Retroelements are genomic parasites which make up ~42% of the human genome and 38% of the mouse genome. Most are degenerate, but a large number have relatively intact promoter elements, suggesting that they are capable of transcription. Transcriptionally active retroelements can perturb normal transcription units in their vicinity through a variety of mechanisms, leading to phenotypic effects and in some cases disease. This phenomenon of transcriptional interference has been observed in organisms as diverse as maize, Drosophila, and the mouse. We analysed the extent of retroelement transcription in normal and diseased tissues, by searching the mouse and human EST databases for transcripts originating in retroelement promoters, and found a large number of transcripts from LINEs, SINEs and ERVs. Retroelement transcripts were found to be initiated in both sense and antisense orientations, and to be equally as common in normal and diseased tissue. Several of these transcripts were chimeric, appearing to initiate in retroelements and reading through to cellular genes, suggestive of transcriptional interference. We have used transposon display to identify and recover retroelement transcripts in the mouse. Transcripts initiated in LINE, SINE and ERV promoters are numerous, and many are chimeric with cellular genes. Although the numbers of recovered chimeric transcripts are too large to permit rigorous analysis of more than a small proportion, some of those we have studied further appear to be authentic transcripts that may represent interference with the canonical promoters of the genes in question. Our results suggest that transcriptional interference by retroelements may be a relatively common occurrence in mammals.

Ricin toxin is a very deadly plant protein that is synthesized by the plant Ricinus communis. The molecular structure of ricin toxin places it in a group of similar proteins classified as a Type II RIP due to its heterodimeric construction; it is composed of a toxic A-chain possessing enzymatic action, and a receptor binding B-chain. Monoclonal antibodies were obtained with binding activities against either the A-chain or B-chain, and a surrogate non-toxic ricin analogue, TST10114, was determined to be suitable for characterization of the anti-ricin monoclonal antibodies. One potent anti-ricin A-chain neutralizing monoclonal antibody was chosen for chimerization, RAC18, which exhibited strong binding affinity and neutralizing properties. The constant regions of a human immunoglobulin G2 (IgG2) were used as the backbone for the recombinant chimeric antibody. The resulting chimeric RAC18-huG2 was transiently expressed in human-derived HEK 293F cells, purified, and assessed for binding characteristics and functional attributes.

Ricin toxin is a very deadly plant protein that is synthesized by the plant Ricinus communis. The molecular structure of ricin toxin places it in a group of similar proteins classified as a Type II RIP due to its heterodimeric construction; it is composed of a toxic A-chain possessing enzymatic action, and a receptor binding B-chain. Monoclonal antibodies were obtained with binding activities against either the A-chain or B-chain, and a surrogate non-toxic ricin analogue, TST10114, was determined to be suitable for characterization of the anti-ricin monoclonal antibodies. One potent anti-ricin A-chain neutralizing monoclonal antibody was chosen for chimerization, RAC18, which exhibited strong binding affinity and neutralizing properties. The constant regions of a human immunoglobulin G2 (IgG2) were used as the backbone for the recombinant chimeric antibody. The resulting chimeric RAC18-huG2 was transiently expressed in human-derived HEK 293F cells, purified, and assessed for binding characteristics and functional attributes.

BACKGROUND: Patients with relapsed or refractory large B-cell lymphoma (LBCL) who have relapsed after at least 2 lines of therapy had a poor prognosis before the introduction of chimeric antigen receptor (CAR) T-cell therapy. The FDA approved three CD19 CAR T-cell products, axicabtagene ciloleucel (axi-cel), tisagenlecleucel, and lisocabtagene maraleucel (liso-cel), based on the results of pivotal phase 2 clinical trials. High response rates and long-term remissions in these multiply relapsed patients led to randomized trials as a second-line therapy against the current standard of care for primary refractory and early relapsing LBCL. Axi-cel and liso-cel are now approved as second-line treatments for patients with relapsed or refractory large B-cell lymphoma based on these trials, while tisagenlecleucel failed to improve upon second-line standard of care. This has led to greater axi-cel and liso-cel usage as compared with tisagenlecleucel. Clinical trials and real-world trials show a higher toxicity profile for axi-cel as compared to liso-cel with similar efficacy outcomes, leading to selection of liso-cel for older patients with more medical comorbidities. However, axi-cel manufacturing is faster and more reliable making it a preferred choice for rapidly progressive lymphomas. No direct comparison has been made between the two in order to optimally inform product selection. OBJECTIVE: We aimed to compare the toxicity profile and efficacy outcomes between two cohorts, one treated with axi-cel and the other with liso-cel, ideally well matched, during the same period of time. METHODS: We retroactively gathered patient data for patients treated between June 2021 to September 2022 with both products. We compared the cohorts for patient characteristics that are proven to affect the toxicity and efficacy in order to identify significant differences that could influence our results and to increase the likelihood that the two cohorts were well matched. We then assessed associated toxicities and long-term efficacy outcomes. RESULTS: The two cohorts were comparable for all patient and disease variables other than age (median age of 62 years old in axi-cel compared to 71 years old liso-cel [p < 0.001]). There was no significant difference between high-grade cytokine release syndrome (CRS) (3% vs 5% for axi-cel vs. liso-cel cohorts, respectively; p = 0.58), high-grade immune effector cell-associated neurotoxicity syndrome (ICANS) (18% [ASTCT] or 19% [CTCAE], 14% [ASTCT] or 12% [CTCAE] for axi-cel vs. liso-cel cohorts, respectively, p = 0.055). There were higher rates of any grade CRS with axi-cel, and duration of hospitalization was longer for axi-cel vs. liso-cel (10 vs. 14 days, respectively). Best overall response rates (ORR) (93% vs. 84% axi-cel vs. liso-cel, respectively) and complete response (CR) rates (71% vs. 56% axi-cel vs liso-cel, respectively) did not statistically differ between the two groups. 12-month overall survival (OS) (76% vs. 81% axi-cel vs. liso-cel, respectively) and progression free survival (PFS) (61% vs. 45% of patients axi-cel vs. liso-cel, respectively) did not statistically differ between the two groups (p =0.94, p =0.51 for OS and PFS, respectively). CONCLUSIONS: Our study showed both products are similar in their high-grade toxicity profile as well as their efficacy. While axi-cel has more any grade CRS and ICANS, the lack of significantly higher high-grade toxicities likely reflects better and more aggressive toxicity mitigation strategies when patients present with low grade side effects. As a result, axi-cel in our study was found to be less toxic than previously seen in past clinical trials as well as real-world studies. Many factors go into selection of a CAR T-cell product, ranging from product performance attributes like safety and efficacy, to product manufacturing qualities like turnaround time and fidelity of manufacturing. With equivalency with regards to product performance, manufacturing qualities may then be most important in guiding product selection for LBCL patients.

Cancer is an uncontrolled growth of the body's own cells. While cancer rates increase with age, this disease afflicts both young and old. Traditional cancer therapy has had three major facets: 1) chemotherapy, which can non-specifically damage healthy tissue, 2) radiation, which can make some types of cancer more likely in the future, and 3) surgery, which can be physically traumatic and is not effective in removing unseen microtumors or circulating metastases. Immunotherapy, by its very nature, is drastically different. Immunotherapy seeks to employ cells or molecules from the immune system, in their original or a modified form, to augment, assist or replace missing elements of the native functioning immune system. Our immunotherapeutic approach has been to develop novel chimeric antigen receptors (CAR) and deliver the engineered transgene into hematopoietic stem cells (HSC). We have developed a novel single chain TCR (scTCR) in which the TCR V-alpha and V-beta segments are joined by a flexible linker. In addition to our scTCR we developed a single chain antibody molecule (scFv) to increase avidity to the tumor antigen and avoid the potential limitation of MHC restriction. Our lab has previously developed a signaling cassette based on the CD3 zeta chain, CD28 and p56Lck proteins which are prominent in the T-cell signaling pathway. The single chain specificities are linked to the signaling cassette that we have shown to function in T-cells. With specificity and signaling coupled, the chimeric antigen receptor can be transduced into hematopoietic stem cells (HSC) via a lentivirus vector. This adoptive immunotherapy can potentially eliminate malignant cells or supplement traditional therapies by providing engineered specificity and a useful method to transfer and expand tumor specific T-cells. We show in this study that the CAR can be delivered effectively to HSC and that the introduced transgene is expressed in multiple cell lineages. We also have developed a novel method of increasing lentiviral transduction efficiency. Both transduced fraction of cells and overall expression can be increased by proper timing and coordination of cell growth, cell cycle phase, vector addition and treatment with heat shock.

Vectors based on lentivirus backbones have revolutionized our ability to transfer genesinto many cell types. Lentiviral vectors integrate into the chromatin of target cells and do not transfer any viral genes causing vector replication. Both of these features arecommonly used in gene therapy and have been used clinically in individuals sufferingfrom cancer, infections and genetic diseases. It has been discovered that T-cells can be genetically modified to be used as effective weapons against cancer: therefore virus mustbe produced to deliver the gene of interest into the T-cells. In this project, lentiviralvectors have been produced to transfer the gene coding for a chimeric antigen receptor(CAR) which is directed to CD19 on B-cells. The vectors will, hence, be used to generateCD19 retargeted T-cells in purpose to kill CD19 cells such as B-cell lymphoma andleukemia. We have evaluated two production protocols to determine a feasible method toculture these vectors. We have also stimulate T-cells with two different antibodies (anti-CD3 and anti-CD28) and transduced T-cells. Our results demonstrate that theconcentration of virus was higher after prolonged incubation in 4˚C, which can not beexplained. The stimulation demonstrated that bound anti-CD3 was the best stimulator,and moreover the FACS-analysis showed that addition of anti-CD28 gave a highertransduction level. In conclusion, the viral vectors may be kept in 4˚C for two days beforeconcentrating the virus, and bound anti-CD3 is a better choice than soluble anti-CD3 forstimulation of T-cells.

Lentiviral gene therapy has held great promise for treating a wide range of neurological disorders due to its ability to stably integrate into the genome of nondividing cells like neurons, in addition to dividing cells. The nervous system is a complex and highly heterogeneous system, and while a therapeutic intervention may have beneficial effects in one population of cells it may have severe side effects in another. For this reason, specific targeting of lentiviral vectors is crucial for their ultimate utility for research and clinical research use. Two different approaches for focusing the targeting of lentiviral vectors were employed in these studies. The first method involved assessing the effects of vector production strategies on the resulting virus’s tropism both in vivo and in vitro. The changes in vector transduction were determined via flow cytometry on cells in culture and immunohistochemistry following brain injections. Results from these experiments suggest that while the production conditions do impact the vectors efficacy, there is not a distinct effect on their tropism. A unique characteristic of retroviral and lentiviral vectors is their capacity for being pseudotyped, conferring a new tropism on the vector. Native tropisms are generally not specific beyond very broad cell types, which may not be sufficient for all applications. In this case, chimeric targeting molecules can provide an even more refined targeting profile compared to native pseudotypes. The second approach utilizes novel chimeric glycoproteins made from nerve growth factor and the vesicular stomatitis virus glycoprotein. These chimeras are designed to pseudotype lentiviral vectors to target nociceptive sensory neurons for a variety of disorders. While these chimeras were successfully produced as protein, they were misfolded and sequestered in the endoplasmic reticulum and therefore unavailable to produce lentivirus. While neither strategy was completely successful, they do provide interesting information for the design and creation of lentiviral vectors. This research shows that small differences in the steps followed as part of a lentivirus production protocol can greatly impact the resulting vectors efficacy. It also shows that while VSV has been used to create chimeric glycoproteins, not all targeting molecules are suitable for this purpose.

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Diss. (sammanfattning) Stockholm : Karol. inst., 2002. / Härtill 4 uppsatser.

Few cancers arising in fluid organ systems can be cured with localized therapeutic modalities, such as radiation or surgical organ removal. Chemotherapy and hematopoietic stem cell transplants have long been employed as the standard of care for patients diagnosed with leukemias and lymphomas. Though research continues to propose new, more potent chemotherapeutic agents, a new paradigm of treating cancerous malignancies with tumor-specific monoclonal antibodies, adoptively transferred tumor-fighting cells, and other exogenously administered immunomodulatory agents, has emerged over the past decade. These immunotherapies have dramatically improved the outcomes of patients diagnosed with cancers of B lymphocytes, referred to as B-cell malignancies. Though curative FDA-approved therapies for patients diagnosed with B-cell malignancies have yet to be established, recent research in the field of adoptive T-cell therapy has produced promising results. Tumor infiltrating lymphocyte therapy (TIL therapy), T-cell Receptor Therapy (TCR therapy) and Chimeric Antigen Receptor T-cell Therapy (CAR T-cell therapy) are the three most extensively studied adoptive T-cell immunotherapies in the context of B-cell malignancies. TIL and TCR therapies, in which patients are provided with either the patient’s own tumor-specific T-cells or T-cells expressing engineered, tumor-specific TCRs, respectively, enhance the patient’s immune system to mount a more potent, anti-tumor response. However, these adoptive T-cell therapies do not change the mechanisms of the immune response. Cancerous cells can evade immune attack and dampen immune responses to survive and thrive in the body. By down-regulating their expression of human major histocompatibility complex I (MHC I), for example, cancer cells escape T-cell recognition, which is dependent on MHC expression. A chimeric antigen receptor (CAR), is composed of an antibody-derived (B-cell derived) extracellular, antigen-recognition domain, and T-cell derived intracellular domains. CAR T-cells, therefore, exploit the cytotoxic nature of CD8+ T-cells, and the MHC independent recognition of B-cell receptors, to identify and destroy all cells expressing a specific target. Consequently, many of the cancer cell’s mechanisms of immune evasion are less effective in the presence of CAR T-cells. Progressive generations of CAR T-cell designs couple these receptors with costimulatory molecules to amplify the activation, efficacy, and potency of these cells in-vivo. Over the past five years, phase I and IIa clinical trials have produced remarkable results in the treatment of advanced stage, high-risk B-cell malignancies, namely Acute Lymphoblastic Leukemia (ALL), Chronic Lymphocytic Leukemia (CLL), and Non-Hodgkin’s Lymphoma (NHL). However, the significant oncogenic risks and fatal adverse events associated with this therapy necessitate further research to improve safety and reliable clinical efficacy of CAR T-cell therapy. In spite of these risks, the adoptive transfer of CD19-targeting, CAR expressing, cytotoxic T-cells (anti-CD19 CAR-T-cells) has produced sustained, complete remissions in patients diagnosed with progressive, advanced-stage, B-cell malignancies, for whom alternative treatments were not available. The unprecedented results of early clinical trials, as well as ongoing preclinical studies aimed at improving the design and production of CAR T-cells suggest a promising future for CAR T-cell therapy as a cure for B-cell malignancies.