16-F10 melanoma cells, tumors from CD7 KO mice grew more rapidly, and the proportion of CD8+ T cells in the spleen and tumors decreased. In vitro, the infiltration and adhesion of CD8+ T cells from the spleen of CD7 KO mice were weakened. Blocking CD7 in normal T cells did not alter the migration and infiltration, but in Jurkat, CCRF-CEM, and KG-1a tumor cell lines, migration and invasion were significantly reduced after blocking CD7. Therefore, CD7 does not affect hematopoietic system development but plays a crucial role in T cell infiltration into tumors.

B7-H3 chimeric antigen receptor-modified T cell shows potential for targeted treatment of acute myeloid leukaemia.

BACKGROUND AND AIMS: Chimeric antigen receptor (CAR)-T cell therapy is a novel type of immunotherapy. However, the use of CAR-T cells to treat acute myeloid leukaemia (AML) has limitations. B7-H3 is expressed in several malignancies, including some types of AML cells. However, its expression in normal tissues is low. Therefore, B7-H3 is ideal for targeted AML therapy. MATERIALS AND METHODS: First, we constructed B7-H3 CAR that can target B7-H3, and then constructed B7-H3-CAR-T cells in vitro, which were co-incubated with six AML cell lines expressing different levels of B7-H3, respectively. The toxicity and cytokines were detected by flow cytometry. In vivo, AML model was established in B-NSG mice to study the toxicity of B7-H3-CAR T on AML cells. RESULTS: In vitro functional tests showed that B7-H3-CAR-T cells were cytotoxic to B7-H3-positive AML tumor cells and had good scavenging effect on B7-H3-expressing AML cell lines, and the cytokine results were consistent. In vivo, B7-H3-CAR-T cells significantly inhibited tumor cell growth in a mouse model of AML, prolonging mouse survival compared with controls. CONCLUSION: B7-H3-CAR-T cells may serve as a novel therapeutic method for the targeted treatment of AML.

RPRM deletion preserves hematopoietic regeneration by promoting EGFR-dependent DNA repair and hematopoietic stem cell proliferation post ionizing radiation.

Reprimo (RPRM), a target gene of p53, is a known tumor suppressor. DNA damage induces RPRM, which triggers p53-dependent G2 arrest by inhibiting cyclin B1/Cdc2 complex activation and promotes DNA damage-induced apoptosis. RPRM negatively regulates ataxia-telangiectasia mutated by promoting its nuclear-cytoplasmic translocation and degradation, thus inhibiting DNA damage. Therefore, RPRM plays a crucial role in DNA damage response. Moreover, the loss of RPRM confers radioresistance in mice, which enables longer survival and less severe intestinal injury after radiation exposure. However, the role of RPRM in radiation-induced hematopoietic system injury remains unknown. Herein, utilizing a RPRM-knockout mouse model, we found that RPRM deletion did not affect steady-state hematopoiesis in mice. However, RPRM knockout significantly alleviated radiation-induced hematopoietic system injury and preserved mouse hematopoietic regeneration in hematopoietic stem cells (HSCs) against radiation-induced DNA damage. Further mechanistic studies showed that RPRM loss significantly increased EGFR expression and phosphorylation in HSCs to activate STAT3 and DNA-PKcs, thus promoting HSC DNA repair and proliferation. These findings reveal the critical role of RPRM in radiation-induced hematopoietic system injury, confirming our hypothesis that RPRM may serve as a novel target for radiation protection.

Nanobody-based anti-CD22-chimeric antigen receptor T cell immunotherapy exhibits improved remission against B-cell acute lymphoblastic leukemia.

Chimeric antigen receptor (CAR) T-cell immunotherapies targeting CD19 can achieve impressive clinical remission rates in the treatment of B-cell non-Hodgkin lymphoma and B-cell acute lymphoblastic leukemia. However, relapse after CD19-CAR T treatment remains a major issue, with CD19 antigen-negative relapse being one of the main reasons. CD22, another antigen expressed in a B-cell lineage-specific pattern, is retained following CD19 loss. Accordingly, we hypothesized that CD22 could represent an alternative target to alleviate or compensate for the ineffectiveness of CD19-CAR T therapy. To this end, we generated camelid-derived CD22 nanobodies, whose smaller size, greater stability, and lower immunogenicity offer better quality than classical antibodies, and we used them to construct third-generation CD22-CARs containing 4-1BB and ICOS co-stimulatory domains. The novel CD22-CAR T cells exhibited impressive cytotoxicity both in vitro and in vivo and significantly prolonged the overall survival of tumor-bearing NSG mice. These findings provide the basis for further translational studies employing CD22-CARs.

Zkscan3 affects erythroblast development by regulating the transcriptional activity of GATA1 and KLF1 in mice.

ZKSCAN3 encodes a zinc-finger transcription factor that regulates the expression of important genes and plays a significant role in tumor development, pathogenesis, and metastasis. However, its biological functions under normal physiological conditions remain largely unknown. In our previous studies, using flow cytometry, we found that the deletion of Zkscan3 may cause abnormal erythropoiesis. In this study, we found that, in a Zkscan3 knockout mice model, the number of splenic early-stage (basophilic-erythroblasts) and late-stage (chromatophilic-erythroblasts to polychromatophilic-erythroblasts through orthochromatophilic-erythroblasts) erythroblasts increased, whereas the number of late erythroblasts in the bone marrow decreased. Moreover, the phenotype was exacerbated after treating mice with phenylhydrazine (PHZ), which causes severe hemolytic anemia. In the knockout mice treated with PHZ, the percentage of reticulocyte in the peripheral blood conspicuously increased, whereas MCHC and red blood cells decreased. Then, we performed RNA-seq and quantitative-polymerase chain reaction assay and found that the expression of GATA1 and Tiam1 in erythroblasts were upregulated, whereas KLF1 was downregulated. Luciferase assays showed that Zkscan3 inhibited the transcription of GATA1 and Tiam1 and promoted the expression of KLF1. Additionally, ChIP and CO-IP results confirmed that Zkscan3 directly interacts with GATA1 and inhibits its transcriptional activity in MEL cells. Our results demonstrate, for the first time, the significant role of Zkscan3 in physiological erythropoiesis through the interaction with GATA1, both at the DNA and protein level, and with Tiam1 and KLF1 at the DNA level.

Chimeric antigen receptor T cells derived from CD7 nanobody exhibit robust antitumor potential against CD7-positive malignancies.

The great success of chimeric antigen receptor T (CAR-T)-cell therapy in B-cell malignancies has significantly promoted its rapid expansion to other targets and indications, including T-cell malignancies and acute myeloid leukemia. However, owing to the life-threatening T-cell hypoplasia caused by CD7-CAR-T cells specific cytotoxic against normal T cells, as well as CAR-T cell-fratricide caused by the shared CD7 antigen on the T-cell surface, the clinical application of CD7 as a potential target for CD7+ malignancies is lagging. Here, we generated CD7ΔT cells using an anti-CD7 nanobody fragment coupled with an endoplasmic reticulum/Golgi retention domain and demonstrated that these cells transduced with CD7-CAR could prevent fratricide and achieve expansion. Additionally, CD7ΔCD7-CAR-T cells exhibited robust antitumor potiential against CD7+ tumors in vitro as well as in cell-line and patient-derived xenograft models of CD7-positive malignancies. Furthermore, we confirmed that the antitumor activity of CD7-CAR-T cells was positively correlated with the antigen density of tumor cells. This strategy adapts well with current clinical-grade CAR-T-cell manufacturing processes and can be rapidly applied for the therapy of patients with CD7+ malignancies.

MUC1-Tn-targeting chimeric antigen receptor-modified Vγ9Vδ2 T cells with enhanced antigen-specific anti-tumor activity.

Chimeric antigen receptor (CAR) αß T cell adoptive immunotherapy has shown great promise for improving cancer treatment. However, there are several hurdles to overcome for the wide clinical application of CAR-αß T cells therapy, including side effects and a limited T cells source from cancer patients. Therefore, we sought to identify an alternative T cell subset that could avoid these limitations and improve the effectiveness of CAR-T immunotherapy. γδ T cells are a minor subset of T cells, which share the characteristic of innate immune cells and adaptive immune cells. Vγ9Vδ2 T cells are a predominant γδ T subset in the circulating peripheral blood. In this study, we investigated the antigen-specific antitumor activity of CAR-Vγ9Vδ2 T cells targeting MUC1-Tn antigen. Vγ9Vδ2 T cells were expanded from peripheral blood mononuclear cells of healthy volunteers with zoledronic acid and interleukin-2. CAR-Vγ9Vδ2 T cells were generated by transfection of lentivirus encoding MUC1-Tn CAR. Cytotoxicity assays with various cancer cell lines revealed that CAR-Vγ9Vδ2 T cells could effectively lyse tumor cells in an antigen-specific manner, with similar or stronger effects than CAR-αß T cells. However, CAR-Vγ9Vδ2 T cells had shorter persistence, which could be improved with the addition of IL-2 to maintain the function of CAR-Vγ9Vδ2 T cells with consecutive stimulation of tumor cells. Using a xenograft mouse model, we further showed that CAR-Vγ9Vδ2 T cells more effectively suppressed tumor growth in vivo than Vγ9Vδ2 T cells. Therefore, MUC1-Tn CAR-modified Vγ9Vδ2 T cells may represent a novel, promising ready-to-use product for cancer allogeneic immunotherapy.

Research progression on the first-line biological target therapy of advanced / 中国肿瘤生物治疗杂志

@#[Abstract] Objective: To develop a new type of CD7 chimeric antigen receptor modified T cell (CD7-CAR-T) for the treatment of CD7 positive acute myeloid leukemia (AML), and to observe its killing effect on CD7 positive AML cells. Methods: The CD7-CAR lentiviral vector was constructed based on the CD7 Nanobody sequence and costimulatory domain sequence of CD28 and 4-1BB. The lentiviral particles were packaged and used to co-transfect human T cells with protein expression blocker (PEBL), so as to prepare CD7- CAR-T cells. Real time cellular analysis (RTCA) was used to monitor the cytotoxicity of CD7-CAR-T cells on CD7 overexpressed 293T cells. Flow cytometry assay was used to detect the effect of CD7-CAR-T cells on proliferation and cytokine secretion of AML cells with high, medium and low CD7 expressions (KG-1, HEL and Kasumi-1 cells, respectively). Results: CD7-CAR-T cell was successfully constructed and its surface expression of CD7 was successfully blocked. Compared with T cells, CD7-CAR-T cells could significantly inhibit the proliferation of CD7-293T cells and promote the release of TNF, Granzyme B and INF-γ; in addition, CD7-CAR-T cells also significantly promoted the apoptosis (t=147.1, P<0.01; t=23.57, P<0.01) and cytokine release (P<0.05 or P<0.01) in CD7 positive KG-1 and HEL cells, but had little effect on Kasumi-1 cells that only expressed minimal CD7 antigen (t=0.7058, P>0.05). Conclusion: CD7-CAR-T cells can specifically kill CD7-positive AML cells in vitro.

A novel CD7 chimeric antigen receptor-modified NK-92MI cell line targeting T-cell acute lymphoblastic leukemia.

Chimeric antigen receptor (CAR) immunotherapy has recently shown promise in clinical trials for B-cell malignancies; however, designing CARs for T-cell based diseases remain a challenge since most target antigens are shared between normal and malignant cells, leading to CAR-T cell fratricide. CD7 is highly expressed in T-cell acute lymphoblastic leukemia (T-ALL), but it is not expressed in one small group of normal T lymphocytes. Here, we constructed monovalent CD7-CAR-NK-92MI and bivalent dCD7-CAR-NK-92MI cells using the CD7 nanobody VHH6 sequences from our laboratory. Both CD7-CAR-NK-92MI and dCD7-CAR-NK-92MI cells consistently showed specific and potent anti-tumor activity against T-cell leukemia cell lines and primary tumor cells. We observed robust cytotoxicity of the bivalent mdCD7-CAR-NK-92MI monoclonal cells against primary T-ALL samples. In agreement with the enhanced cytotoxicity of mdCD7-CAR-NK-92MI cells, significant elevations in the secretion of Granzyme B and interferon γ (IFN-γ) were also found in mdCD7-CAR-NK-92MI cells in response to CD7-positive primary T-ALL cells compared with NK-92MI-mock cells. Furthermore, we also demonstrated that mdCD7-CAR-NK-92MI cells significantly inhibited disease progression in xenograft mouse models of T-ALL primary tumor cells. Our data suggest that CD7-CAR-NK-92MI cells can be used as a new method or a complementary therapy for treating T-cell acute lymphocytic leukemia.

Expression of PD-1 in T-cell acute lymphoblastic leukemia cells and its clinical significance / 中国肿瘤生物治疗杂志

@# Objective: To investigate the expression and clinical significance of PD-1 molecule in tumor cells (T-ALL cells) derived from the patient with T-cell acute lymphoblastic leukemia (T-ALL). Methods: T-ALL cells from one patient and PBMCs from four healthy volunteers provided by the Department of Hematology in Jiangsu Provincial Hospital of Traditional Chinese Medicine in December 2015, and human 293T/PD-1 cells provided by Persongen Bio Therapeutics (Suzhou) Co., Ltd. were used for this study. The mouse T-ALL xenograft model was constructed by injecting T-ALL cells into tail vein of B-NDG mice, and flow cytometry was used to verify whether the cells obtained from the spleen of transplanted mice were mainly consisted of T-ALL cells. Flow cytometry was used to study the protein expression of PD-1 in T-ALL cells, and RT-PCR was applied to further verify the mRNA expression of PD-1 in T-ALL cells. The PD-1 gene in T-ALL cells was sequenced by SNP genotyping to detect whether the DNA sequence of PD-1 gene changed. PD-1 inhibitor was used in vitro to study their effects on proliferation, apoptosis, and the mRNA expression levels of related factors in T-ALL cells. Results: The mouse T-ALL xenograft model was successfully constructed and verified by flow cytometry as T-ALL. PD-1 was highly expressed at both mRNA and protein levels in T-ALL cells (all P<0.01). A C-to-T mutation was detected in the fifth exon of the PD-1 gene. PD-1 inhibitor had no significant effect on proliferation and apoptosis of T-ALL cells in vitro; PD-1 inhibitor up-regulated the mRNA expression of tumor-suppressor protein IGFBP3 and decreased the mRNA expression of oncoprotein SULT1A3 (all P<0.01). Conclusion: PD-1 is highly expressed in T-ALL cells, and PD-1 could be used as a target for clinical diagnosis and treatment for T-ALL.

Effects of CSF1R-targeted chimeric antigen receptor-modified NK92MI & T cells on tumor-associated macrophages.

Tumor immunotherapy has shown great progress for the treatment of cancer; however, both endogenous and exogenous T cells are inhibited by the immunosuppressive tumor microenvironment. Tumor-associated macrophages (TAMs) in the microenvironment play pivotal and complex roles in tumor development and progression. Macrophages are categorized as M1 and M2 types. Relevant studies suggest that M2 TAMs correlate with poor prognosis. Colony-stimulating factor 1 receptor (CSF1R) controls the formation, differentiation and function of M2 macrophages, which helps tumors grow, metastasize and secrete immunosuppressive cytokines. The objectives of this study were to establish two types of third-generation chimeric antigen receptors (CARs) that could specifically target human CSF1R, and to introduce the CARs into NK92MI cells and normal human peripheral blood T cells through lentiviral transduction to produce CAR-natural killer (NK) and -T cells. We then tested their cytotoxicity against cell lines and peripheral blood monocytes expressing CSF1R. In vitro experiments confirmed that third-generation CARs had good target specificity and cytotoxicity. It was expected that CAR-NK and -T cells could specifically kill M2 TAMs in the tumor microenvironment and remove their inhibitory effect. Therefore, CSF1R-targeting CAR-NK and -T cells could represent a novel cellular immunotherapy strategy in conjunction with other antibody-based drugs and targeted therapeutics.

La/SSB chimeric autoantibody receptor modified NK92MI cells for targeted therapy of autoimmune disease.

It has been long sought to specifically eliminate B-cell clones that generate autoreactive antibodies, while sparing the immune system when combating autoimmune disease. Although it was impossible to achieve this goal before, newly developed techniques have made it feasible today. Autoantibodies against La/SSB were involved in several autoimmune diseases. Here, we aimed to introduce La/SSB epitope-based chimeric autoantibody receptors (CAAR) into NK92MI cells enabled it to destroy the corresponding La/SSB-specific B cell receptor (BCR) -bearing lymphoma cells (LaA-BCR-Romas, LaA-BCR-Maver-1, and LaA-BCR-Jurkat cells). Such cell lines could eliminate a part of the B-cells in the blood of patients positive for anti-La/SSB antibodies. The CAAR we used in this study was constructed by fusing fragments from the nucleus protein, La/SSB, with the TCR signaling molecules, CD28, CD137, and CD3ζ. Thus, this method could specifically destroy the La/SSB autoreactive B-cell clones. Our results might provide a new strategy to combat antibody-mediated autoimmune diseases.

CAR-NK-92MI and CAR-CD19-T cells targeting CD19 antigen exhibit significant in vitro killing activity on mantle cell lymphoma / 中国肿瘤生物治疗杂志

@#[Abstract] Objective: To investigate the in vitro cytotoxicity of the chimeric antigen receptor-modified T cells and NK-92MI cells (CAR-NK-92MI cells) and CAR-CD19-T cells against mantle cell lymphoma (MCL). Methods: CAR-T cell technology, successfully obtained in clinical trial of B-lineage acute lymphoblastic leukemia (B-ALL) treatment, was used in this study. In the case of high expression of CD19 antigen in MCL, CAR- CD19-T cells and CAR- CD19-NK-92MI cells targeting CD19 antigen were generated, respectively. Then, their cytotoxicity against MCL cell lines was detected by LDH release assay and the results were verified by flow cytometry. Results: Compared with control group, both CAR-NK-92MI and CAR-CD19-T cells exhibited prominent killing effect against MCLcells（all P<0.01）; in addition, the two CAR cells exhibited high cytotoxicity against K562-CD19 cells but not on K562 cells（all P <0.01）. The death rate of MCL cells from CAR-NK-92MI group was 30%-40% more than that of control group, and the death rate of MCL from CAR-CD19-T group was 40%-50% more than that of control group. Conclusion: Both CAR-NK-92MI and CAR-CD19-T cells exhibited potent cytotoxicity against MCLcells in vitro.

Effects of IL-7 and IL-21 modified NK-92MI cells on themselves and T cells from normal human peripheral blood / 中国肿瘤生物治疗杂志

@#[Abstract] Objective：To investigate whether the proliferation and cytotoxicity of NK-92MI cells can be improved by IL-7 and IL-21 genes modification, and determine the effects of this genetically modified NK-92MI cells on T cells from normal human peripheral blood. Methods：IL-7 and IL-21 gene fragments were constructed into electroporation vector by genetic engineering method, and NK92MI/IL-21 and NK-92MI/IL-7&21 cells were constructed by electroporation transfection. The in vitro proliferation and cytotoxicity of NK-92MI, NK-92MI/IL-21 and NK-92MI/IL-7&21 cells were measured by cell count and flow cytometry assays. Then, normal human PBMCs were co-cultured with NK-92MI, NK-92MI/IL-21 and NK-92MI/IL-7&21 cells in vitro respectively, and the phenotype change of T cells was measured by flow cytometry. In addition, the cytotoxicity between the activated T cells and three NK-92MI cell lines (NK-92MI, NK-92MI/IL-21 and NK-92MI/IL-7&21 cells) as well as the cytotoxicity of the three NK-92MI cells on tumor cells after co-incubation with activated T cells were detected. Results: NK-92MI/IL-21 cell line (highly expressing IL-21) and NK-92MI/IL-7&21 cell line (highly expressing both IL-7 and IL-21) were successfully constructed. The toxicity of NK-92MI, NK-92MI/IL-21 and NK92MI/IL-7&21 cells on Jurkat and K562 cells showed no difference, while the proliferation of NK-92MI/IL-21 and NK-92MI/IL-7&21 cells was increased compared with NK-92MI cells. Furthermore, NK-92MI/IL-21 and NK-92MI/IL-7&21 cells promoted the activation of T cells to a certain degree, and the activated T cells showed merely no cytotoxicity on NK-92MI, NK-92MI/IL-21 and NK-92MI/IL7&21 cells; Meanwhile, the activated T cells did not affect the cytotoxicity of the three NK cells (NK-92MI, NK-92MI/IL-21, and NK92MI/IL-7&21 cells) on K562 cells under their co-existence. Conclusion: The in vitro proliferation of NK-92MI/IL-21 and NK-92MI/ IL-7&21 cells were enhanced after gene modification, which could also stimulate and promote the activation of T cells from peripheral blood. The cytotoxicity assay showed that the activated T cells had no cytotoxicity on NK-92MI, NK-92MI/IL-21, and NK-92MI/IL-7& 21 cells. Meanwhile, the presence of the activated T cells did not affect the cytotoxicity of NK-92MI cells.

Gene-modified NK-92MI cells expressing a chimeric CD16-BB-ζ or CD64-BB-ζ receptor exhibit enhanced cancer-killing ability in combination with therapeutic antibody.

Natural killer (NK) cells play a pivotal role in monoclonal antibody-mediated immunotherapy through the antibody-dependent cell-mediated cytotoxicity (ADCC) mechanism. NK-92MI is an interleukin-2 (IL-2)-independent cell line, which was derived from NK-92 cells with superior cytotoxicity toward a wide range of tumor cells in vitro and in vivo. Nonetheless, the Fc-receptor (CD16) that usually mediates ADCC is absent in NK-92 and NK-92MI cells. To apply NK-92MI cell-based immunotherapy to cancer treatment, we designed and generated two chimeric receptors in NK-92MI cells that can bind the Fc portion of human immunoglobulins. The construct includes the low-affinity Fc receptor CD16 (158F) or the high-affinity Fc receptor CD64, with the addition of the CD8a extracellular domain, CD28 transmembrane domains, two costimulatory domains (CD28 and 4-1BB), and the signaling domain from CD3ζ. The resulting chimeric receptors, termed CD16-BB-ζ and CD64-BB-ζ, were used to generate modified NK-92MI cells expressing the chimeric receptor, which were named NK-92MIhCD16 and NK-92MIhCD64 cells, respectively. We found that NK-92MIhCD16 and NK-92MIhCD64 cells significantly improved cytotoxicity against CD20-positive non-Hodgkin's lymphoma cells in the presence of rituximab. These results suggest that the chimeric receptor-expressing NK-92MI cells may enhance the clinical responses to currently available anticancer monoclonal antibodies.

Novel CD7-specific nanobody-based immunotoxins potently enhanced apoptosis of CD7-positive malignant cells.

Various CD7-targeting immunotoxins have been tested for its potential in treating CD7+ malignant patients but none of those immunotoxins was approved clinically because of lacking enough efficacy and safety. Here we successfully constructed the monovalent and bivalent CD7 nanobody-based immunotoxins PG001 and PG002, both conjugated with a truncated derivative of Pseudomonas exotoxin A respectively. The prokaryotic system expressed immunotoxins not only maintained their binding specificity for CD7-positive cells with a Kd of 16.74 nM and 3.6 nM for PG001 and PG002 respectively, but also efficiently promoted antigen-restricted apoptosis of the CD7-positive leukemia cell lines Jurkat and CEM, and primary T-cell acute lymphoblastic leukemia (T-ALL) and acute myeloid leukemia (AML) cells with an in vitro cytotoxic activity (EC50) in the range of 23-30 pM for PG002. In NOD/SCID mice transplanted with CEM cells, PG001 and PG002 prevented engraftment of the cells and markedly prolonged mouse survival. Owing to the efficient antigen-restricted anti-leukemic activity of PG002, this CD7 nanobody-based immunotoxin exhibited a superior anti-CD7 positive malignancies activity than previously reported immunotoxins, and may represent a promising therapeutic strategy in treating CD7-positive leukemia and lymphoma, which still remain a significant clinical challenge.

Anti-human CD138 monoclonal antibodies and their bispecific formats: generation and characterization.

Syndecan-1 (CD138), a heparan sulfate proteoglycan, acts as a co-receptor for growth factors and chemokines and is a molecular marker associated with the epithelial-mesenchymal transition during development and carcinogenesis. In this study, we generated two specific mouse anti-human CD138 monoclonal antibodies (mAbs, clone ID: 480CT5.4.3, 587CT7.3.6.5) using hybridoma technology and identified their immunological characteristics. After hybridoma sequencing, the single-chain variable fragments (ScFvs) cloned from two hybridoma cells were combined with anti-CD3 OKT-3 ScFv to generate two recombinant bispecific antibodies (h-STL002, m-STL002) against CD138 and CD3 molecules, respectively. The bispecific antibodies were able to specifically target CD138 + multiple myeloma (MM) cells and CD3 + T cells, and showed the potent cytotoxicity against MM RPMI-8226 cell line through T cell activation. However, these bispecific antibodies without T cells did not cause toxic side effect on MM cells. Overall, the two hybridoma clones and their bispecific formats have great potential to promote diagnosis and immunotherapy of plasma cell malignancy.