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Chimeric antigen receptor T-cell therapy (CAR-T-cell therapy) has revolutionized the treatment of relapsed and refractory hematological malignancies. Targeting of the CD19 antigen on B cells has yielded high rates of remission induction and sustained remission in patients with acute lymphoblastic leukemia and B-cell lymphomas. Despite these remarkable responses, many escape mechanisms from CAR-T cell therapy have been identified, with the most common being target antigen deficiency. This paper focuses on CD19 CAR-T cell therapies, which are currently the most clinically used, and describes new strategies to overcome resistance using multi-targeted CAR-T cells, such as CD19-CD20 CAR-T cells and CD19-CD22 CAR-T cells, which are being developed in preclinical and clinical trials.

Chimeric antigen receptor-transduced autologous T (CAR-T) cell therapy targeting CD19 has revolutionized the treatment of CD19-positive hematological tumors, including acute lymphoblastic leukemia and large B-cell lymphoma. However, despite the high response rate, many problems such as exceedingly high cost, complex logistics, insufficient speed, and manufacturing failures have become apparent. One solution for these problems is to use an allogeneic cell as an effector cell for genetic modification with CAR. Allogeneic, or "off-the-shelf", CAR-expressing immune-effector cells include 1) genome-edited, T-cell receptor (TCR) gene-deleted CAR-T cells generated using healthy adult donor T cells, 2) induced pluripotent stem cell-derived CAR-T cells, and 3) CAR NK cells. NK cells are notorious for their poor ex-vivo expansion and low susceptibility to genetic modification. In this article, I will review the current state and future prospects of allogeneic CAR cell therapies, with special reference to CAR NK cells.

CAR-T cell therapy targeting CD19 and BCMA for relapsed or refractory hematopoietic tumors has been adopted in routine practice and has shown dramatic results. However, half of patients who achieve remission with CAR-T therapy eventually relapse, and thus efforts to improve the efficacy of CAR-T therapy are gaining momentum. Notably, studies have described innovative technologies that enable control of cell kinetics after infusion, which is not possible with conventional CAR-T therapies. In this article, we review the challenges of CAR-T cell therapy and the development of new technologies.

Chimeric antigen receptor (CAR)-T cell therapy is among the most promising immunotherapies for hematological malignancies and can be used to treat myeloid malignancies in practice. However, developing CAR-T therapies for such diseases is particularly challenging due to the heterogeneity of target antigen expression across leukemic cells and patients, the difficulty in excluding on-target/off-target tumor effects, and the immunosuppressive tumor microenvironment. To date, various targets, including CD33, NKG2D, CD123, CLL-1, and CD7, have been actively studied for CAR-T cells, especially for acute myeloid leukemia (AML). Although no CAR-T cell products have been approved, several clinical trials have shown promising results, particularly for those targeting CLL-1 and CD123. Furthermore, new ideal targets and use of allogeneic or off-the-shelf CAR-T cell products are under investigation. Meanwhile, it remains unknown whether CAR-T therapy would be effective for other myeloid malignancies, including myelodysplastic syndromes and myeloproliferative diseases. This review discusses challenges in the development of CAR-T therapy for myeloid malignancies, especially for AML, from the perspectives of target antigen characteristics and disease-specific on-target/off-tumor toxicity. Moreover, it discusses the clinical development and prospects of CAR-T cells for these diseases.

Chimeric antigen receptor (CAR) T-cell therapy has revolutionized the treatment paradigm for refractory/relapsed (R/R) hematologic malignancies, with six products approved for B-cell tumors and multiple myeloma as of the end of 2023. However, adoptive cell therapy (ACT) for solid tumors is hindered by critical challenges in multiple areas, including (1) lack of appropriate tumor-specific antigens, (2) inefficient T-cell trafficking and infiltration into the tumor microenvironment, and (3) immunosuppressive signals within the tumor milieu that induce T-cell dysfunction. This review examines the existing clinical trial data on ACT for solid tumors to elucidate the current landscape of ACT development for solid tumors. It also outlines the trajectory of ACT for solid tumors and integrative approaches to overcoming the complex tumor microenvironment.

T cell malignancies pose several unique issues for CAR-T cell therapy that were not significant concerns with CAR-T cells for B-cell malignancies. A general problem to consider in the production of CAR-T cells is "on target-off tumor toxicity." This occurs when the antigen targeted by the CAR-T cells is also expressed on normal cells, not just tumor cells, which causes CAR-T cells to damage these normal cells. In CAR-T cell therapy for T cell tumors, antigens expressed on T cells (such as CD5, CD7, etc.) are the targets, which leads to a problem known as "fratricide," where CAR-T cells kill each other. Other issues include T cell aplasia and contamination of CAR-T cell products with tumor cells. However, several recent clinical trials have shown excellent outcomes for CAR-T cell therapy when genome editing technology is used to overcome these issues by knocking out target antigens or T cell receptors. This review article outlines these challenges and their solutions and discusses the results of recent clinical trials.

Prostate cancer is basically hormone sensitive tumor. However, once it turns to metastatic castration-resistant prostate cancer(mCRPC), it is hard to suppress tumor growth and metastasis. Despite chemotherapy and novel androgen-receptor signalling inhibitors, mCRPC remains a lethal disease with poor clinical outcomes. Immunotherapy with chimeric antigen receptor T(CAR-T)cells redirects a patient's immune cells against the tumor antigen. CAR-T cell therapy has demonstrated promise in treating patients with several haematological malignancies. On the other hand, solid tumors impose immunologic and physical barriers to the efficacy of CAR-T cell therapy. As a new strategy to mCRPC, CAR-T cells with targeting prostate-specific membrane antigen(PSMA)has been developed. Several clinical trials using anti PSMA-CAR-T cells against mCRPC are going on overseas. A few of the trials showed promising results on the safety and efficacy of this therapy in mCRPC. Herein, we review strategies of constructing CAR-T cells to mCRPC, and the latest reports of international clinical trials of anti PSMA-CAR-T therapy.

CD19-targeted chimeric antigen receptor T-cell (CAR-T) therapy has shown promise as treatment of relapsed or refractory B-cell malignancies. However, the clinical utility of early CAR-T monitoring within 1 month after infusion has not been elucidated. In this study, we quantitatively measured CAR-T kinetics in peripheral blood on days 2, 4, 7, 9, 11, 14, 21, and 28 post-infusion using flow cytometry and quantitative polymerase chain reaction in 13 patients with relapsed refractory diffuse large B-cell lymphoma (DLBCL) treated with tisagenlecleucel (tisa-cel). No relationships were identified between bulk CAR-T kinetics and treatment outcomes. Interestingly, the magnitude of CD4 + CAR-T expansion was higher in responders than in nonresponders, while CD8 + CAR-T expansion was minimal in responders. Additionally, CAR-T proliferation was more pronounced in patients with cytokine release syndrome. Our results suggest that CD4 + CAR-T cellular kinetics within 1 month after CAR-T infusion may predict its efficacy after tisa-cel therapy in adult patients with DLBCL.

The clinical application of chimeric antigen receptor T-cell therapy (CAR-T therapy) has significantly altered the therapeutic strategy for B-cell tumors and is now being used to treat myeloid and solid tumors. Nonetheless, the efficacy of CAR-T cell therapy for myeloid and solid tumors has been limited, and several studies are being conducted to understand and overcome the underlying mechanisms. Recent research achievements have revealed that the properties of CAR-T cells, particularly their memory function, which can be continuously amplified in the body without exhaustion after administration, are closely related to CAR-T cell clinical efficacy. Furthermore, because the characteristics of CAR-T cells are greatly influenced by the quality of peripheral blood mononuclear cells, the raw material of CAR-T cells, and the T-cell used during the manufacturing process, attention has been drawn to the development of high-quality CAR-T cell manufacturing methods and combination therapies that maintain CAR-T cell memory function and suppress immune exhaustion. This article provides an overview of the current state of CAR-T cell development and clinical application to cancer, particularly emphasizing the development of manufacturing processes and efforts to improve CAR-T cell efficacy in combination therapy with molecular-targeting drugs.

CAR-T cell therapy is a twenty-first-century immunotherapy breakthrough. Another breakthrough in immunotherapy is checkpoint antibody therapy, which can treat multiple cancers with a single antibody, whereas CAR-T cell therapy requires an appropriate target for each cancer type. Other than B-cell malignancies, no good target antigen for cancers has been discovered. We have been working on the development of CAR-T cells that target differences in post-translational changes such as conformation or glycosylation, between cancer and normal cells rather than differences in protein expression levels. CAR-T cells specific for activated integrins, which are consistently overexpressed in multiple myeloma, are one of the achievements. We recently discovered an antibody with myeloma specificity despite binding to a ubiquitously expressed protein, CD98hc, and hypothesized that this specificity may be owing to altered N-glycosylation of CD98hc.

The use of novel agents has improved the outcomes of patients with multiple myeloma. However, almost all patients eventually relapsed, became resistant to available treatments, and, thus, required further therapy. To improve the outcomes of relapsed and/or refractory, the best efficacy in each line of treatment should be achieved. Currently, the prognosis of patients who became refractory to triple-class agents including proteasome inhibitors, immunomodulatory drugs, and anti-CD38 antibodies is extremely poor. Moreover, the best treatment regimen for these patients remains unclear. In Japan, the use of B-cell maturation antigen-targeting chimeric antigen receptor T-cell therapy for this patient group was approved. Furthermore, bispecific T-cell engagers and B-cell maturation-targeting antibody-drug conjugate are currently developed. Since it is challenging to identify the optimal sequences, it is important to apply each treatment individually based on clinical trial results. In the educational session, the framework to choose the most optimal treatment based on evidence of relapsed multiple myeloma therapies in each treatment line will be discussed.

The frequency of the manufacturing failure of chimeric antigen receptor (CAR)-T cell therapy in clinical practice is unknown. To clarify the current state of how likely CAR-T cell production is to succeed or fail for B-cell acute lymphoblastic leukemia (B-ALL), we analyzed cases in which the production of tisagenlecleucel was performed for patients with B-ALL at 15 facilities in Japan from October 2019 to March 2022. Total 81 patients (47 males and 34 females) were analyzed. The median age at apheresis was 13 years (1-25) with a median number of prior treatments of 4 (1-9). The numbers of patients with histories of allogeneic transplantation, inotuzumab ozogamicin, or blinatumomab treatments were 51 (63.0%), 26 (32.1%), and 37 (45.7%), respectively. The median blast percentage and CD3 + cell counts in peripheral blood were 0% (0-91.5), and 611/µl (35-4,210) at apheresis, and the median number of CD3 + cells shipped was 2.2×10 9 (0.5-8.3). While cases with a history of heavy prior treatment before apheresis were included, no manufacturing failures were observed. Continuing to monitor the status of manufacturing failures is necessary as the number of B-ALL cases treated with CAR-T cell therapy increases.

Multiple myeloma (MM) is still one of the most difficult hematological diseases. It is distinguished by recurrent relapses despite successful treatment. Proteasome inhibitors, immunomodulatory imide drugs, and monoclonal antibodies have all been developed in the last 10 years as treatment options for MM. However, obtaining a long-term treatment-free period for relapsed and refractory multiple myeloma (RRMM) remains difficult. The most recent and intriguing research is on new BCMA-targeting therapies. CAR-T cell therapy, in particular, has shown promising results in the treatment for triple class refractory MM patients. BCMA CAR-T cell therapy is gaining attention as a potentially game-changing treatment for multiple myeloma. Fortunately, CAR-T cell therapy will be available in Japan in January 2022. However, numerous issues must be addressed. Many RRMM patients receive CAR-T cell therapy, for example, relapse, and progression-free survival are short. This section provides an overview of clinical study results for CAR-T cell therapy targeting BCMA, and the antibody-drug conjugate, bispecific antibodies, selinexor, and venetoclax.

Chimeric antigen receptor (CAR) T-cell therapy has become the standard of care for some B-cell malignancies due to its high antitumor efficacy. However, adverse events specific to CAR T-cell therapy, such as cytokine release syndrome (CRS), are frequently observed. Typical clinical manifestations of CRS include hyperthermia, hypotension, hypoxemia, and increased vascular permeability. Coagulopathy may also be observed along with to the typical symptoms of CRS. Previous studies, including ours, have revealed that the increase in interleukin-6 levels associated with CRS promotes production of plasminogen activator inhibitor-1 from the vascular endothelium, resulting in suppressed fibrinolysis and hypercoagulability and an exhaustible decrease in fibrinogen. Continuous monitoring of coagulation parameters is necessary during CAR T-cell therapy, and adequate provision of fresh-frozen plasma or cryoprecipitate is recommended if the patient is evaluated to be at high risk for severe CRS.

For over a decade, various chimeric antigen receptor (CAR)-modified T-cells targeting myeloid antigens have been researched and developed overseas for relapsed/refractory acute myeloid leukemia (AML). However, none of them is domestically and internationally nearing approval. Clinical trial results on CAR T-cells targeting LeY, CD33, NKG2D ligands, CD38, or CD123 have been reported; however, they have not shown significant clinical benefit. More recently, several promising studies in CLL1 CAR T-cells have been reported in China, which attracted attention. We started a first-in-human clinical trial of GMR CAR T-cells in patients with CD116-positive myeloid neoplasms, particularly AML and juvenile myelomonocytic leukemia, in 2021. CAR T-cells can be a promising and practical treatment option for patients with relapsed/refractory AML.

Adoptive cell therapy using tumor-infiltrating T cells has shown durable responses in patients with melanoma, and immunotherapy using genetically engineered T cells (TCR-T or CAR-T) is rapidly emerging as a promising treatment, especially for hematological malignancies. However, the progress is limited because of the lack of readily available good-quality human T cells. Although the efficacy of adoptive cell therapy correlates with the quality of infusing T cells, most antigen-specific T cells in patients with cancer have been exhausted. To overcome this, we have reprogrammed donor (or original) T cells to iPS cells (T-iPS) and differentiated these into rejuvenated antigen-specific cells (T-iPS-T). Moreover, iPS cells provide an unlimited source of genetically engineered T cells such as TCR/CAR-T or PD-1 knockout T cells. The iPS cells' potential for immune cell therapy is infinite.

Recently, promising clinical outcomes of cancer immunotherapy including administration of an anti PD-1 antibody targeting for T cell reactivation has gained particular attention worldwide. Adoptive cell therapy with tumor infiltrating lymphocytes and TCR/CAR (Chimeric Antigen Receptor) transgenic T cells are also under development. Although it has become clearer that the efficacy of adoptive cell therapy correlate with the quality of infusing T cells, antigen specific T cells in patients with chronic infection and cancer have been exhausted. We have succeeded to generate rejuvenated antigen specific T cells by reprogramming to pluripotency and differentiation. In this article, we introduce fundamentals of this technology and describe its potential for adoptive cell therapy in the future.

Adoptive T-cell therapy using chimeric antigen receptor (CAR) technology is a novel approach to cancer immuno-gene therapy. CARs are hybrid proteins consisting of target-antigen-specific single-chain antibody fragment fused to intracellular T-cell activation domains (CD28 or CD137/CD3 zeta receptor). CAR-expressing engineered T lymphocytes can directly recognize and kill tumor cells in an HLA independent manner. In the United States, promising results have been obtained in the clinical trials of adoptive immuno-gene therapy using CD19-CAR-T lymphocytes for the treatment of refractory B-cell malignancies, including chronic lymphocytic leukemia (CLL) and acute lymphoblastic leukemia (ALL). In this review article, CD19-CAR-T gene therapy for refractory B-cell non-Hodgkin lymphoma is discussed.

Based on the concept of living with cancer, wherein the goal is to help patients with highly advanced solid cancers maintain a high quality of life(QOL)without adverse events and drug resistance, we developed a new immunocyte therapy based on BRM-activated killer(BAK)cells, which are primarily CD56 positive lymphocytes. In a previous report, we documented the disappearance of liver metastases, as assessed by positron emission tomography-computed tomography(PET-CT), in patients with metastatic liver cancers into which BAK immunocytes had been administered via injection into the hepatic artery. Herein, upon the patient's request, we locally injected BAK lymphocytes into an abdominal tumor. In BAK therapy, 20 mL of peripheral blood are collected from a patient. Lymphocytes from this blood sample are subsequently activated and multiplied with immobilized anti-CD3 antibodies and IL-2 and are cultured for 2 weeks with E(bina)and serum-free ALys media to yield approximately 10 billion autologous lymphocytes. On the final day of incubation, the lymphocytes are treated with 1,000 units/mL of interferon(IFN)-a for 15 minutes to enhance their therapeutic killing effects. During the second week, approximately 10 billion isolated autologous lymphocytes are suspended in 200 mL of Ringer's solution and are then drip-infused into the patient over a period of 1 hour. We injected approximately 10 billion BAK lymphocytes suspended in 50 mL of Ringer's solution into a 2-cm abdominal tumor in a single 60-year-old woman under ultrasonography guidance. This procedure was repeated every 3 weeks. After the third administration, we collected a biopsy specimen and examined it using PAS staining and microscopy. The 3 separate local injections of approximately 10 billion activated autologous lymphocytes each, primarily CD56 positive cells, into the tumor led to tumor fragmentation, leaving approximately 10 lymphocytes surrounding each cancer cell. These results suggest that BAK therapy is efficacious and show that locally administered BAK lymphocytes can reach cancer tissues and effectively kill cancer cells.

To enable "life with cancer" while maintaining quality of life(QOL) and freedom from adverse effects, we have devised biological response modifier (BRM)-activated killer (BAK) therapy, a new immune cell therapy using CD56-positive lymphocytes designed to treat advanced solid cancers. This treatment is yet to be applied to liver cancer, because BAK cells administered by intravenous infusion are unlikely to reach lesions via the complex vascular and lymphatic systems. Here, we report a case in which the patient, a surgeon, requested that we attempt to deliver BAK cells to cancer lesions in the liver via hepatic intra-arterial infusion. In 2005, with the patient's consent, we injected autologous lymphocytes intra-arterially into the liver using a catheter and confirmed the absence of adverse effects. In December 2008, we began injecting BAK cells intra-arterially into the liver of the patient, a male physician aged 52 years at the time, after identifying liver metastases following surgery for rectal cancer. We injected 10 billion cells intra-arterially into the liver via a catheter once a month for 6 months, and then undertook conventional BAK therapy by monthly intravenous infusion. Based on images obtained by positron emission tomography-computed tomography, BAK therapy led to complete remission and disappearance of the liver metastases. This case highlights the effectiveness of hepatic intra-arterial infusion of BAK cells in cases of liver cancer.

False positive elevation of carcinoembryonic antigen (CEA) was observed in a 65-year-old woman who was treated with dendritic cell therapy (DCT) and activated lymphocyte therapy (ALT) for intrahepatic cholangiocarcinoma. Three months after the initiation of these therapies, her CEA value measured by AIA (TOSOH) began to increase without any evidence of worsening of cholangiocarcinoma. CEA was measured by several different methods, and only the result measured by AIA was high, indicating the presence of a false positive phenomenon. To clarify this phenomenon, we evaluated the patient's serum precisely. Gel filtration chromatography of her serum showed that CEA was detected in the elution fraction of IgG, which was different from the reference samples. Furthermore, this peak disappeared after incubation of patient's CEA and HBR-1. The immunoglobulin absorption test revealed that CEA value was decreased only after absorption of IgG and absorption tests using HBR-1 and MAK-absorbents showed a dramatic decrease in CEA value. These findings indicated the presence of IgG type human anti-mouse antibodies (HAMA), which interfered the measurement by AIA. Although we could not identify the reason why HAMA was produced in this patient, the facts that the false positive phenomenon was observed after the initiation of DCT and ALT, and that CEA value decreased after theses therapies were discontinued, indicated that immuno-modulaton by DCT and ALT may have a close relationship to HAMA production. It was probable that DCT and ALT activated preexisting heterophile-antibody-producing cells, which stimulated HAMA production. The incidence of such false positive reaction of CEA by HAMA in patients with DCT and ALT was low, but as the number of the patient with immuno-cell therapy increases, the incidence of such phenomenon surely increases. Because HAMA reacts to all types of immunoassay, careful attention should be paid to the evaluation of laboratory findings in patients undergoing with such immuno-cell therapies.

We enrolled in this study 14 immunosuppressed non-small cell lung cancer (NSCLC) outpatients whose IAP level in serum were over 580 microg/ml and 26 immunoreactive outpatients whose IAP level in serum were under 580 microg/ml. After giving informed consent, patients were treated with BAK therapy on an outpatient basis. Treated with BAK therapy, the mean survival of immunosuppressed patients was 5.2 months, on the other hand, one of immunoreactive patients was 26.3 months. The difference in survival between 2 groups was significant (p<0.01). A stage IV NSCLC patient whose serum IAP is under 580 microg/ml is good indication for BAK therapy. The favorable clinical response in lung cancer patients to BAK treatment may be due to the fact that lungs are the first exposed to BAK cells via the blood stream. BAK therapy has a life prolonging effect with no apparent adverse effects for advanced NSCLC patients.

Autologous tumor cells stimulated with T lymphocytes (AuTL) were generated ex vivo from peripheral blood lymphocytes over a two-week co-culturing process with autologous tumor cells. These AuTLs were capable of lysing established tumor cell lines and may have a potential for efficacy as an adoptive immunotherapy (IT) in advanced and metastatic refractory cancer patients (pts). We investigated the feasibility of a combination of AuTL transfer and chemotherapy (ChT) based on the conventional conditioning regimen in order to take advantage by both the anticancer effects and reconstruction of antitumor immunity. Nineteen patients were enrolled in a pilot clinical trial. The two administrations of AuTL were given prior to chemotherapy (ChT) for one treatment cycle. The treatment was repeated at least for three cycles over a one-week interval. The conventional ChT regimen was based on the standard dosage. The pts consisted of 3 of gastric cancer, colon cancer, lung adenocarcinoma, respectively, 6 of esophageal cancer, and 2 of breast and pancreas carcinoma, respectively. AuTLs were administered 1x/2 weeks using direct injection or intraarterial infusion. The median duration of the treatment was over 11.5 months, and the median survival time was 14.8 months. Adverse events related to both the ChT and AuTL transfers at all dosages were minimal. Four of the 13 pts achieved major tumor responses (2 CR: complete regression and 2 PR: partial regression) in this study. Three pts showed progressive disease, and 6 pts had stable disease for over 90 days. PBMC were evaluated for cytokine production prior to the treatment and after 3 treatments. Two and one of 4 CR/PR pts had increased IFN-gamma and TNF-alpha production with no TGF-beta1 responses by their PBMC after 3 treatments, respectively. Two out of 6 pts who experienced stable disease after the treatment had high IFN-gamma and TNF-alpha responses and no TGF-beta1 or IL-4 response. TGF-beta1 and IL-4 secretion increased in parallel in 3 out of 3 pts that experienced progressive disease after the treatment. These data show that combination therapy of AuTL transfer and non-myeloablative ChT is a feasible option for patients with refractory advanced cancers without serious adverse events and without reducing Th1 cytokine responses in peripheral blood for most of the pts that responded to the treatment. According to each mechanism of IT and ChT, a more stringent evaluation of AuTL transfer combined with non-myeloablative ChT for various kinds of cancers should be performed to manage the immunodeficiency in the pts with advanced cancer and to improve the effect of antitumor AuTLs.

Adoptive immunotherapy was performed for patients with multiple hepatocellular carcinoma (HCC) using LAK cells via the hepatic arterial catheter. One case study: A 45-year-old male patient was performed an absolute-non-cure operation. The operation was a success. However, some residual tumors remained in the liver. After the operation, adoptive immunotherapy was performed for 7 times in 5 weeks. The total inoculated LAK cells were 6.9x10(9). After the adoptive immunotherapy, no residual tumors were detected in the liver. He survived for 7 years in remission. Our results show that adoptive immunotherapy with LAK cells is useful and effective for patients with multiple HCC.

Although an expression of MHC molecules and tumor associated antigens of the cancer are not uniform, we consider that the cancer immunotherapy for some specific tumor antigens cannot correspond to molecular biological varieties of the cancer. Consequently, we tried to develop a method to separate dendritic cells (DC), T-cells and natural killer (NK) cells from peripheral blood mononuclear cells (PBMC) obtained from healthy volunteers. PBMC were separated by centrifugation on Ficoll-Hypaque gradients from peripheral blood obtained from healthy volunteers. After separating these cells, the cells were put into a plastic flask, and we isolated monocyte fraction (dendritic cells), NK cell fraction and T-cell fraction one after another by the difference in its ability to adhere to a plastic flask. We analyzed surface markers and activation states of these groups. We could induce dendritic cells from the monocyte fraction, CD3-activated T-cells (CAT) from the T-cell fraction, and adherent lymphokine activated-killer cells (A-LAK) from the NK cell fraction. Therefore, we indicate the possibility of the combined cell therapy with three immune cell fractions in which we can induce from the same blood at once.

Objectives: Autologous tumor-cell stimulated cytotoxic T lymphocytes (AuTLs) were prepared from peripheral blood T cells and the T cells were activated ex vivo over a 2-week culturing process in the presence of IL-2 and autologous biopsied tumor-cells from advanced cancer patients. These AuTLs may have potential for efficacy as a locoregional therapy in patients with refractory cancer., Methods: Twenty-nine of 35 cancer patients (13 esophagus, 5 lung, 3 stomach, 4 breast, 1 melanoma) were enrolled in an early Phase II clinical trial. The patients received direct locoregional intratumoral injection of AuTLs biweekly through medical endoscope or intraarterial infusion reservoir system. Mean 0.25 x 10(9) AuTLs were injected 1 x /2 weeks for 6 weeks., Results: Adverse events related to AuTL were minimal. AuTLs specific for autologous tumor cells were observed in 12 of 29 patients. Seven of these 12 patients (58.3%) had partial response (PR) or stable disease (SD). In contrast, 8 of 23 (34.8%) remaining patients treated by non-specific AuTLs had SD. Infiltration of T effector cells was significantly increased on the biopsied tumor specimens in the immunohistological studies. Furthermore, 11 patients receiving systemic infusion of non-specific LAK/NK T cells without autologous tumor-cell stimulation only had 2 SDs (18.2%)., Conclusions: Our results demonstrated the clinical potential of intra-peritumoral administration of AuTLs. Thus, locoregional immunotherapy with autologous tumor specific AuTLs may be more effective than systemic adoptive immunotherapy using intravenous infusion of autologous tumor non-specific LAK/NK T cells for the treatment of solid cancers.

In induction of autologous tumor-reactive antigen (TRA) specific cytotoxic T lymphocytes (CTLs) using antigenic peptides and cultured dendritic cells (DCs), identification of the adequate tumor antigens and HLA typing of individuals are required. These restrictions have promoted the use of tumor cells themselves, including tumor cell lysates and tumor cell-DC fusion cells. However, it is very difficult to obtain enough tumor cells for treatment in the clinical setting. We have studied the use of RNA derived from tiny tumor cells. RNA was reverse-transcribed into cDNA, after which T7-amplification and in vitro transcription were carried out. The amplified RNA was successfully electroporated into DCs, and polyclonal polyspecific CTLs could be generated. EBV transformed B cells were also good candidates to be electroporated with the RNA. This suggests that tumor RNA amplification followed by introduction into DCs or EBV transformed B cells is a feasible and practical method to prepare potent APCs.

The field of bone marrow transplantation has undergone dramatic changes over the past few decades. Not only has the terminology changed (e.g., hematopoietic stem-cell transplantation), but the role of allogeneic transplantation has been modified from supportive to immunotherapeutic and the applications have expanded from hematologic malignancies to solid tumors. The development of nonmyeloablative conditioning regimen has greatly increased the number of patients eligible for this kind of treatment. Use of hematopoietic stem-cell transplantation as a form of adoptive immunotherapy in the treatment of cancer depends on advances in tumor immunology, particularly the identification of tumor antigens and mechanisms of immunotherapy. The earliest use of allogeneic transplantation of immunogenic cells for the treatment of solid tumors in the late 1960s and early 1970s produced no definite graft-versus-tumor effects. However, as conventional allogeneic hematopoietic stem-cell transplantation methods for the treatment of hematologic malignancies have matured, these methods have reestablished the foundation for expanding their application to solid tumors. From the first case reports on medulloblastoma and breast cancer to subsequent case series reports on breast cancer and renal cell carcinoma, allogeneic hematopoietic stem-cell transplants have demonstrated graft-versus-tumor effect. At present, the most common solid tumor for which this treatment is used is advanced renal cell carcinoma, but allogeneic hematopoietic stem-cell transplants have proven feasible for other solid tumors as well. Directions for future study include the identification of the definitive tumor antigens involved in the graft-versus-tumor effect and means of selecting those patients who will benefit the most from this form of treatment. This review summarizes the peer-reviewed literature on the use of allogeneic transplantation for solid tumors based on US studies.

We investigated beneficial effects of AIT with anticancer agents on survival of subcutaneous tumor-bearing mice and suppression of artificial lung metastasis, and optimal schedule of administration of each treatment in vivo. 7-8 weeks old C 57 BL/6 mice were inoculated s.c. with 5 x 10(6) B 16 melanoma cells, or i.v. with 2 x 10(5) B 16-F 10 melanoma cells. Mouse splenocytes were cultured with 3.5 x 10(3) JRU/ml interleukin 2 for 14 days, and induced LAK cells were harvested. Anticancer agents (Cx), CDDP or MMC were given i.p. in mice. 1 x 10(7) or 5 x 10(7) LAK cells were given either s.c. around the tumor or i.v. respectively, and 1.4 x 10(5) JRU/mouse IL-2 was administered s.c. for 6 days after LAK cell injection. Therapy groups were as follows #1: Cx day 3, AIT day 3-8. #2: Cx day 3, AIT day 6-11, #3: Cx day 8, AIT day 3-8. In therapy groups #1 and #2, we observed additive effects of AIT and anti-cancer agents in life-prolongation and suppression of lung metastasis. It was also shown that LAK induction in vivo was augmented by anticancer agents in groups #1 and #2, which might represent one of the mechanisms behind observed additive effects. Furthermore, our results suggest that therapeutic effects of the combination of AIT and anticancer agents depend on the schedule of administration.

The Epstein-Barr virus (EBV) has been implicated in the etiology of many lymphoid and other malignancies. Understanding of the immune control of the virus appears to be very important to establish strategy to overcome those disease. Recent advance of T cell biology and the culture technic have made it possible to apply adoptive immune transfer of EBV-specific cytotoxic T lymphocytes (CTL) in clinical settings. This review summarizes immunotherapy using EBV-specific CTL for the treatment of EBV-associated lymphoproliferative disorders in patients receiving bone marrow transplantation and for a patient with severe chronic active EBV infection. Its future application is discussed.

Activated and expanded autologous peripheral blood lymphocytes by cultivation with immobilized anti-CD3 antibody and IL-2 have been infused to the patients with hepatocellular carcinoma after culative operation as a randomized clinical trial. This study is on-going and primary efficacy endpoints of this study were disease-free survival and overall survival, and the sample size for the study was designed as a minimum of 146 patients. From interim analysis at the second year from start of the study, Eligible cases were 101, 49 cases in treated group and 52 cases in control. Recurrences were confirmed 13 cases from treated group and 22 cases from control group. Minor adverse reaction were observed in 28 cases (57%).

A 54-year-old woman who had undergone radical nephrectomy for renal cell cancer nine months before was admitted to our hospital because of difficulty in breathing. X-ray films of the chest showed massive pleural effusion on the left side and cytological examination of the effusion revealed malignant cells which may have originated from renal cell cancer. Intrapleural instillations of interleukin-2 and of tumor infiltrating lymphocytes isolated from the pleural effusion were performed. After the initiation of the treatment, the pleural effusion decreased and malignant cells disappeared from the pleural fluid. Partial response defined by the criteria provided by the Japan Lung Cancer Society was achieved. No serious side effects were observed. This would be a useful treatment for pleuritis carcinomatosa by renal cell cancer.

We developed CTL therapy against cancer as an cancer specific immunotherapy. The primary CTL therapy showed certain efficacy on advanced cancer patients. However, when ratio of monocyte population in peripheral blood lymphoid cells (PBL) of advanced cancer patients increases more than that of CD8+ T cell population, so that CD8+ T cell/M3 ratio is less than 1, CTL directed to cancer cannot be induced in PBL and the primary CTL therapy is not materialized for these patients. Monocyte-macrophages in these patients release a suppressive factor(s) capable of inhibiting CTL induction and proliferation at induction phase but not inhibiting cytotoxic activity in target cell lysis at effector phase. Furthermore, these monocytes expressed strong message of TGF beta. If most of increased monocytes of PBL from advanced cancer patients are removed by a nylon wool column method from the PBL, CTL directed to cancer cells can be induced even in PBL of advanced cancer patients with high frequency, this improved CTL therapy against cancer showed remarkable efficacy as the cancer specific immunotherapy in various advanced cancer patients without any side effects.

A patient with pulmonary tuberculosis caused by bacteria resistant to various anti-microbial agents was treated with adoptively transferred autologous peripheral blood leucocytes (PBL) sensitized with killed Mycobacterium tuberculosis organisms in vitro. The 32-year-old man was admitted to our hospital from National Sanitarium Okinawa Hospital with weight loss, high fever, and rapid aggravation on chest X-ray. Patient's PBL obtained by leukapheresis and separated with Ficoll-Hypaque solution were cultured with killed Mycobacterium tuberculosis bacteria of 0.4 microgram per ml at 1 x 10(6) cells per ml for 7 days in media containing 0.5 U recombinant 1L-2 per ml. After incubation, PBL were layered and centrifuged on Ficoll-Hypaque solution and washed three times with saline. PBL (1-3 x 10(8)) were combined and concentrated for infusion in 20 to 30 ml saline. After injection, patient displayed fever and transitory drop of PaO2. Although the patient did not have an improved on chest X-ray, his fever was alleviated, weight was increased, accelerated ESR was slightly improved, and the number of organisms in sputum (Number of Gaffky) temporarily decreased. Adoptive immunotherapy using the autologous PBL which were sensitized with killed bacteria may be an effective anti-tuberculous immunotherapy.