Recent Progress In Immunotherapy Research Articles

Characteristics and trends of globally registered malignant pleural mesothelioma clinical trials in the past 27 years.

e20544 Background: Malignant pleural mesothelioma (MPM), arising from the serosal lining of the thoracic cavity, is a rare malignancy with limited treatment options and very poor prognosis. Recent progress in immunotherapy and innovative devices have advanced treatment fields of MPM. To help investigators understand the landscape of MPM clinical research, characteristic and trends of globally registered trials in the past decades were analyzed in our study. Methods: This is a cross-sectional analysis of MPM trials registered on ClinicalTrials.gov between January 1996 and December 2022. Characteristics pertaining to trail phase, enrollment number, study design, type, funding source, age of population, trial purpose, and participating country were extracted, and chronological shifts were analyzed. Clinical trial variables were enumerated using descriptive statistics and categorical data were presented as frequencies and percentages. Chi-square trend analysis (Cochran–Armitage trend test) was used to evaluate the trend of MPM trials. Results: 267 registered MPM trials were analyzed involving 40 participating countries among which the United States, the United Kingdom, and Italy were top 3. 8.2% trials were phase 3 and 18.7% were randomized. 74 trials had publications by the December 2022. The trial purposes concerning surgery, radiotherapy, chemotherapy, target therapy, immunotherapy, photodynamic therapy, cryotherapy, tumor treating fields （TTF）and non-anticancer research accounted for 2.6%, 5.2%, 20.2%, 25.5%, 31.1%, 1.1%, 0.7%, 0.7% and 12.7% respectively. In the past 27 years, the numbers of chemotherapy and targeted therapy trials declined while immunotherapy trails steadily escalated (all above ptrend < 0.05). There are no significant changes for the trends of each trial phase. 205 drug therapy trials involved 26 cytotoxic drugs, 44 targeted drugs and 11 immune checkpoint inhibitors and 34 other drugs. Pemetrexed-cisplatin with TTF and ipilimumab-nivolumab have changed the frontline treatment space while improvement for regulatory approvals has been far slower in salvage treatment, with the median overall survival all less than 11 months in phase 3 trials. There are no definitive trials that justify the use of predictive biomarkers for systemic therapy in MPM. Conclusions: Immunotherapy and TTF have reshaped the standard of care landscape over the past 27 years. With the enhanced understanding of molecular characteristics and mechanism of MPM, it is anticipated that ongoing immunotherapy combined with target therapy, chemotherapy or other novel treatment can improve the frontline and post-line therapeutic effect.

A perspective of immunotherapy for acute myeloid leukemia: Current advances and challenges.

During the last decade, the underlying pathogenic mechanisms of acute myeloid leukemia (AML) have been the subject of extensive study which has considerably increased our understanding of the disease. However, both resistance to chemotherapy and disease relapse remain the principal obstacles to successful treatment. Because of acute and chronic undesirable effects frequently associated with conventional cytotoxic chemotherapy, consolidation chemotherapy is not feasible, especially for elderly patients, which has attracted a growing body of research to attempt to tackle this problem. Immunotherapies for acute myeloid leukemia, including immune checkpoint inhibitors, monoclonal antibodies, dendritic cell (DC) vaccines, together with T-cell therapy based on engineered antigen receptor have been developed recently. Our review presents the recent progress in immunotherapy for the treatment of AML and discusses effective therapies that have the most potential and major challenges.

Recent Progress in Immunotherapy for Gastric Cancer

Gastric cancer (GC) is the fourth leading cause of cancer-related deaths worldwide. Under the standard of care, patients with advanced GC (AGC) have a median survival time of approximately 12-15 months. With the emergence of immunotherapy as a key therapeutic strategy in medical oncology, relevant changes are expected in the systemic treatment of GC. In the phase III ATTRACTION-2 trial, nivolumab, a monoclonal anti-programmed cell death 1 (PD-1) antibody, as a third- or later-line treatment improved overall survival (OS) compared with placebo in patients with AGC. Furthermore, nivolumab in combination with 5-fluorouracil and platinum as a first-line treatment improved OS in patients with human epidermal growth factor receptor-2 (HER2)-negative AGC in the global phase III CheckMate-649 study. Another anti-PD-1 antibody, pembrolizumab, in combination with trastuzumab and cytotoxic chemotherapy as a first-line treatment, significantly improved the overall response rate in patients with HER2-positive AGC. Therefore, immune checkpoint inhibitors (ICIs) are essential components of the current treatment of GC. Subsequent treatments after ICI combination therapy, such as ICI rechallenge or combination therapy with agents having other modes of action, are being actively investigated to date. On the basis of the success of immunotherapy in the treatment of AGC, various clinical trials are underway to apply this therapeutic strategy in the perioperative and postoperative settings for patients with early GC. This review describes recent progress in immunotherapy and potential immunotherapy biomarkers for GC.

Advances in CAR T cell immunotherapy for paediatric brain tumours.

Brain tumours are the most common solid tumour in children and the leading cause of cancer related death in children. Current treatments include surgery, chemotherapy and radiotherapy. The need for aggressive treatment means many survivors are left with permanent severe disability, physical, intellectual and social. Recent progress in immunotherapy, including genetically engineered T cells with chimeric antigen receptors (CARs) for treating cancer, may provide new avenues to improved outcomes for patients with paediatric brain cancer. In this review we discuss advances in CAR T cell immunotherapy, the major CAR T cell targets that are in clinical and pre-clinical development with a focus on paediatric brain tumours, the paediatric brain tumour microenvironment and strategies used to improve CAR T cell therapy for paediatric tumours.

Recent progress in immunotherapy for skin cancer

Skin cancer is a global health concern due to its growing incidence and high mortality rate. The most common therapeutic modalities in skin cancer include surgery, radiotherapy, and chemotherapy. However, those therapies do not specifically target cancer cells and may damage healthy tissues. Cancer induces immune response by releasing soluble antigens and danger signals caused by tumor cellular stress or death, while the immune system continuously monitor and control malignant proliferation through cancer immunoediting. Therefore, targeting this mechanism is a promising approach to manage cancer, especially those unresponsive to conventional therapies. Immunotherapy is a specific therapy that manipulates the immune system to fight the disease. Previous studies have shown promising results in its clinical use in melanoma and non-melanoma skin cancer (NMSC). However, its potential toxicity and tolerability may pose significant obstacles in developing effective cancer immunotherapy. Biomedical, immunological, and clinical research in skin cancer is still needed to elaborate further on its pathogenesis and design safe and effective therapy for each skin cancer. Keywords: immunotherapy, melanoma, NMSC, toxicity

Dissecting the Role of N6-Methylandenosine-Related Long Non-coding RNAs Signature in Prognosis and Immune Microenvironment of Breast Cancer.

Breast cancer (BC) represents a molecularly and clinically heterogeneous disease. Recent progress in immunotherapy has provided a glimmer of hope for several BC subtypes. The relationship between N6-methyladenosine (m6A) modification and long non-coding RNAs (LncRNAs) is still largely unexplored in BC. Here, with the intention to dissect the landscape of m6A-related lncRNAs and explore the immunotherapeutic value of the m6A-related lncRNA signature, we identified m6A-related lncRNAs by co-expression analysis from The Cancer Genome Atlas (TCGA) and stratified BC patients into different subgroups. Furthermore, we generated an m6A-related lncRNA prognostic signature. Four molecular subtypes were identified by consensus clustering. Cluster 3 preferentially had favorable prognosis, upregulated immune checkpoint expression, and high level of immune cell infiltration. Twenty-one m6A-related lncRNAs were applied to construct the m6A-related lncRNA model (m6A-LncRM). Survival analysis and receiver operating characteristic (ROC) curves further confirmed the prognostic value and prediction performance of m6A-LncRM. Finally, high- and low-risk BC subgroups displayed significantly different clinical features and immune cell infiltration status. Overall, our study systematically explored the prognostic value of the m6A-related LncRNAs and identified a high immunogenicity BC subtype. The proposed m6A-related LncRNA model might serve as a robust prognostic signature and attractive immunotherapeutic targets for BC treatment.

Combined with interventional therapy, immunotherapy can create a new outlook for tumor treatment.

Recent progress in immunotherapy provides hope of a complete cure to cancer patients. However, recent studies have reported that only a limited number of cancer patients with a specific immune status, known as "cold tumor", can benefit from a single immune agent. Although the combination of immune agents with different mechanisms can partially increase the low response rate and improve efficacy, it can also result in more side effects. Therefore, discovering therapies that can improve tumors' response rate to immunotherapy without increasing toxicity for patients is urgently needed. Tumor interventional therapy is promising. It mainly includes transcatheter arterial chemoembolization, ablation, radioactive particle internal irradiation, and photodynamic interventional therapy based on a luminal stent. Interventional therapy can directly kill tumor cells by targeted drug delivery in situ, thus reducing drug dosage and systemic toxicity like cytokine release syndrome. More importantly, interventional therapy can regulate the immune system through numerous mechanisms, making it a suitable choice for immunotherapy to combine with. In this review, we provide a brief description of immunotherapies (and their side effects) on tumors of different immune types and preliminarily elaborate on interventional therapy mechanisms to improve immune efficacy. We also discuss the progress and challenges of the combination of interventional therapy and immunotherapy.

Recent progress in immunotherapy of breast cancer targeting the human epidermal growth factor receptor 2 (HER2).

Breast cancer is responsible for most of the cancer-induced deaths in women around the world. The current review will discuss different approaches of targeting HER2, an epidermal growth factor overexpressed in 30% of breast cancer cases. We conducted a search on Pubmed and Scopus databases to find studies relevant to HER2+ breast cancers and targeting HER2 as means of immunotherapy. Out of 1043 articles, 105 studies were included in this review. As well as the introduction of HER2 and breast cancer subtypes, we discussed various aspects of HER2-targeting immunotherapy including monoclonal antibodies, Antibody-drug conjugates (ADCs), Chimeric Antigen Receptor (CAR) T-cells and vaccines. Despite several ways of controlling breast cancer, the need to investigate new drugs and approaches seems to be much significant as this cancer still has a heavy burden on people's health and survival.

Abstract 6121: Using the chicken chorioallantoic membrane in vivo model to study metastatic kidney cancer: A novel drug screening approach with more sustainable patient-derived xenografts

Abstract Clear cell renal cell carcinoma (ccRCC) is the most common subtype of kidney cancers. Annually, 74,000 Americans are newly diagnosed with the disease, and more than 14,000 patients succumb to death, especially due to the advanced-stage ccRCC developing distant metastases. Unfortunately, there is currently no effective therapy for the deadly stage. Despite the recent progress in immunotherapy or anti-angiogenesis kinase inhibitors, the efficacy varies from one patient to another. Therefore, gaining further insight into the metastatic mechanism and establishing more effective and personalized treatments are of an urgent need to overcome the deadly outcome of metastatic ccRCC. Patient-derived xenografts (PDXs) have been a great resource to study clinical cases for oncologic pathways and therapeutics. However, the cost and time to establish PDXs in immunocompromised mice, such as SCID mice, have been significant burdens. Here, we propose an alternative PDX in the chicken chorioallantoic membrane (CAM) model. This model has a short incubation time, an inherently immunodeficient state, direct tumor visibility, and is both cost and labor efficient in comparison to the traditional mouse model. In the pursuit to streamline the in vivo cancer research and drug development, we explored the feasibility of the CAM PDXs of metastatic ccRCC. First, we sought to develop an optimized protocol for engrafting ccRCC from patients into the CAM model. Patient specimens were implanted either as primary tumor cells or small pieces of tumor chunks. The rate of successful PDXs engraftment was incredibly high, at 70% or above. Our preliminary data using the established cancer cell lines validated the CAM model's ability to recapitulate the tumor biology, including the growth and metastasis, of both mouse and human cell lines. By implanting cells stably transduced with Firefly luciferase, bioluminescence imaging can be used for sensitive detection of tumor growth on the membrane. FDG-PET can also be performed, showing promises of the model for further implementation of imaging and therapeutics. Taken together, the CAM model serves as a valuable platform of novel PDXs to accelerate the study of tumor biology and the development of individualized medicine. Many of the advantages of this model, however, could potentially also be limitations. For example, the short incubation time and immunodeficient state complicate the observation and interpretation of therapeutic effects. To overcome these limitations and make the model more clinically relevant, we are trying to propagate PDXs over multiple generations of CAM, as well as to incorporate autologous immune cells from patients. With these flexible modifications, the CAM PDXs could be even more promising to advance personalized medicine and improve the adverse prognosis of metastatic ccRCC. Citation Format: Moe Ishihara, Junhui Hu, Celine Cano-Ruiz, Anthony Wong, Lily Wu. Using the chicken chorioallantoic membrane in vivo model to study metastatic kidney cancer: A novel drug screening approach with more sustainable patient-derived xenografts [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 6121.

CAR T cells for brain tumors: Lessons learned and road ahead.

Malignant brain tumors, including glioblastoma, represent some of the most difficult to treat of solid tumors. Nevertheless, recent progress in immunotherapy, across a broad range of tumor types, provides hope that immunological approaches will have the potential to improve outcomes for patients with brain tumors. Chimeric antigen receptors (CAR) T cells, a promising immunotherapeutic modality, utilizes the tumor targeting specificity of any antibody or receptor ligand to redirect the cytolytic potency of T cells. The remarkable clinical response rates of CD19‐targeted CAR T cells and early clinical experiences in glioblastoma demonstrating safety and evidence for disease modifying activity support the potential of further advancements ultimately providing clinical benefit for patients. The brain, however, is an immune specialized organ presenting unique and specific challenges to immune‐based therapies. Remaining barriers to be overcome for achieving effective CAR T cell therapy in the central nervous system (CNS) include tumor antigenic heterogeneity, an immune‐suppressive microenvironment, unique properties of the CNS that limit T cell entry, and risks of immune‐based toxicities in this highly sensitive organ. This review will summarize preclinical and clinical data for CAR T cell immunotherapy in glioblastoma and other malignant brain tumors, including present obstacles to advancement.

Plant Virus-Like Particle In Situ Vaccine for Intracranial Glioma Immunotherapy.

Despite aggressive multi-modality treatment with surgery, radiation and chemotherapies, malignant glioma inevitably recurs and has dismal survival rates. Recent progress in immunotherapy has led to a resurgence of interest, and immunotherapies are being investigated for treatment of glioma. However, the unique brain anatomy and a highly immunosuppressive glioma microenvironment pose significant challenges to achieving efficacy. Thus, there is a critical need for assessment of next-generation immunotherapies for glioma. In this study, we have investigated the efficacy of the nanoparticle platform technology based on plant-derived Cowpea mosaic virus like particles (empty CPMV or eCPMV) to instigate a potent immune response against intracranial glioma. CPMV immunotherapy has been shown to efficiently reverse the immunosuppressive tumor microenvironments in pre-clinical murine models of dermal melanoma and metastatic melanoma, metastatic breast cancer, intraperitoneal ovarian cancer and in canine patients with oral melanoma. In the present study, we demonstrate that in situ administration of CPMV immunotherapy in the setting of glioma can effectively recruit unique subset of effector innate and adaptive immune cells to the brain parenchyma while reducing immune suppressive cellular population, leading to regression of intracranial glioma. The in situ CPMV nanoparticle vaccine offers a potent yet safe and localized immunotherapy for intracranial glioma.

Advances in Designing and Developing Vaccines, Drugs and Therapeutic Approaches to Counter Human Papilloma Virus.

Human papillomavirus (HPV) is a viral infection with skin-to-skin based transmission mode. HPV annually caused over 500,000 cancer cases including cervical, anogenital and oropharyngeal cancer among others. HPV vaccination has become a public-health concern, worldwide, to prevent the cases of HPV infections including precancerous lesions, cervical cancers, and genital warts especially in adolescent female and male population by launching national programs with international alliances. Currently, available prophylactic and therapeutic vaccines are expensive to be used in developing countries for vaccination programs. The recent progress in immunotherapy, biotechnology, recombinant DNA technology and molecular biology along with alternative and complementary medicinal systems have paved novel ways and valuable opportunities to design and develop effective prophylactic and therapeutic vaccines, drugs and treatment approach to counter HPV effectively. Exploration and more researches on such advances could result in the gradual reduction in the incidences of HPV cases across the world. The present review presents a current global scenario and futuristic prospects of the advanced prophylactic and therapeutic approaches against HPV along with recent patents coverage of the progress and advances in drugs, vaccines and therapeutic regimens to effectively combat HPV infections and its cancerous conditions.

New developments in immunotherapy for pediatric leukemia.

Immunotherapy for the treatment of cancer has advanced at a tremendous pace over the last decade. In this review, we provide an overview of recent progress in immunotherapy for the treatment of leukemia, focusing on antibody-drug conjugates (ADC), bi-specific T-cell engagers (BiTE), and chimeric antigen receptor (CAR) T cells. Ongoing clinical trials of CAR T cells directed against CD19 have produced complete remission rates as high as 93%, prompting global multicenter phase 2 trials and the first FDA approval of a CAR T-cell therapy. Insights into cytokine release syndrome, a toxicity of CAR T-cell therapy, and the cause for relapse after CAR T-cell therapy are evolving. The bispecific antibody blinatumomab and the ADCs inotuzumab and gemtuzumab have also recently received FDA approval for ALL and AML, respectively, moving these agents into a more prominent role in the relapse setting. The use of immunotherapy for leukemia has been successful in creating durable remissions for multiply relapsed and refractory patients who previously had little chance of cure. The ongoing clinical and preclinical work continues to advance our understanding of these immune-based therapies, and will shape the next generation of clinical trials.

The progress and current status of immunotherapy in acute myeloid leukemia.

Recently, there has been remarkable progress in basic and preclinical studies of acute myeloid leukemia (AML). The improved outcomes of AML can largely be attributed to advances in supportive care and hematopoietic cell transplantation as opposed to conventional chemotherapy. However, as the 5-year survival rate remains low due to a high incidence of relapse, novel and effective treatments are urgently needed. Increasing attention is focusing on identifying suitable immunotherapeutic strategies for AML. Here, we describe the immunological features, mechanisms of immune escape, and recent progress in immunotherapy for AML. Problems encountered in the clinic will also be discussed. Although current outcomes may be limited, ongoing preclinical or clinical efforts are aimed at improving immunotherapy modalities and designing novel therapies, such as vaccines, monoclonal antibody therapy, chimeric antibody receptor-engineered T cells (CAR-T), TCR-engineered T cells (TCR-T), and checkpoint inhibitors, which may provide promising and effective therapies with higher specificity and efficacy for AML.

Multicenter, real-life experience with checkpoint inhibitors and targeted therapy agents in advanced melanoma patients in Switzerland.

Metastatic melanoma is a highly aggressive disease. Recent progress in immunotherapy (IT) and targeted therapy (TT) has led to significant improvements in response and survival rates in metastatic melanoma patients. The current project aims to determine the benefit of the introduction of these new therapies in advanced melanoma across several regions of Switzerland. This is a retrospective multicenter analysis of 395 advanced melanoma patients treated with standard chemotherapy, checkpoint inhibitors, and kinase inhibitors from January 2008 until December 2014. The 1-year survival was 69% (n=121) in patients treated with checkpoint inhibitors (IT), 50% in patients treated with TTs (n=113), 85% in the IT+TT group (n=66), and 38% in patients treated with standard chemotherapy (n=95). The median overall survival (mOS) from first systemic treatment in the entire study cohort was 16.9 months. mOS of patients treated either with checkpoint or kinase inhibitors (n=300, 14.6 months) between 2008 and 2014 was significantly improved (P<0.0001) compared with patients treated with standard chemotherapy in 2008–2009 (n=95, 7.4 months). mOS of 61 patients with brain metastases at stage IV was 8.1 versus 12.5 months for patients without at stage IV (n=334), therefore being significantly different (P=0.00065). Furthermore, a significant reduction in hospitalization duration compared with chemotherapy was noted. Treatment with checkpoint and kinase inhibitors beyond clinical trials significantly improves the mOS in real life and the results are consistent with published prospective trial data.

Recent progress in immunotherapy for urological cancer

Cancer immunotherapy for urological tumors had made progress for several decades, but recent advances in immunotherapy, as therapeutic vaccines or immune checkpoint inhibitors, have drastically changed the present treatment strategy. Recently, nivolumab and atezolizumab have been approved by the Food and Drug Administration for treatment of urological cancers. Additional immune checkpoint inhibitors and vaccines are being tested in clinical trials. Despite advances in these therapeutic modalities, benefits are limited to a subset of patients. New agents and novel combinations will also continue to create new immunotherapy strategies. Further development of biomarkers for predicting response is required to achieve optimal efficacy with these therapeutic interventions.

Immunotherapy for the treatment of multiple myeloma.

Immunotherapy has recently emerged as a promising treatment for multiple myeloma (MM). There are now several monoclonal antibodies that target specific surface antigens on myeloma cells or the checkpoints of immune and myeloma cells. Elotuzumab (targeting SLAMF7), daratumumab (targeting CD38), and pembrolizumab (targeting PD-1) have shown clinical activity in clinical studies with relapsed/refractory MM. Dendritic cell vaccination is a safe strategy that has shown some efficacy in a subset of myeloma patients and may become a crucial part of MM treatment when combined with immunomodulatory drugs or immune check-point blockade. Genetically engineered T cells, such as chimeric antigen receptor T cells or T cell receptor-engineered T cells, have also shown encouraging results in recent clinical studies of patients with MM. In this paper, we discuss recent progress in immunotherapy for the treatment of MM.

Identification of melanoma cells and lymphocyte subpopulations in lymph node metastases by FTIR imaging histopathology

While early stages of melanoma are usually cured by surgery, metastatic melanomas are difficult to treat because the widely available options have low response rates. Careful and precise diagnosis and staging are essential to determine patient's risk and to select appropriate treatments. Fortunately, the recent progress in immunotherapy is very encouraging. In this context, it is important to characterize the intratumoral infiltration of immune cells in each patient, which is however not done routinely due to the lack of standardized methods. In this study, we used Fourier transform infrared (FTIR) imaging combined with multivariate statistical analyses to investigate non-metastatic and metastatic lymph nodes from melanoma patients. Our results show that the different cell types have different infrared spectral features allowing automated identification of these cell types. High recognition rates were obtained using a supervised partial least square discriminant analysis (PLS-DA) model. Melanoma cells were recognized with 87.1% sensitivity and 85.7% specificity, showing that FTIR spectroscopy has similar detection power as immunohistochemistry. Besides, FTIR imaging could also distinguish lymphocyte subpopulations (B and T cells). Finally, we investigated the changes in lymphocytes due to the presence of metastases. Interestingly, specific features of spectra of lymphocytes present in metastatic or tumor-free lymph nodes could be evidenced by PCA. A PLS-DA model was capable of predicting whether lymphocytes originated from invaded or non-invaded lymph nodes. These data demonstrate that FTIR imaging is capable to distinguish known and also novel biological features in human tissues, with potential practical relevance for histopathological diagnosis and biomarker assessment.

Present and future perspectives on immunotherapy for advanced renal cell carcinoma: Going to the core or beating around the bush?

Metastatic lesions of renal cell carcinoma (RCC) occasionally regress spontaneously after surgical removal of the primary tumor. Although this is an exceptionally rare occurrence, RCC has thus been postulated to be immunogenic. Immunotherapies, including cytokine therapy, peptide-based vaccines, and immune checkpoint inhibitors have therefore been used to treat patients with advanced, metastatic RCC. We review the history, trends, and recent progress in immunotherapy for advanced RCC and discuss future perspectives, with consideration of our experimental work on galectin 9 and PINCH as promising specific immunotherapy targets.

Abstract 2796: Engineering multiple chimeric antigen receptors in T cells for the treatment of multiple myeloma

Abstract Multiple myeloma (MM) is an incurable plasma cell neoplasm accounting for 10% of all hematologic malignancies. With advanced treatments, the average survival has significantly improved. Recent progress in immunotherapy based on antibody and T cell receptor (TCR) engineering has shown great potential for treating cancer including MM. We hypothesize that immune cell engineering that overcomes immune suppression in the BM may completely eradicate residual cancer cells. To test our hypothesis, we have developed a streamlined approach to generate lentiviral constructs with chimeric antigen receptors (CAR) of T cells based on single chain antibodies that target multiple MM antigens including CD138, CD269 (mature B cell marker BCMA, or TNFRSF17), CD317 (hm1.24), and CD319 (CS1). To increase safety, we also incorporated a self-destructive, inducible suicide caspase 9 gene, which is fused with the CAR. Memory CD8 T cells were isolated from BM specimens of MM patients and immortalized for prolonged in vitro studies. These primary BM T cells were transduced using lentiviral vectors encoding the MM-targeting CARs, and co-cultured with MM cell lines to measure killing effect. The MM-specific killing activity was demonstrated based on annexin V and propidium iodide staining and flow cytometry analysis for quantitation of cell death. In transient (24 hours) killing assays, both CD138 and CD 319 CARs displayed moderate MM killing activities, at 48% and 36%, respectively, when using a CAR T effector to MM Target (E/T) ratio of 0.5/1. Interestingly, a synergistic MM killing effect (a five fold increase at an E/T ratio of 1/1) was observed when these two CARs were used in combination. On the other hand, CD269 and CD317 CAR T cells displayed marked MM killing activities, approximately 80% for both at an E/T ratio of 0.5/1 in 24 hours. In a long term CAR T/MM co-culture setting at E/T ratios of 0.5/1 and 1/1, we observed near complete MM killing (100%) by all four MM-targeting CAR T cells in two weeks. The self-destructive function of the inducible suicide gene in the CAR T cells was also demonstrated in the long-term co-cultures. Interestingly, upon killing of the CAR T cells by apoptotic induction, residual MM cells rebounded from the CD319 CAR T/MM co-culture, but not the other three CAR T/MM co-cultures, suggesting that rapid and efficient killing of MM cells by CAR T cells is necessary for complete eradication of MM cells. In summary, all four CARs demonstrated MM killing activities in short term and long term T cell co-cultures. Combination of multiple CARs may be necessary for effective suppression of MM residual disease in vivo and to prevent antigenic escape phenomenon. Together with the inducible apoptotic gene design, increased safety and efficiency for the CAR-based immunotherapy may be attained in future clinical trials. Citation Format: Lung-Ji Chang, Yuchen Liu, Jan S. Moreb. Engineering multiple chimeric antigen receptors in T cells for the treatment of multiple myeloma. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2796. doi:10.1158/1538-7445.AM2014-2796