No description available.

Multiple myeloma is an aggressive plasma cell cancer that consistently acquires multi-drug resistance and relapses despite initial treatment successes. Patients may go through greater than 10-lines of therapy, highlighting the need for more effective treatment options. Immunotherapies are the latest evolution in targeted cancer treatments, and thus far have displayed impressive results in several hematological cancers, including multiple myeloma. T cells possess robust anti-tumor functions which can be harnessed and refined for the treatment of cancers. Genetic engineering of T cells to express a chimeric antigen receptor (CAR) confers antigen-specific tumor-targeting, and adoptive transfer of patient-derived CAR-engineered T (CAR T) cells has been efficacious in relapsed/refractory multiple myeloma. Despite the high efficacy, CAR T cell therapy for myeloma is associated with serious adverse events, which limits dose levels and patient eligibility. We have developed a novel synthetic antigen receptor platform, called the T cell antigen coupler (TAC) receptor, which has shown comparatively higher efficacy with a reduced pro-inflammatory profile compared with CAR T cells in pre-clinical models. The TAC receptor was purpose-built to co-opt the natural T cell activation machinery and lacks the costimulatory signaling typically incorporated in CAR designs. This thesis investigates strategies to augment TAC T cell function against for multiple myeloma through the evaluation of ancillary pharmacological and protein stimuli that would complement the anti-tumor functions of TAC T cells without modifying the TAC receptor design. In chapter 2, I investigated a strategy combining TAC T cells with the SMAC mimetic LCL161 to provide transient costimulatory effects. While LCL161 boosted TAC T cells survival and proliferation, the drug also enhanced susceptibility of TAC T cells to apoptosis and offered no advantage to the TAC T cells when challenged with myeloma. In chapter 3, I engineered TAC T cells to secrete IL-27 in an attempt to modulate the myeloma microenvironment and support T cell cytolytic function. IL-27 did not enhance the anti-tumor activity of TAC T cells but forced expression of IL-27 led to a reduction in the production of pro-inflammatory cytokines without altering cytotoxicity. In appendix I, I describe the process of optimizing CRISPR/Cas9 editing of primary TAC T cells. This methodology was required for much of the work in chapter 2. Ph.D. Thesis – Arya Afsahi McMaster University – Biochemistry and Biomedical Sciences v In appendix II, I describe an assessment of mRNA-engineering as a method to produce TAC T cells. This approach proved to be therapeutically futile and was not pursued beyond the work described herein. The work presented here highlights methods of combining TAC T cells with a clinically relevant SMAC mimetic, or the cytokine IL-27, and provides insights into the biological mechanisms that are affected by these approaches. / Thesis / Doctor of Philosophy (PhD)

Multiple myeloma, or myeloma, is a bone marrow cancer which characterizes by uncontrolled proliferation of mutant plasma cells. It is a disease that claims many lives every year, mostly due to the absence of curative treatment. Finding a suitable treatment is therefor of great importance. One way to study different diseases is to use a gene editing method for knockdown or knockout of specific genes. The main aim of this project was to design guide RNAs, to be able to use CRISPR/Cas9 for knockout of the two genes BMPR1A and BMPR2 in different myeloma cell lines (KJON, INA-6 and IH-1). This, to be able to study the expression and function of these genes. Further aim of the project was to investigate potential SMAD activation by treatment with different bone morphogenetic proteins (BMPs). However, due to limited time this could not be carried through. Six guide RNAs were designed and ligated into pLentiCRISPRv2. Plasmid amplification was done by transformation of Escherichia coli. To check the quality of the plasmids, PCR, gel electrophoresis and Sanger sequencing was performed. The results from the gel electrophoresis showed that nine of the twelve samples for BMPR1A and seven of the thirteen samples for BMPR2, that were tested, were positive. The results from the Sanger sequencing confirmed that all guides that were tested (BMPR1A 3.2.3, BMPR1A 4.2.2, BMPR2 1.1.4 and BMPR2 2.1.2), were properly ligated into the plasmids. The main aim of the project was successfully accomplished, but additional work is needed for any further conclusions.

No description available.

Daratumumab är en antikropp som används vid behandling av multipelt myelom. Daratumumab är specifik för CD38, ytproteinet som uttrycks i stor mängd på myeloma plasmaceller. Vid antikroppsscreen orsakar daratumumab panreaktivitet då erytrocyter också uttrycker CD38. Dithiothreitol (DTT) behandling av testerytrocyter har visats kunna eliminera panreaktiviteten med daratumumab-antikropparna, detta genom att DTT klyver bort epitopet på CD38 som daratumumab reagerar mot utan att förstöra andra kliniskt relevanta antigen. På Södra Älvsborgs sjukhus skickas prover från daratumumab-behandlade patienter till Sahlgrenska sjukhuset för genotypning för att hitta kompatibelt blod. Förhoppningen är att den nya metoden ska möjliggöra antikroppsscreen på daratumumab-behandlade patienter vid Södra Älvsborgs sjukhus.Syftet med examensarbetet var att verifiera metoden för användning av DTT-behandlade testerytrocyter på plasma från daratumumab-behandlade patienter för att underlätta val av erytrocyter för blodtransfusion.Provmaterialet var venöst tagen patientplasma från 16 daratumumab-behandlade patienter testades mot 0,2 M DTT-behandlade- och obehandlade-testerytrocyter. DTT-behandlade testerytrocyter testades även mot kända antikroppar på plasmaprover från patienter som ej behandlats med daratumumab. Hållbarhetsstudie utfördes med behandlade BAS-testceller.Panreaktivitet sågs hos samtliga patientprover med obehandlade testerytrocyter. Vid test med DTT-behandlade testerytrocyter blev samtliga prover negativa. Behandlade testerytrocyter som testades mot kända antikroppar gav resultat som var oförändrat jämfört med originalscreen. Behandlade testceller var brukbara 25 dagar.DTT behandling av testerytrocyter är effektivt och billigt, resultatet var pålitligt då samtliga patientprover inte uppvisade panreaktivitet efter DTT-behandling av testerytrocyter. De DTT-behandlade erytrocyterna behöll kliniskt relevanta antigen efter behandling och var hållbara 25 dagar. Metoden anses som användbar för Södra Älvsborgs sjukhus. / Daratumumab is an antibody used for treatment of multiple myeloma. The antibody is specific for the surface protein CD38 which is being expressed in high quantity on myeloma plasma cells. Daratumumab is causing pan-reactivity during antibody screening due to regular erythrocytes also express CD38. Dithiothreitol (DTT) treatment of test erythrocytes has shown to eliminate the pan-reactivity caused by the daratumumab antibodies by cleaving the epitope on CD38 that daratumumab is specific to. Södra Älvsborgs hospital are currently sending patient samples from patients treated with daratumumab to Sahlgrenska hospital in Gothenburg for genotyping to find compatible blood.The purpose of the project was to verify the method for use of DTT-treated test erythrocytes on plasma from daratumumab treated patients to screen for antibodies and easier find compatible erythrocytes for blood transfusion at Södra Älvsborgs hospital.Plasma samples from 16 patients treated with daratumumab were tested with DTT-treated and untreated test erythrocytes. DTT-treated test erythrocytes were tested against samples with known antibodies from patients not treated with daratumumab. The cells durability was also tested.Pan-reactivity was shown with all daratumumab samples with non-treated test erythrocytes. Tests with DTT-treated test erythrocytes showed no pan-reactivity. Results from treated test erythrocytes tested against known antibodies were unchanged from original screening. The cells were durable for 25 days.DTT-treatment of test erythrocytes is effective and cheap, test results were reliable, all patient samples had their pan-reactivity eliminated. DTT-treated erythrocytes kept clinically significant antigens. The method is useful for clinical use at Södra Älvsborgs hospital.

INTRODUCTION: Multiple myeloma (MM) is a disease that results in the production of ineffective immunoglobulins and monoclonal proteins in the blood and urine, leading to insufficient organ function or death. Currently, there is a 5-year survival rate of 47% for patients diagnosed with MM, with a proportion of patients ultimately succumbing to the disease. The current standard of care for MM includes toxic combinations of chemotherapy. The evolution of chimeric antigen receptor (CAR) T-cell therapy for hematologic cancers such as lymphoma, leukemia, and now myeloma has provided another effective treatment option for patients who have relapsed after standard treatments for MM. Idecabtagene Vicleucel (ide-cel), was approved in March 2021 for patients with relapsed and refractory MM. While CAR T-cell treatment appears to be far less toxic than standard chemotherapy, this therapy comes with its own associated toxicities, mainly cytokine release syndrome (CRS) and neurotoxicity (NT). In clinical trials, ide-cel demonstrated to be an effective treatment in some patients, leading to the FDA approval for patients who have exhausted multiple other lines of therapy. Currently, it is unclear why patients respond differently to CAR T-cell treatment and why some patients present with more severe toxicity than others. Therefore, this study aims to examine patient factors such as demographics, age, and treatment history to determine if such characteristics may influence the CAR T-cell response; also, we assess the efficacy of ide-cel in a real-world experience outside of a clinical trial. METHODS: In this study, 14 patients’ medical records were reviewed after receiving commercial CAR T-cell therapy between August 2021 and January 2022. Eligible patients for the therapy were determined by strict inclusion criteria, including having a confirmed diagnosis of MM and exhausting at least four prior lines of therapy, as well as exclusion criteria, such as excluding individuals who have received CAR T-cells prior in a clinical trial setting. Approximately one month before preparation lymphodepletion chemotherapy, eligible patients underwent leukapheresis and had their blood sent to a laboratory to extract T-cells and genetically modify them to express the CAR for reinfusion. On 3 and 5 days prior to CAR T-cell infusion, patients underwent lymphodepletion using fludarabine and cyclophosphamide. Patients remained in the hospital for approximately one week following infusion, pending adverse reactions. After discharge, patients returned to the hospital for routine follow-ups. Data analysis was then performed on collected clinical readouts such as: prior treatments, bone marrow biopsies, response rates, laboratory values from blood samples, and pre- and post-infusion scans of various tissues within the body. RESULTS: At a median follow-up time of 15 weeks, six patients (43%) achieved a complete response (CR), three patients demonstrated a partial response (PR, 21%), and four patients showed disease progression (PD, 28%). Post-infusion scans were not available for one subject (7%) as they were still in the hospital. These results are similar to the phase I and phase II trials in which 45% and 33% of patients demonstrated a CR post-infusion, respectively. As for associated toxicities, 10 patients (71%) experienced CRS and one patient (7%) presented with ICANS. All patients that achieved a CR experienced ide-cel related toxicities, compared with only 38% of those with less favorable or unknown outcomes, which indicates that systemic immune system activation which causes CRS may be required to achieve a CR but CRS is not always linked with a CR outcome. There were 28 different chemotherapy regimens used as the standard of care treatment prior to ide-cel therapy. We assessed the most recent chemotherapeutic regimen in each patient to assess whether there is an association with most recent treatment and response. Of the six patients that achieved a CR to ide-cel, all were previously treated with RVD or CyBorD regimens, compared to the four patients who had disease progression who were mainly treated with salvage DCEP chemotherapy. Four patients (29%) received DCEP as their final chemotherapy regimen, and 3 of these 4 (75%) demonstrated progressive disease after ide-cel. Two patients received Belantamab-Mafodotin prior to ide-cel treatment, with one patient presenting with disease progression and the other patient achieving CR. 71% of patients experienced CRS following ide-cel infusion, which is resembles the phase II trial of ide-cel in which 84% of patients demonstrated CRS. In this study, only 7% of patients experienced neurological toxicity, which is comparable to the 18% of patients that demonstrated to have ICANS in the phase II study. CONCLUSIONS: We found similar performance of the ide-cel CAR-T therapy in the real world setting as in the clinical trial. Also, the complete responses were achieved by subjects with an array of characteristics, including varying recent chemotherapeutic treatments, IgG, IgA, and light-chain only subtypes of MM, and diverse demographics and other characteristics. The characteristic that demonstrated the most predictability and somewhat unique to subjects with CR was the associated toxicities from ide-cel. Development of these associated toxicities may attest that substantial immune activation, of CAR T-cells and other immune cells, leads to the efficacy of the product in eliminating cancer cells. Further analysis will need to be completed as more individuals enroll in this study to be able to determine if there are significant associations between demographics and prior lines of treatment with response to ide-cel CAR-T therapy. Lastly, future studies should assess the immune cell effector functions that are generated in CR patients that will help to specify the association between ide-cel activation, experienced associated toxicities, and its efficacy.

Multiple myeloma and other plasma cell disorders infiltrate the bone marrow in different patterns. While some patients show a homogeneous distribution of the clonal plasma cells others present with focal accumulations, commonly called focal lesions. Novel imaging techniques can provide information on these infiltration patterns and, due to their low invasiveness, can be performed repeatedly and therefore be used for monitoring. Conventional magnetic resonance imaging (MRI) has a high sensitivity for bone marrow assessment but cannot safely differentiate between active and inactive lesions. Therefore, positron emission tomography, especially combined with computed tomography (PET/CT), has been more widely used, at least for the monitoring of treatment response. Comparative, but mostly retrospective studies, have shown that functional MRI techniques, namely diffusion-weighted imaging (DWI), which assesses the movement of water molecules, can evaluate tissue cellularity with high sensitivity, which challenges the dominance of PET/CT in treatment response assessment. This review will discuss the benefits and challenges of DWI and compare it to other available imaging techniques used in patients with monoclonal plasma cell disorders

No description available.

京都大学 / 新制・論文博士 / 博士(医学) / 乙第13603号 / 論医博第2313号 / 新制||医||1073(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 森田 智視, 教授 佐藤 俊哉, 教授 永井 純正 / 学位規則第4条第2項該当 / Doctor of Medical Science / Kyoto University / DFAM

<p>Multiple myeloma (MM) is an incurable B-cell malignancy mainly localized to the bone marrow. Our aim was to examine the growth- and survival-promoting role of the IGF-IR and its downstream signaling components in MM cells to identify potential targets for therapy. </p><p>Octreotide, a somatostatin analog that has been demonstrated to interfere with the actions of IGF-I, induced growth inhibition in both IL-6-dependent and IL-6-independent MM cell lines expressing the somatostatin receptors sst2, sst3 and sst5. Additionally, a slight pro-apoptotic effect could be observed in a few cell lines. In primary MM cells octreotide induced apoptosis, an effect that was abrogated by exogenously added IGF-I, but not by IL-6.</p><p>Inhibition of IGF-I signaling in Karpas 707 cells, using either the anti-IGF-IR antibody αIR3 or the PI 3-K inhibitors LY294002 and wortmannin, increased sensitivity to apoptosis induced by dexamethasone. Exogenously added IGF-I prevented dexamethasone-induced apoptosis, an effect that could partly be mimicked by the pharmacological GSK-3β inhibitors LiCl and SB415286. Thus, we suggest the GSK-3β as an important mediator of the anti-apoptotic effects of IGF-IR signaling in MM.</p><p>Using rapamycin we selectively inhibited mTOR, a phosphoprotein downstream of the IGF-IR. In MM cell lines rapamycin induced G0/G1-arrest, an effect being associated with an increase of the cyclin-dependent kinase inhibitor p27 and a decrease of the cyclins D2, D3 and E. Interestingly, in primary MM cells rapamycin induced apoptosis. Moreover, rapamycin potentiated dexamethasone-induced apoptosis, an effect that was associated with a downregulation of the anti-apoptotic protein survivin. Strikingly, the combinatorial treatment with rapamycin and dexamethasone suppressed the anti-apoptotic effects of exogenously added IGF-I and IL-6, thus suggesting this drug-combination to be active also in vivo. </p><p>Two newly developed, selective IGF-I RTK inhibitors proved to be very effective in MM cell lines and in primary MM cells providing 50-90% growth inhibition within 48 h of incubation. The inhibitors induced massive apoptosis together with a prominent cell cycle arrest in the G2/M-phase. Importantly, the IGF-I RTK inhibitors downregulated the tyrosine phosphorylation of the IGF-IR β-chain but not of the insulin receptor β-chain. </p><p>In conclusion, the IGF-IR potently promotes growth and survival of MM cells. Therefore, interfering with the IGF-IR signaling pathway might be a suitable strategy to improve MM treatment.</p>