The inhibitory receptors PD1, Tim3, and A2aR are highly expressed during mesoCAR T cell manufacturing in advanced human epithelial ovarian cancer.

BACKGROUND: Chemotherapy and surgery have been the mainstays of epithelial ovarian cancer (EOC) treatment so far. Cellular immunotherapies such as CAR T cell therapy have recently given hope of a cure for solid tumors like EOC. However, extrinsic factors associated with the CAR T cell manufacturing process and/or intrinsic dysregulation of patient-derived T cells, which could be associated with cancer itself, cancer stage, and treatment regimen, may hamper the efficacy of CAR T cell therapy and promote their exhaustion or dysfunction. METHODS: To investigate the association of these factors with CAR T cell exhaustion, the frequency of T and CAR T cells expressing three immune inhibitory receptors (i.e., TIM3, PD1, A2aR) generated from T cells of EOC patients and healthy controls was measured during each stage of CAR T cell production. RESULTS: Our findings revealed that primary T cells from EOC patients show significantly elevated expression of immune inhibitory receptors, and this increase was more prominent in patients undergoing chemotherapy and those with advanced cancer. In addition, the CAR T cell manufacturing process itself was found to upregulate the expression of these inhibitory receptors and more importantly increase the population of exhausted mesoCAR T cells. CONCLUSIONS: Our observations suggest that intrinsic characteristics of patient-derived T cells and extrinsic factors in CAR T cell production protocols should be considered and properly counteracted during CAR T cell manufacturing process. In addition, mitigating the signaling of immune inhibitory receptors through pharmacological/genetic perturbation during CAR T cell manufacturing might profoundly improve CAR T cells function and their antitumor activity in EOC and other solid tumors.

Epigenetic strategies to boost CAR T cell therapy.

Chimeric antigen receptor (CAR) T cell therapy has led to a paradigm shift in cancer immunotherapy, but still several obstacles limit CAR T cell efficacy in cancers. Advances in high-throughput technologies revealed new insights into the role that epigenetic reprogramming plays in T cells. Mechanistic studies as well as comprehensive epigenome maps revealed an important role for epigenetic remodeling in T cell differentiation. These modifications shape the overall immune response through alterations in T cell phenotype and function. Here, we outline how epigenetic modifications in CAR T cells can overcome barriers limiting CAR T cell effectiveness, particularly in immunosuppressive tumor microenvironments. We also offer our perspective on how selected epigenetic modifications can boost CAR T cells to ultimately improve the efficacy of CAR T cell therapy.

Hersintuzumab: A novel humanized anti-HER2 monoclonal antibody induces potent tumor growth inhibition.

Humanized monoclonal antibodies (mAbs) against HER2 including trastuzumab and pertuzumab are widely used to treat HER2 overexpressing metastatic breast cancers. These two mAbs recognize distinct epitopes on HER2 and their combination induces a more potent blockade of HER2 signaling than trastuzumab alone. Recently, we have reported characterization of a new chimeric mAb (c-1T0) which binds to an epitope different from that recognized by trastuzumab and significantly inhibits proliferation of HER2 overexpressing tumor cells. Here, we describe humanization of this mAb by grafting all six complementarity determining regions (CDRs) onto human variable germline genes. Humanized VH and VL sequences were synthesized and ligated to human γ1 and κ constant region genes using splice overlap extension (SOE) PCR. Subsequently, the humanized antibody designated hersintuzumab was expressed and characterized by ELISA, Western blot and flow cytometry. The purified humanized mAb binds to recombinant HER2 and HER2-overexpressing tumor cells with an affinity comparable with the chimeric and parental mouse mAbs. It recognizes an epitope distinct from those recognized by trastuzumab and pertuzumab. Binding of hersintuzumab to HER2 overexpressing tumor cells induces G1 cell cycle arrest, inhibition of ERK and AKT signaling pathways and growth inhibition. Moreover, hersintuzumab could induce antibody-dependent cell cytotoxicity (ADCC) on BT-474 cells. This new humanized mAb is a potentially valuable tool for single or combination breast cancer therapy.

Simultaneous targeting of Tim3 and A2a receptors modulates MSLN-CAR T cell antitumor function in a human cervical tumor xenograft model.

Introduction: Chimeric antigen receptor (CAR) T cell therapy has transformed the treatment of hematological malignancies. However, its efficacy in solid tumors is limited by the immunosuppressive tumor microenvironment that compromises CAR T cell antitumor function in clinical settings. To overcome this challenge, researchers have investigated the potential of inhibiting specific immune checkpoint receptors, including A2aR (Adenosine A2 Receptor) and Tim3 (T cell immunoglobulin and mucin domain-containing protein 3), to enhance CAR T cell function. In this study, we evaluated the impact of genetic targeting of Tim3 and A2a receptors on the antitumor function of human mesothelin-specific CAR T cells (MSLN-CAR) in vitro and in vivo. Methods: Second-generation anti-mesothelin CAR T cells were produced using standard cellular and molecular techniques. A2aR-knockdown and/or Tim3- knockdown anti-mesothelin-CAR T cells were generated using shRNA-mediated gene silencing. The antitumor function of CAR T cells was evaluated by measuring cytokine production, proliferation, and cytotoxicity in vitro through coculture with cervical cancer cells (HeLa cell line). To evaluate in vivo antitumor efficacy of manufactured CAR T cells, tumor growth and mouse survival were monitored in a human cervical cancer xenograft model. Results: In vitro experiments demonstrated that knockdown of A2aR alone or in combination with Tim3 significantly improved CAR T cell proliferation, cytokine production, and cytotoxicity in presence of tumor cells in an antigen-specific manner. Furthermore, in the humanized xenograft model, both double knockdown CAR T cells and control CAR T cells could effectively control tumor growth. However, single knockdown CAR T cells were associated with reduced survival in mice. Conclusion: These findings highlight the potential of concomitant genetic targeting of Tim3 and A2a receptors to augment the efficacy of CAR T cell therapy in solid tumors. Nevertheless, caution should be exercised in light of our observation of decreased survival in mice treated with single knockdown MSLN-CAR T cells, emphasizing the need for careful efficacy considerations.

PX-478, an HIF-1α inhibitor, impairs mesoCAR T cell antitumor function in cervical cancer.

Introduction: Chimeric Antigen Receptor (CAR) T cell therapy has demonstrated remarkable success in treating hematological malignancies. However, its efficacy against solid tumors, including cervical cancer, remains a challenge. Hypoxia, a common feature of the tumor microenvironment, profoundly impacts CAR T cell function, emphasizing the need to explore strategies targeting hypoxia-inducible factor-1α (HIF-1α). Methods: In this study, we evaluated the effects of the HIF-1α inhibitor PX-478 on mesoCAR T cell function through in-silico and in vitro experiments. We conducted comprehensive analyses of HIF-1α expression in cervical cancer patients and examined the impact of PX-478 on T cell proliferation, cytokine production, cytotoxicity, and exhaustion markers. Results: Our in-silico analyses revealed high expression of HIF-1α in cervical cancer patients, correlating with poor prognosis. PX-478 effectively reduced HIF-1α levels in T and HeLa cells. While PX-478 exhibited dose-dependent inhibition of antigen-nonspecific T and mesoCAR T cell proliferation, it had minimal impact on antigen-specific mesoCAR T cell proliferation. Notably, PX-478 significantly impaired the cytotoxic function of mesoCAR T cells and induced terminally exhausted T cells. Discussion: Our results underscore the significant potential and physiological relevance of the HIF-1α pathway in determining the fate and function of both T and CAR T cells. However, we recognize the imperative for further molecular investigations aimed at unraveling the intricate downstream targets associated with HIF-1α and its influence on antitumor immunity, particularly within the context of hypoxic tumors. These insights serve as a foundation for the careful development of combination therapies tailored to counter immunosuppressive pathways within hypoxic environments and fine-tune CAR T cell performance in the intricate tumor microenvironment.

PGE2-EP2/EP4 signaling elicits mesoCAR T cell immunosuppression in pancreatic cancer.

Introduction: For many years, surgery, adjuvant and combination chemotherapy have been the cornerstone of pancreatic cancer treatment. Although these approaches have improved patient survival, relapse remains a common occurrence, necessitating the exploration of novel therapeutic strategies. CAR T cell therapies are now showing tremendous success in hematological cancers. However, the clinical efficacy of CAR T cells in solid tumors remained low, notably due to presence of an immunosuppressive tumor microenvironment (TME). Prostaglandin E2, a bioactive lipid metabolite found within the TME, plays a significant role in promoting cancer progression by increasing tumor proliferation, improving angiogenesis, and impairing immune cell's function. Despite the well-established impact of PGE2 signaling on cancer, its specific effects on CAR T cell therapy remain under investigation. Methods: To address this gap in knowledge the role of PGE2-related genes in cancer tissue and T cells of pancreatic cancer patients were evaluated in-silico. Through our in vitro study, we manufactured fully human functional mesoCAR T cells specific for pancreatic cancer and investigated the influence of PGE2-EP2/EP4 signaling on proliferation, cytotoxicity, and cytokine production of mesoCAR T cells against pancreatic cancer cells. Results: In-silico investigations uncovered a significant negative correlation between PGE2 expression and gene signature of memory T cells. Furthermore, in vitro experiments demonstrated that the activation of PGE2 signaling through EP2 and EP4 receptors suppressed the proliferation and major antitumor functions of mesoCAR T cells. Interestingly, the dual blockade of EP2 and EP4 receptors effectively reversed PGE2-mediated suppression of mesoCAR T cells, while individual receptor antagonists failed to mitigate the PGE2-induced suppression. Discussion: In summary, our findings suggest that mitigating PGE2-EP2/EP4 signaling may be a viable strategy for enhancing CAR T cell activity within the challenging TME, thereby improving the efficacy of CAR T cell therapy in clinical settings.

Crosstalk between autophagy and metabolic regulation of (CAR) T cells: therapeutic implications.

Despite chimeric antigen receptor (CAR) T cell therapy's extraordinary success in subsets of B-cell lymphoma and leukemia, various barriers restrict its application in solid tumors. This has prompted investigating new approaches for producing CAR T cells with superior therapeutic potential. Emerging insights into the barriers to CAR T cell clinical success indicate that autophagy shapes the immune response via reprogramming cellular metabolism and vice versa. Autophagy, a self-cannibalization process that includes destroying and recycling intracellular components in the lysosome, influences T cell biology, including development, survival, memory formation, and cellular metabolism. In this review, we will emphasize the critical role of autophagy in regulating and rewiring metabolic circuits in CAR T cells, as well as how the metabolic status of CAR T cells and the tumor microenvironment (TME) alter autophagy regulation in CAR T cells to restore functional competence in CAR Ts traversing solid TMEs.

Genetic Modification of Cytokine Signaling to Enhance Efficacy of CAR T Cell Therapy in Solid Tumors.

Chimeric antigen receptor (CAR) T cell therapy has shown unprecedented success in treating advanced hematological malignancies. Its effectiveness in solid tumors has been limited due to heterogeneous antigen expression, a suppressive tumor microenvironment, suboptimal trafficking to the tumor site and poor CAR T cell persistence. Several approaches have been developed to overcome these obstacles through various strategies including the genetic engineering of CAR T cells to blunt the signaling of immune inhibitory receptors as well as to modulate signaling of cytokine/chemokine molecules and their receptors. In this review we offer our perspective on how genetically modifying cytokine/chemokine molecules and their receptors can improve CAR T cell qualities such as functionality, persistence (e.g. resistance to pro-apoptotic signals) and infiltration into tumor sites. Understanding how such modifications can overcome barriers to CAR T cell effectiveness will undoubtedly enhance the potential of CAR T cells against solid tumors.

Chimeric Antigen Receptor (CAR) T Cell Therapy for Metastatic Melanoma: Challenges and Road Ahead.

Metastatic melanoma is the most aggressive and difficult to treat type of skin cancer, with a survival rate of less than 10%. Metastatic melanoma has conventionally been considered very difficult to treat; however, recent progress in understanding the cellular and molecular mechanisms involved in the tumorigenesis, metastasis and immune escape have led to the introduction of new therapies. These include targeted molecular therapy and novel immune-based approaches such as immune checkpoint blockade (ICB), tumor-infiltrating lymphocytes (TILs), and genetically engineered T-lymphocytes such as chimeric antigen receptor (CAR) T cells. Among these, CAR T cell therapy has recently made promising strides towards the treatment of advanced hematological and solid cancers. Although CAR T cell therapy might offer new hope for melanoma patients, it is not without its shortcomings, which include off-target toxicity, and the emergence of resistance to therapy (e.g., due to antigen loss), leading to eventual relapse. The present review will not only describe the basic steps of melanoma metastasis, but also discuss how CAR T cells could treat metastatic melanoma. We will outline specific strategies including combination approaches that could be used to overcome some limitations of CAR T cell therapy for metastatic melanoma.

Development of a Novel Inhibitory Chimeric Anti-HER2 Monoclonal Antibody.

BACKGROUND: We have recently produced an inhibitory mouse anti-human HER2 mAb (2A8) which displayed potent anti-tumor activity in combination with trastuzumab. OBJECTIVE: To describe chimerization and functional characterization of 2A8 mAb. METHODS: The VH and VL genes of 2A8 mAb were amplified from cDNA of the mouse hybridoma, ligated to constant regions of human immunoglobulin, and expressed in CHO cell line. Reactivity with four members of human HER family, the inhibitory effects and antibody-dependent cell cytotoxicity (ADCC) of purified chimeric mAb (c2A8) were assessed by ELISA, XTT, H3-tymidine incorporation and lactate dehydrogenase assays. Inhibition of ERK and AKT downstream signaling pathways by the chimeric antibody were analyzed by Western blotting. RESULTS: Chimeric 2A8 mAb bound to recombinant human HER2 and did not cross-react with the other members of HER family. Moreover, c2A8 was able to recognize HER2-overexpressing cancer cell line and inhibited growth and proliferation of these cells. The binding affinity of c2A8 was comparable to the mouse parental mAb. ADCC and Western blotting results showed that the mouse 2A8 mAb was successfully chimerized and could significantly inhibit phosphorylation of AKT in combination with trastuzumab. CONCLUSION: The c2A8 mAb is potentially a valuable tool for targeted immunotherapy of HER2 positive cancers.

Differential Effects of Inhibitory and Stimulatory Anti-HER2 Monoclonal Antibodies on AKT/ERK Signaling Pathways

Objective: Homo- and heterodimerization of the receptor tyrosine kinase HER2 hyperactivate several downstream signaling pathways, leading to uncontrolled growth and proliferation of tumor cells. Anti-HER2 monoclonal antibodies (mAbs) may induce different effects on HER2 dimerization and signaling. Methods: The effect of two inhibitory (2A8, 1T0) and one stimulatory (1H9) anti-HER2 mAbs either alone or in combination with trastuzumab was investigated on AKT and ERK signaling pathways and HER2 degradation in a human breast cancer cell line (BT-474) by Western blotting. Result: While 1H9 mAb had no significant effect on AKT and ERK signaling pathways, 1T0 and 2A8 mAbs inhibited phosphorylation of both pathways. Combination of 1T0 mAb with trastuzumab resulted in significant synergistic inhibition of both pathways and HER2 degradation, much more potently than the combination of trastuzumab and pertuzumab. Conclusion: Our data indicate that anti-HER2 mAbs may induce different signaling pathways depending on their effect on tumor cell growth and proliferation. The significant inhibition of ERK and AKT phosphorylation by 1T0 alone or particularly in combination with trastuzumab suggests its potential therapeutic application for targeted immunotherapy of HER2 overexpressing malignancies.

Polyclonal Antibody against Different Extracellular Subdomains of HER2 Induces Tumor Growth Inhibition in vitro.

BACKGROUND: Human epidermal growth factor receptor 2 (HER2) has a crucial role in several malignancies. The extracellular domain of HER2 (HER2-ECD) has been extensively employed as an important target in passive and active immunotherapy. Isolated recombinant prokaryotic HER2-ECD subdomains were previously found to be ineffective in inducing anti-tumor antibody response. OBJECTIVE: To employ recombinant eukaryotic HER2-ECD subdomains to raise anti-HER2 antibodies and determine their anti-tumor activity in vitro. METHODS: Two paired subdomains of HER2-ECD (DI+II and DIII+IV), representing Pertuzumab and Trastuzumab binding domains, respectively, along with the full extracellular domain of HER2 were generated in CHO-K1 cells. Polyclonal antibodies were raised against these subdomains and characterized using ELISA, flow cytometry, and immunoblot and their anti-tumor activity was assessed by XTT assay. The cross-reactivity of these antibodies was specified along with other members of the human HER family. RESULTS: Similar to Trastuzumab and anti-HER2-ECD antibody, anti-DI+II and DIII+IV polyclonal antibodies reacted with recombinant HER2-ECD and native HER2 expressed on tumor cells. These two polyclonal antibodies were able to inhibit the binding of Pertuzumab and Trastuzumab to HER2, respectively, and did not cross-react with other members of HER family. These antibodies were able to inhibit tumor cell growth in vitro, similar to Trastuzumab. CONCLUSION: The high immunogenicity of human HER2 DI+II and DIII+IV subdomains in rabbits and the tumor inhibitory activity of the purified specific antibodies imply that they might be suitable for active immunotherapy in formulation with appropriate adjuvants and in combination with other HER2 specific therapeutics.

Epitope Mapping of Human HER2 Specific Mouse Monoclonal Antibodies Using Recombinant Extracellular Subdomains

Background: Human epidermal growth factor receptor 2 (HER2) is overexpressed in several human malignancies and numerous studies have indicated that it plays important roles in the development and maintenance of the malignant phenotype. Targeting of HER2 molecules with monoclonal antibodies (mAbs) is a promising therapeutic approach. However, anti-HER2 mAbs affect cancer cells differently, depending on the distinct epitopes which are the targets. Methods: Reactivity of a panel of 8 mouse anti-HER2 mAbs was investigated by ELISA and Western blotting using different subdomains of the extracellular domain (ECD) of HER2. All subdomains, including I, II, III, IV, I+II, III+IV and full HER2-ECD were constructed and expressed in CHO cells. Cross-reactivity of the mAbs with other members of the human HER family and Cynomolgus HER2 was also studied by ELISA. The mAbs were also tested by immunohistochemistry (IHC) using HER2 positive breast cancer tissues. Results: Our results demonstrated that 3 out of 8 mAbs detected conformational epitopes (1T0, 2A8 and 1B5), while 5 mAbs identified linear epitopes (1F2, 1H9, 4C7, 1H6 and 2A9). Three of the mAbs recognized subdomain I, one reacted with subdomain I+II, 2 recognized either subdomain III or IV and 2 recognized subdomain III+IV. However, none of our mAbs recognized the subdomain II alone. The mAbs displayed either inhibitory or stimulatory effects on HER2-overexpressing tumor cells and did not react with other members of the human HER family. The pattern of IHC results implied better reactivity of the mAbs recognizing linear epitopes. Conclusions: Our findings suggest that paired subdomains of HER2 are essential for mapping of mAbs recognizing conformational epitopes. Moreover, there seems to be no association between subdomain specificity and antitumor activity of our anti-HER2 mAbs.