Barnabas & Raphatuel.pptx cell biology seminar

stem cell presentation for master of science in health biothechnology and molecular biology

1. T Cell-Mediated Immunity| Implications in Health and Disease By Barnabas Tefera & Raphatuel Sija Addis Ababa, 2024 Addis Ababa University, Institute of Biotechnology; Department of Molecular Biology

2. Contents of the presentation Our Presentation Exclusively Focuses On Discussing These Topics Principles of adoptive T cell therapy in cancer Met Ö, Jensen KM, Chamberlain CA, Donia M, Svane IM. Principles of adoptive T cell therapy in cancer. Semin Immunopathol. 2019 Jan;41(1):49-58. The current state of T cell-based vaccines against human papillomaviruses Yang A, Farmer E, Lin J, Wu TC, Hung CF. The current state of therapeutic and T cell-based vaccines against human papillomaviruses. Virus Res. 2017 Mar 2;231:148-165. 

3. Özcan Met (PhD.) & Others Principles of adoptive T cell therapy in cancer

4. Introduction Cancer immunotherapy involves stimulating the immune system to target and combat cancer cells. Two effective types of immunotherapy are immune checkpoint inhibitors, which enhance natural antitumor activity, and adoptive cell therapy (ACT), which involves administering specific antitumor immune cells. Monoclonal antibodies, particularly those targeting regulatory immune checkpoint molecules like CTLA-4, PD-1, and PD-L1, have shown significant benefits in clinical trials for various cancers. While immune checkpoint therapy works well in many tumors, it may fail in poorly immunogenic cancers. ACT, specifically using tumor-recognizing T cells, could address this limitation and enhance responses in tumors already responsive to immune checkpoint therapy. The presentation outlines the basic principles of ACT, focusing on tumor-infiltrating lymphocytes (TILs) and genetically engineered T cells.

5. ACT Modalities The primary objective of adoptive cell therapy (ACT) is to induce a strong immune-mediated response against tumors by infusing T cells that have been manipulated outside the body. ACT strategies for tumor destruction involve two main approaches: isolating naturally occurring tumor-specific T cells from existing tumor masses, known as tumor-infiltrating lymphocytes (TILs), and genetically modifying T cells derived from blood to enable specific recognition of tumor cells. In both cases, T cells undergo ex vivo manipulation, followed by expansion and subsequent reintroduction into the patient after lymphodepletion, as illustrated in Figure 1 .

6. Fig. 1: Different adoptive T cell transfer (ACT) approaches to harness the immune system to treat cancer (a) Adoptive transfer of anti-tumor T cells isolated from within a patient’s tumor. Tumor infiltrating T cells (TILs) are extracted from surgically resected tumor samples, then expanded in vitro, followed by re-infusion into the lymphodepleted patient. (b) T cells from patient peripheral blood are isolated and expanded in culture and genetically modified to express either a T cell receptor (TCR) or a chimeric antigen receptor (CAR) that confers the ability to specifically recognize and destroy tumor cells when re-infused into the lymphodepleted patient.

7. Naturally Occurring tumor specific T cells Tumor-Infiltrating Lymphocytes (TILs) are a diverse group of lymphocytes, mainly comprising T cells and natural killer cells, that naturally migrate into tumors and are found in various solid tumors. Clinical evidence, dating back to a 1972 case report, indicates that the presence of TILs in tumors correlates with favorable prognoses. Adoptive Cell Therapy (ACT) involving TILs has been successful, particularly in metastatic melanoma, following methods developed for large-scale in vitro expansion using interleukin-2 (IL-2). This personalized immunotherapy involves the infusion of expanded TILs, preceded by nonmyeloablative lymphodepletion and followed by IL-2 administration.

8. …Continued The process includes fragmenting resected tumors, growing individual fragments in IL-2, and isolating TILs, which are then rapidly expanded using a protocol pioneered by Steven Rosenberg. While the traditional approach involves selecting TIL cultures based on specific attributes, such as tumor reactivity, a more streamlined method utilizes minimally cultured TILs for mass expansion, reducing complexity and cost. Patients undergo preconditioning with cyclophosphamide and fludarabine before TIL infusion to enhance treatment efficacy. ACT with TILs has shown remarkable success, with objective response rates of 40-50%, including complete tumor regression in 10-25% of patients. Notably, durable responses are observed, even in late-stage metastatic melanoma cases. Comparatively, other treatments, such as immune checkpoint blockade, have lower rates of complete tumor regression.

9. TIL-based ACT appears effective even in patients who have failed prior immunotherapies, suggesting distinct mechanisms of action. While associated with lower treatment-associated mortality than conventional treatments, TIL-based ACT can cause significant toxicities, primarily related to the preconditioning regimen and high-dose IL-2 administration. Strategies such as an attenuated IL-2 decrescendo regimen, have been explored to manage toxicities. Predictive criteria for treatment response, like tumor mutational burden or neoepitope burden, are still under investigation. Interest in TILs extends beyond melanoma to other solid cancers, but clinical responses with TIL-based ACT have been more moderate. Ongoing research aims to improve efficacy, identify cancer mutations, and extend the application of TIL-based ACT to other common cancers. …Continued

10. Genetically Modified Cells In contrast to TIL-based Adoptive Cell Therapy (ACT), the second approach for creating tumor-specific T cell therapies involves genetically modifying T cells to enhance their antitumor immune function. This strategy is employed when natural tumor-specific immune responses have proven ineffective. The genetic modification is achieved by introducing genetic material encoding either a cloned T cell receptor (TCR) or a synthetic chimeric antigen receptor (CAR) targeting tumor-specific antigens. CARs are engineered by combining the antigen-binding regions of an antibody molecule with the signaling components of various immunoreceptors and costimulatory molecules, making them highly specific and reactive against tumor cells. The general process of generating genetically modified T cells is illustrated in Fig. 1b. Peripheral blood T cells, usually obtained after leukapheresis, are activated, genetically altered, and expanded before being reintroduced into the patient.

11. …Continued Prior to T cell infusion, patients typically undergo a preconditioning regimen similar to that used in TIL-based ACT. Various gene transfer methods are employed for the genetic engineering of T cells, including transient mRNA transfection, retroviral vectors, lentiviral vectors, transposons, and, more recently, homologous recombination after gene editing. These techniques facilitate the introduction of the desired genetic material into T cells, enabling them to specifically target and attack tumor cells.

12. TCR Modified Cells To sum up, T-cell receptors (TCRs) are natural receptors on T cells that recognize peptide antigens presented by the major histocompatibility complex (MHC)/human leukocyte antigen (HLA) system on host cells. Genetically modified TCR therapy involves altering T cell specificity by expressing a new TCR alpha and beta chain pair that targets tumor antigens. This is achieved by isolating tumor-specific TCRs that recognize naturally processed tumor antigens, cloning them into retro- or lentiviral vectors, and transducing peripheral blood T cells ex vivo for expansion and infusion into patients. Tumor antigen-specific T cells, isolated from cancer patients, often have low affinity due to central tolerance. Strategies to address this include engineering high-affinity TCRs through affinity maturation, generating murine TCRs through immunization, and isolating TCRs in an allogeneic setting to bypass thymic selection limitations.

13. …Continued In a pioneering study using genetically modified TCRs, T cells from metastatic melanoma patients were transduced with a TCR directed against specific peptides, leading to sustained objective responses and persistent TCR-modified T cells for over a year in some patients. Other trials have demonstrated significant and prolonged tumor regression using TCRs against various tumor antigens. However, there are safety risks associated with genetically modified T cell therapies, including on-target off-tumor toxicity, off-target reactivity, and cytokine-release syndrome (CRS), Which involves a sudden increase in inflammatory cytokines induced by infused T cells. These risks highlight the importance of careful consideration in the development and application of genetically modified T cell therapies.

14. CAR-modified T cells The genetic modification of T cells with Chimeric Antigen Receptors (CARs) combines the specificity of antibody-like recognition with the cytotoxic potency and activation potential of T cells. Unlike TCR modification, CAR recognition does not rely on peptide processing or presentation by MHC molecules, making all surface-expressed target molecules potential CAR-triggering epitopes. First-generation CARs consist of an antigen-binding region (single-chain antibody variable fragment) fused to T cell signaling domains, providing only activation signal 1 and leading to CAR-T cell anergy upon repeated antigen stimulation. Second-generation CARs include an additional co-stimulatory domain, such as CD28 or 4-1BB, offering a second activation signal, resulting in enhanced clinical response rates compared to the first generation.

15. …Continued Third-generation CARs, incorporating another co-stimulatory domain, are in development to further potentiate CAR-T cell persistence and activity. CAR-T cells targeting CD19, a B cell-lineage antigen, have shown robust efficacy and frequent durable responses in clinical trials, particularly in treating B cell malignancies like chronic lymphocytic leukemia (CLL) and acute lymphoblastic leukemia (ALL). Success has also been observed with CAR-T cell therapy targeting the B cell maturation antigen (BCMA) for multiple myeloma. However, CAR-T cell therapy against solid tumors faces challenges, including inefficient T cell localization, physical barriers preventing tumor infiltration, high antigen heterogeneity, risk of on-target, off-tumor toxicity, and potent immunosuppressive factors in the tumor microenvironment. Ongoing research aims to overcome these obstacles through modified gene transfer methods, novel CAR designs, and the development of new targets for various solid cancers.

16. Figure 2 : Genetically modified T cells T cells recognize their target by the TCR complex, which is composed of the TCR α and β chain for recognition and the CD3 chains for signaling. T cells can be genetically engineered with defined specificity by expression of recombinant TCR αβ chains of known specificity. CARs are composed of a single-chain fragment of variable region (scFv) derived from the antigen-binding domain of antibodies, fused to the CD3ζ transmembrane and intracellular signaling domains from the TCR complex. Additional intracellular signaling domains are added for costimulatory signals, such as the CD28 and 4-1BB signaling domains, to yield second- and third-generation CARs.

17. Yang A. (PhD.) & Others The current state of T cell based vaccines against human papillomaviruses

18. Introduction Human Papillomavirus (HPV) is a causative agent of various conditions, including warts, cancer, and other diseases. Notably, it is responsible for nearly 100% of cervical cancer cases and has been linked to other cancers like penile, vaginal, vulvar, anal, and oropharynx cancers. Over 200 types of HPV have been identified, categorized into high and low-risk groups based on their oncogenic capacity. HPV infections are widespread, posing a significant global health burden. Most sexually active women experience HPV infection, usually asymptomatic and cleared by the immune system. However, persistent infections can lead to cervical intraepithelial neoplasia (CIN) and cervical carcinoma. The identification of HPV as a causative factor for malignancies provides an opportunity for control through immunization and targeted therapies.

19. …Continued Prophylactic vaccines targeting the major capsid protein L1 have been successful in preventing HPV infections, But evidence for their therapeutic effects in treating established infections and associated lesions is limited.

20. Therapeutic Vaccines In light of the global prevalence of HPV infections, there is an urgent need to develop effective treatments for established infections and associated diseases. Therapeutic vaccines, unlike preventative vaccines, aim to stimulate cell-mediated immune responses to target and eliminate infected cells. In cases where HPV-associated lesions progress to cancer, the integration of HPV viral DNA into the host's genome renders prophylactic vaccines ineffective. The deletion of certain genes during integration, particularly L1, L2, and E2, contributes to the increased expression of oncoproteins E6 and E7, considered key factors in the development and maintenance of HPV-associated malignancies. The constant expression of E6 and E7 in transformed cells makes them ideal targets for therapeutic HPV vaccines, as they can overcome immune tolerance challenges presented by many other cancers.

21. …Continued Various types of therapeutic HPV vaccines, such as live vector, protein or peptide, nucleic acid, and cell-based vaccines, have been developed and tested in preclinical and clinical trials. These vaccines predominantly target E6 and E7 antigens, aiming to stimulate CD8+ cytotoxic T cell or CD4+ helper T cell responses through antigen presentation via major histocompatibility complex (MHC) class I and MHC class II. The presentation of E6 and E7 antigens on MHC class I involves their processing by proteasomes in antigen-presenting cells (APCs). Only certain short peptides containing antigenic fragments (epitopes) can bind to MHC molecules with high affinity and interact with the T cell receptor (TCR) for effective immune response elicitation. Most therapeutic vaccines focus on provoking immune responses against the better-characterized E7 antigen in preclinical models.

22. Live vector-based Live vector-based vaccines, categorized as either bacterial or viral vectors, have the capacity to elicit robust cellular and humoral immune responses, as extensively reviewed by Yang et al. (2016). These vaccines are highly immunogenic, offering a variety of vectors for effective antigen delivery. However, a significant drawback of live vector-based vaccines is their inherent safety risk, particularly for individuals with compromised immune systems. This safety concern must be carefully considered and addressed in the development and administration of such vaccines.

23. Bacterial Vectors Bacterial vectors, such as Listeria monocytogenes , Lactobacillus lactis , Lactobacillus plantarum , and Lactobacillus casei , are extensively studied for therapeutic HPV vaccines. Listeria , with its unique ability to escape phagosomal lysis, proves promising. Listeria -based E7 vaccines effectively reduce tumors expressing E6/E7 and limit spontaneous tumor growth in mice. The inclusion of listeriolysin O (LLO) enhances the vaccine's therapeutic effects by decreasing regulatory T cells. Attenuated bacterial vectors, including mutant strains of Salmonella, Shigella, and Escherichia coli, can be generated for delivering plasmid-encoded genes to antigen-presenting cells. These findings underscore the potential of bacterial vectors in developing effective therapeutic HPV vaccines.

24. Viral Vectors Viral vectors present an appealing option for therapeutic HPV vaccines due to their ability to infect and express encoded antigens. Various viral vectors, including adenoviruses, adeno-associated viruses, alphaviruses, and vaccinia viruses, have been explored for delivering HPV E6 and E7 antigens. Vaccinia virus, a double-stranded DNA virus, is particularly promising due to its large genome, high infectivity, and low risk of irregular DNA integration. Vaccinia-based vaccines, such as those encoding E7 fused to calreticulin (CRT), E7 fused to LLO, and E7 linked to sorting signals and lysosomal-associated membrane protein (SigE7-LAMP-1), show potential. Adenoviruses have been investigated in preclinical studies, with promising results.

25. …Continued Replication-deficient adenoviruses encoding CRT/E7 fusion protein successfully eliminated E7-expressing tumors in mice. Another adenovirus vaccine encoding chimeric hepatitis B virus surface antigen (HBsAg) and HPV-16 E7 proteins increased E7-specific antibody and cytotoxic T lymphocyte (CTL) responses in vaccinated mice. An alphavirus vector-based vaccine, SFVeE6,7, encoding a fusion protein of HPV-16 E6 and E7 packaged into recombinant Semliki Forest virus (SFV) particles, shows potential for HPV treatment without requiring additional immune interventions to modulate regulatory T cell activity.

26. Peptide-based and Protein-based Peptide-based and protein-based vaccines for HPV are safe, stable, and easily produced. Peptides and proteins derived from HPV antigens are processed by dendritic cells (DCs) and presented on MHC class I or class II molecules to stimulate CD8+ or CD4+ T cell responses. Peptide-based vaccines, while stable and safe, suffer from poor immunogenicity. To address this, peptides are linked to lipids and adjuvants like chemokines, cytokines, and Toll-like receptor (TLR) ligands to enhance immune responses. However, these vaccines are MHC specific, limiting their suitability for large-scale production and treatment. Overlapping long-peptide vaccines have been proposed to improve potency and induce antigen-specific T cell responses.

27. …Continued Protein-based vaccines contain all possible human leukocyte antigen (HLA) epitopes, addressing a concern in peptide-based vaccines. However, they also exhibit poor immunogenicity, primarily activating antibody production through the MHC class II pathway. Adjuvants and immuno-stimulating molecules are added to protein-based vaccines to enhance endogenous processing, uptake, and presentation by MHC class I molecules.

28. Nucleic acid-based DNA vaccines are a potentially effective approach for antigen-specific immunotherapy, being safe, stable, and easy to produce. However, they may carry a risk of cellular transformation when encoding HPV oncogenes E6 and E7. This risk can be addressed by modifying the DNA to express non-oncogenic proteins. DNA vaccines involve injecting plasmid DNA into the host's cells, typically administered intramuscularly. Myocytes transfected with the DNA express the target antigen but are not efficient activators of the immune response. Dendritic cells (DCs) are crucial for presenting antigens to CD8+ cytotoxic T cells, either through exogenous antigen uptake or direct transfection.

29. …Continued Naked DNA has low immunogenicity as it cannot amplify or spread in vivo. Various strategies have been developed to enhance DNA vaccine potency. RNA replicon vaccines, derived from RNA viruses, offer sustained antigen expression and increased immunogenicity. They do not form viral particles, allowing repeated administration and avoiding the risk of chromosomal integration and cellular transformation. However, RNA replicons face challenges like low stability. Combining RNA replicons with DNA in a "suicidal DNA" approach has been attempted to trigger apoptosis in cells that take up the DNA, preventing integration and transformation. Despite promising preclinical results, RNA vaccines targeting HPV antigens and associated diseases have not been tested in clinical settings.

30. Whole cell-based Dendritic cell-based HPV vaccines involve loading dendritic cells (DCs) with HPV antigens and delivering them to patients. DCs can serve as natural adjuvants, enhancing antigen-specific immunotherapy against cancer. Strategies like transfecting DCs with siRNAs aim to prolong DC survival and improve vaccine efficacy. However, challenges include technical complexity, potential inconsistencies in vaccine quality due to varying culture techniques, and uncertainty about the most effective administration route. Tumor cell-based HPV vaccines seek to enhance tumor cell immunogenicity by increasing immune modulatory protein expression. While cytokine genes like IL-2, IL-12, and GMCSF have been used, the approach faces challenges.

31. …Continued Tumor antigens need not be well identified, allowing protection against more antigens, but for HPV-associated cervical cancers with well-known tumor-specific antigens, this method becomes impractical. Additionally, the risk of implanting new cancers into patients limits the use and testing of tumor cell-based vaccines in clinical trials against HPV and associated malignancies.

32. Methods to Enhance Therapeutic Vaccines Therapeutic HPV vaccines often exhibit low immunogenicity. Various strategies aim to enhance their effectiveness, including incorporating immunostimulating molecules or adjuvants, employing prime-boost regimens, using immunomodulatory agents, exploring different therapeutic modalities, and optimizing methods and locations for vaccine administration. These approaches are being investigated to improve the therapeutic impact of HPV vaccines, as discussed in a review by Yang et al. (2016).

33. Thanks