Costimulatory Signals Research Articles

Next generation armored CAR-T cells with a drug inducible cytokine circuit

Immunotherapies which direct the bodys own immune system against tumors are a safer and more efficacious alternative to other types of cancer treatments. Chimeric Antigen Receptor or CAR T cell therapy has emerged at the forefront of contemporary cancer immunotherapy research and is highly specific for a wide range of cancer types, resulting in improved patient outcomes. Early generations of CAR T cells were ineffective and failed to proliferate due to lack of costimulatory signals and immunosuppression within the tumor microenvironment (TME), but later generations of armored CAR T cells added costimulatory receptor domains and constitutive expression of stimulatory immunocytokines to ameliorate this. However, increased aggressiveness in T cells comes with issues such as off-site toxicity, fails to address relapse due to immune evasion, and combined with the prohibitive cost of engineering CAR T cells limit the efficacy of the treatment. Next generation CAR T-cells address these problems with the engineering of synthetic biological circuits that provide selective control over immune function in response to inducers. In this research proposal, we design a next generation armored CAR T cell with a small molecule inducible cytokine circuit, combining different synthetic biology approaches from previous research on CAR T cells to enhance the safety of the therapy by providing a reversible, safe, and rapid method of modulating CAR T cell stimulation.

Targeting colorectal cancer cells using AND-gated adaptor RevCAR T-cells.

Despite the success of chimeric antigen receptor (CAR) T-cells especially for treating hematological malignancies, critical drawbacks, such as "on-target, off-tumor" toxicities, need to be addressed to improve safety in translating to clinical application. This is especially true, when targeting tumor-associated antigens (TAAs) that are not exclusively expressed by solid tumors but also on hea9lthy tissues. To improve the safety profile, we developed switchable adaptor CAR systems including the RevCAR system. RevCAR T-cells are activated by cross-linking of bifunctional adaptor molecules termed target modules (RevTM). In a further development, we established a Dual-RevCAR system for an AND-gated combinatorial targeting by splitting the stimulatory and co-stimulatory signals of the RevCAR T-cells on two individual CARs. Examples of common markers for colorectal cancer (CRC) are the carcinoembryonic antigen (CEA) and the epithelial cell adhesion molecule (EpCAM), while these antigens are also expressed by healthy cells. Here we describe four novel structurally different RevTMs for targeting of CEA and EpCAM. All anti-CEA and anti-EpCAM RevTMs were validated and the simultaneous targeting of CEA+ and EpCAM+ cancer cells redirected specific in vitro and in vivo killing by Dual-RevCAR T-cells. In summary, we describe the development of CEA and EpCAM specific adaptor RevTMs for monospecific and AND-gated targeting of CRC cells via the RevCAR platform as an improved approach to increase tumor specificity and safety of CAR T-cell therapies.

The preprogrammed anti-inflammatory phenotypes of CD11chigh macrophages by Streptococcus pneumoniae aminopeptidase N safeguard from allergic asthma

BackgroundEarly microbial exposure is associate with protective allergic asthma. We have previously demonstrated that Streptococcus pneumoniae aminopeptidase N (PepN), one of the pneumococcal components, inhibits ovalbumin (OVA) -induced airway inflammation in murine models of allergic asthma, but the underlying mechanism was incompletely determined.MethodsBALB/c mice were pretreated with the PepN protein and exposed intranasally to HDM allergen. The anti-inflammatory mechanisms were investigated using depletion and adoptive transfer experiments as well as transcriptome analysis and isolated lung CD11chigh macrophages.ResultsWe found pretreatment of mice with PepN promoted the proliferation of lung-resident F4/80+CD11chigh macrophages in situ but also mobilized bone marrow monocytes to infiltrate lung tissue that were then transformed into CD11high macrophages. PepN pre-programmed the macrophages during maturation to an anti-inflammatory phenotype by shaping the metabolic preference for oxidative phosphorylation (OXPHOS) and also inhibited the inflammatory response of macrophages by activating AMP-activated protein kinase. Furthermore, PepN treated macrophages also exhibited high-level costimulatory signaling molecules which directed the differentiation into Treg.ConclusionOur results demonstrated that the expansion of CD11chigh macrophages in lungs and the OXPHOS metabolic bias of macrophages are associated with reduced allergic airway inflammation after PepN exposure, which paves the way for its application in preventing allergic asthma.

A dual-receptor T-cell platform with Ab-TCR and costimulatory receptor achieves specificity and potency against AML

AbstractChimeric antigen receptor T-cell (CAR T) therapy has produced remarkable clinical responses in B-cell neoplasms. However, many challenges limit this class of agents for the treatment of other cancer types, in particular the lack of tumor-selective antigens for solid tumors and other hematological malignancies, such as acute myeloid leukemia (AML), which may be addressed without significant risk of severe toxicities while providing sufficient abundance for efficient tumor suppression. One approach to overcome this hurdle is dual targeting by an antibody–T-cell receptor (AbTCR) and a chimeric costimulatory signaling receptor (CSR) to 2 different antigens, in which both antigens are found together on the cancer cells but not together on normal cells. To explore this proof of concept in AML, we engineered a new T-cell format targeting Wilms tumor 1 protein (WT1) and CD33; both are highly expressed on most AML cells. Using an AbTCR comprising a newly developed TCR-mimic monoclonal antibody against the WT1 RMFPNAPYL (RMF) epitope/HLA-A2 complex, ESK2, and a secondary CSR comprising a single-chain variable fragment directed to CD33 linked to a truncated CD28 costimulatory fragment, this unique platform confers specific T-cell cytotoxicity to the AML cells while sparing healthy hematopoietic cells, including CD33+ myelomonocytic normal cells. These data suggest that this new platform, named AbTCR-CSR, through the combination of a AbTCR CAR and CSR could be an effective strategy to reduce toxicity and improve specificity and clinical outcomes in adoptive T-cell therapy in AML.

Human CD4+CD25+ T cells expressing a chimeric antigen receptor against aberrant superoxide dismutase 1 trigger antigen-specific immunomodulation

Background aimsAmyotrophic lateral sclerosis (ALS) is a fatal disease associated with motor neuron degeneration, accumulation of aggregated misfolded proteins and neuroinflammation in motor regions of the central nervous system (CNS). Clinical trials using regulatory T cells (Tregs) are ongoing because of Tregs’ immunomodulatory function, ability to traffic to the CNS, high numbers correlating with slower disease in ALS and disease-modifying activity in ALS mouse models. In the current study, a chimeric antigen receptor (CAR) was developed and characterized in human Tregs to enhance their immunomodulatory activity when in contact with an ALS protein aggregate. MethodsA CAR (DG05-28-3z) consisting of a human superoxide dismutase 1 (hSOD1)-binding single-chain variable fragment, CD28 hinge, transmembrane and co-stimulatory domain and CD3ζ signaling domain was created and expressed in human Tregs. Human Tregs were isolated by either magnetic enrichment for CD4+CD25hi cells (Enr-Tregs) or cell sorting for CD4+CD25hiCD127lo cells (FP-Tregs), transduced and expanded for 17 days. ResultsThe CAR bound preferentially to the ALS mutant G93A-hSOD1 protein relative to the wild-type hSOD1 protein. The CAR Tregs produced IL-10 when cultured with aggregated G93A-hSOD1 proteins or spinal cord explants from G93A-hSOD1 transgenic mice. Co-culturing DG05-28-3z CAR Tregs with human monocytes/macrophages inhibited production of tumor necrosis factor alpha and reactive oxygen species. Expanded FP-Tregs resulted in more robust Tregs compared with Enr-Tregs. FP-Tregs produced similar IL-10 and less interferon gamma, had lower Treg-specific demethylated region methylation and expressed higher FoxP3 and CD39. ConclusionsTaken together, this study demonstrates that gene-modified Tregs can be developed to target an aggregated ALS-relevant protein to elicit CAR-mediated Treg effector functions and provides an approach for generating Treg therapies for ALS with the goal of enhanced disease site-specific immunomodulation.

Abstract B034: CD27 is a new promising T cell co-stimulatory target for cancer immunotherapy

Abstract Members of the tumor necrosis factor receptor superfamily (TNFRSF) are key co-stimulators of T cells. CD27, a member of the TNFRSF, is expressed only on the surface of lymphocytes, including naive and activated CD4+ and CD8+ T cells as well as NK cells. It enhances T cell activation, proliferation, and differentiation of effector and memory T cells after stimulation with its ligand, CD70. The costimulatory signal of CD27 is mediated via the NFkB pathway but also via the phosphatidylinositol 3 kinase and the protein kinase B pathways. CD27 signaling also influences the innate immune response via direct activation of NK cells and subsequent secretion of interferon-gamma (IFNg). Several published preclinical studies demonstrated that anti-CD27 agonistic monoclonal antibodies can promote T-cell activation and antitumor immunity making CD27 an attractive cancer immunotherapy target. Here we describe the characterization, preclinical development, and selection of our anti-CD27 fully human monoclonal antibody (mAb) lead candidate. We selected this candidate from a library of 147 anti-CD27 mAbs generated after immunization of humanized Trianni® mice with soluble human CD27 extracellular domain (hCD27-ECD). Anti-CD27 mAbs were tested in an accelerated stability study and showed excellent stability parameters for up to 7 days at 4 and 37°C in commonly used formulation buffers. The selected agonist anti-CD27 mAb demonstrated high affinity binding to both human and cynomolgus monkey CD27 and not to mouse CD27. It also demonstrated high specificity against CD27 with no cross-reactivity detected against other members of the TNFRSF. This lead candidate did not block the binding of CD27 natural ligand, CD70 and induced strong NFkB-mediated CD27 signaling in the absence or presence of cross-linking by Fc gamma receptors or secondary cross-linking antibodies. Moreover, this anti-CD27 mAb mediated NFkB activation is significantly potentiated by the addition of a sub-optimal amount of soluble CD70. The anti-CD27 lead mAb induced T cell proliferation and secretion of pro-inflammatory cytokines only in the presence of sub-optimal TCR stimulation in vitro using primary human T cells. It also activated NK cells demonstrated by CD69 expression induction. The anti-CD27 mAb lead candidate showed extended serum half-life in hCD27-KI mice. It also demonstrated a significant antitumor effect as a single agent in human CD27-Knockin mice (hCD27-KI) subcutaneously implanted with MC38 or in NOD-SCID mice subcutaneously implanted with Raji. These preclinical results establish that the selected anti-CD27 mAb is a promising drug candidate and we are actively pursuing its development. Citation Format: Thierry Guillaudeux, Yulia Ovechkina, Kurt Lustig, Shawn Iadonato. CD27 is a new promising T cell co-stimulatory target for cancer immunotherapy [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Tumor Immunology and Immunotherapy; 2023 Oct 1-4; Toronto, Ontario, Canada. Philadelphia (PA): AACR; Cancer Immunol Res 2023;11(12 Suppl):Abstract nr B034.

CD80-Fc fusion protein as a potential cancer immunotherapy strategy.

The activation of T lymphocytes is a crucial component of the immune response, and the presence of CD80, a membrane antigen, is necessary for T-cell activation. CD80 is usually expressed on antigen-presenting cells (APCs), which can interact with cluster of differentiation 28 (CD28) or programmed cell death ligand 1 (PD-L1) to promote T-cell proliferation, differentiation and function by activating costimulatory signal or blocking inhibitory signal. Simultaneously, CD80 on the APCs also interacts with cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) on the surface of T cells to suppress the response of specific effector T cells, particularly in the context of persistent antigenic stimulation. Due to the pivotal role of CD80 in the immune response, the CD80-Fc fusion protein has emerged as a promising approach for cancer immunotherapy. This review primarily focused on the crucial role of CD80 in the cancer immunotherapy. We also reviewed the current advancements in the research of CD80-Fc fusion proteins. Finally, we deliberated on the challenges encountered by CD80-Fc fusion proteins and proposed the potential strategies that could yield the benefits for patients.

T Cell Dysfunction and Exhaustion in Patients with CLL: The Impact of Long Term Ibrutinib Treatment

Introduction: Chronic lymphocytic leukemia (CLL) is a B-cell malignancy associated with defects in the immune system and is dependent on the microenvironmental niche for survival. Ibrutinib, the first irreversible inhibitor of Bruton's tyrosine kinase targets B-cell receptor signaling to kill tumor cells. In addition, ibrutinib also appears to regulate the tumor microenvironment and T-cell immunity, but this mechanism unknown. Increased expression of inhibitory receptors, and functional defects such as reduced proliferative capacity and cytotoxicity are common in T cells from patients with CLL. The effect of ibrutinib on T-cell immunity has not been extensively studied in the setting of long term ibrutinib treatment. Therefore, we investigated the phenotype and function of CD4+ and CD8+ T-cells from patients with CLL treated with ibrutinib, treatment naïve CLL and age-matched healthy controls. Methods: CLL patient samples were obtained from treatment naïve patients at diagnosis, and at the 3- and 5-year timepoints. Samples from patients on continuous ibrutinib who had not relapsed at the 3- and 5-year timepoints; and pre-ibrutinib were obtained as well. Age matched healthy donor-derived peripheral blood mononuclear cells (PBMCs) were isolated by density gradient centrifugation using Ficoll. PBMCs from CLL samples and healthy donors were labeled with CellTrace Violet and treated with anti-CD3 and anti-CD28 (ThermoFisher) for 7 days. T-cells were stimulated to identify proliferation capacity and to identify immune profiles. Results: Efficient T-cell activation and proliferation requires multiple signals such as T-cell receptor (TCR) stimulation, a costimulatory signal, and cytokine production. We analyzed the proliferation of T cells in-vitro upon stimulation with anti-CD3/CD28 beads using CellTrace Violet on PBMCs and found that this stimulation resulted in increased expansion of CD4+ T cells and CD8+ T cells in all the samples. We also found significant difference in the proliferation between untreated and treated CLL samples. Interestingly, proliferating T cells had different expression pattern of the exhaustion markers and inhibitory molecules such as TIM3, TIGIT, LAG3, CD160, KLRG1, CD244 and PD-1 at different phases of the disease. Increased expression of CD160, CD244, and PD1 are the key features of T-cell exhaustion. We found significant increase in the percentage of T cells from CLL patients expressing CD244, CD160 and PD1 compared to healthy donor controls but not between naïve and ibrutinib treated samples. Long term ibrutinib treatment in 3yr and 5yr samples does not change the exhaustion state in patients. We also found T-cells from ibrutinib treated samples express higher levels of CD57 which is known as a marker of senescence (Figure 1A). The phenotypes of untreated CLL samples are CCR7+CD45RA- central memory T-cells (35%) and CCR7+CD45RA+ naïve T cells (25%) (p<0.05) after stimulation in-vitro on day 3. In the same setting, ibrutinib treated samples had higher levels of effector memory T-cells (p<0.05) (80%) compared to untreated CLL samples (Figure 1B). These results suggest that the phenotype has changed to central memory phenotype in untreated samples. Functional T-cell exhaustion in CLL also leads to failure in cytokine production. Therefore, we measured different cytokine levels after stimulation in all the patient samples and in healthy controls. We found significant increase in the levels of inflammatory cytokines such as IFN-γ (p<0.05) and TNF-α (p<0.05) in long term ibrutinib treated samples compared to naïve CLL samples and healthy controls. Conclusion: Several studies have showed that T-cell counts are increased after treatment with ibrutinib in patients with CLL but not in extended therapy. Our findings suggest that long term ibrutinib treatment does increase T-cell number and proliferative capacity in CLL. The proliferating T cells express higher levels of exhaustion markers and inhibitory molecules in-vitro making them dysfunctional. However, these T-cells still maintained good proliferative potential, on par to healthy donors and better than untreated patients which can be applied in expanding improved T cell grafts for various types of adoptive T cell therapy, such as TCR gene therapy, and CAR therapy. Also better understanding of these subtypes may lead to new strategies and to improve antitumor function of these cells.

Blockade of Common Gamma Chain Cytokine Signaling with REGN7257, an Interleukin 2 Receptor Gamma (IL2RG) Monoclonal Antibody, in Combination with Costimulatory Blockers Delayed Skin Graft Rejection in Mice

There is a current medical need to identify innovative maintenance therapies to prevent allograft rejection in solid organ transplantation. During transplant rejection, recipient immune cells identify a grafted organ as foreign and attack it: CD4 + T cells orchestrate transplant rejection by recruiting a range of effector cells responsible for the damage of rejection including CD8 + T cells, NK cells, B cells and macrophages. These pathogenic immune cell responses can be driven by cytokines of the common gamma chain (γ c) cytokine family (IL2, IL4, IL7, IL9, IL15, and IL21). γ c cytokines signal through their corresponding receptors, expressed primarily on immune cells, that share a common coreceptor, interleukin 2 receptor subunit gamma (IL2RG) that is required for signaling. To understand the role of γ c cytokines in driving transplant rejection, we generated REGN7257, a fully human IL2RG monoclonal antibody that inhibits γ c cytokine-induced signaling, and we tested its ability to suppress allogeneic immune cell responses in a mouse model of skin graft rejection when delivered as a monotherapy and in combination with mycophenolate mofetil (MMF) or T cell costimulatory pathway blockers. In this model, recipient B6/129 (mixed background) Il2rg hu/hu mice were transplanted with tail skin from BALB/cJ donor mice. Graft survival was analyzed using a log rank Mantel-Cox test to detect statistically significant differences across groups (*=p<0.033; **= p<0.002; ***=p<0.0002; ****=p<0.0001). In a mouse model of skin graft rejection, γ c cytokine signaling blockade with REGN7257 given as maintenance immunosuppression effectively improved overall graft survival (Median Survival Time [MST] IL2RG=13) compared to mice that received a control antibody (MST CTRL=9 [****vs IL2RG Ab]), and with similar efficacy to that observed with the calcineurin inhibitor tacrolimus (MST TAC=12.5 [**vs control, n.s. vs IL2RG Ab]). In treatment combination experiments, while monotherapy treatment with the antiproliferative agent MMF was not as efficient as REGN7257 at delaying skin graft rejection (MST MMF=10 [*vs control] and MST IL2RG=12 [*vs MMF]), simultaneous blockade of cell proliferation and γ c cytokine signaling conferred an advantage over monotherapies (MST MMF+IL2RG=15.5 [****vs control, **vs IL2RG Ab]). Similarly, induction therapy consisting of γ c cytokine signaling blockade followed by maintenance immunosuppression using the CTLA4-Ig fusion protein abatacept, significantly improved overall skin graft survival (MST IL2RG+CTLA4=19.5 [****vs control]) compared to REGN7257 induction only (MST IL2RG=11 [****vs control and combo]) or abatacept maintenance only (MST CTLA4=10 [*vs control, ****vs combo]). Finally, maintenance immunosuppression combining blocking of γc cytokine signaling together with blocking of both CD28 and CD40L costimulatory signals prolonged skin graft survival better than any combination therapies of only 2 pathway blockers (MST TRIPLE=26 [**vs control] and MST DOUBLE=16-21 [n.s.-** vs triple]). Overall, our study showed that γ c cytokine blockade could potentially be an alternative to calcineurin inhibitor-based regimens to prevent rejection by specifically targeting immune cells. Our data also highlighted the inherent complexity of effector mechanisms and pathways involved in allograft rejection and showed that prolonged allograft survival requires simultaneous blockade of multiple T cell activating signals, including inhibition of cytokine signaling and costimulatory signals. These data provide evidence of γ c cytokines as key drivers of allogeneic immune cell responses during acute transplant rejection, offering a potentially novel strategy for the management of transplant patients.

Pre-Clinical Activity of Navitoclax in TCR-Driven and Non-ALCL Mature T-Cell Lymphomas

Background: In contrast to many B-cell lymphomas that highly express BCL-2, BCL-2 expression is observed in <50% of most mature T-cell lymphoma (MTCL) subtypes, although a clinical trial investigating venetoclax in BCL-2 positive relapsed/refractory PTCL is ongoing (NCT03552692). In contrast, BCL-xL and/or MCL-1 are expressed in the majority of MTCL. Consistent with these findings, MCL-1 expressing anaplastic large cell lymphomas (ALCL) and typical (non-ETP) T-ALL, which highly express BCL-xL, have been shown by BH3 profiling to be dependent upon MCL-1 or BCL-xL, respectively. Among the more common MTCL subtypes, including cutaneous T-cell lymphomas (CTCL), the extent to which BCL-2 family members are a therapeutic vulnerability is uncertain. However, we have previously demonstrated that antigen- and costimulatory receptor signaling promote the growth and survival of these lymphomas and confers resistance to chemotherapy, and in conventional (non-malignant) T cells, these same signals upregulate BCL-xL, which is required for their clonal expansion and escape from cell death following T-cell activation. Therefore, given the increasing availability of selective BH3 mimetics and the need for improved therapeutic strategies, we were motivated to examine BCL-2 family member dependencies in these lymphomas. Methods: Peptides generated from BH3-only proteins and selective BH3 mimetics were utilized to perform “BH3 profilng” in MTCL. Cell viability upon exposure to BH3 mimetics was determined using standard approaches. Results: To pre-clinically assess BCL-2 family member dependencies and the mitochondrial apoptotic pathway in T-cell lymphoproliferative neoplasms, we performed BH3 profiling in 17 cell lines. Peptides derived from BIM and PUMA, which are promiscuous, were utilized to assess the extent to which cells are “primed” for apoptosis. Differences in apoptotic priming were not observed across subtypes, and most cell lines examined were highly primed. We next examined the dependence on specific antiapoptotic proteins by BH3 profiling using BAD (antagonizes BCL-2, BCL-xL, and BCL-w), HRK (antagonizes BCL-xL), MS1 (antagonizes MCL-1), and FS1 (antagonizes A1/BFL-1) peptides. BCL-2 dependence was not observed in the T-cell lymphoma cell lines we examined. However, and in stark contrast, most non-ALCL cell lines were BCL-xL dependent, whereas ALCL cell lines were largely MCL-1 dependent. In an effort to further confirm these results, and examine the extent to which peptide-based BH3 profiling predicts sensitivity to selective BH3 mimetics in MTCL, cell lines were treated with venetoclax, A1155463 (a BCL-xL selective antagonist), navitoclax (a BCL-2/BCL-xL antagonist), or S63845 (MCL-1 antagonist), and cell viability determined. Consistent with peptide-based BH3 profiling, all cell lines examined were resistant to venetoclax. ALCL cell lines were largely MCL-1 dependent, whereas the non-ALCL cell lines examined were largely BCL-xL dependent. Therefore, we examined BCL-xL and MCL-1 expression in these cells and observed a strong association between BCL-xL expression and both cytochrome c release in response to HRK peptide and cell viability in the presence of A1155463. A similar association was observed between MCL-1 expression and sensitivity to MS1 and S63845. Transcriptionally profiled MTCL specimens were stratified by MTCL classification (ALCL vs. non-ALCL) and in keeping with the data obtained using cell lines increased BCL-xL expression was observed in non-ALCL MTCL subtypes. Given the observed association between BCL-xL expression and vulnerability to BCL-xL antagonists, we sought to examined mechanisms that promote its expression. Both TCR signaling and CD28 costimulation are exploited by non-ALCL subtypes. We examined the extent to which TCR/CD28 stimulation regulates BCL-xL transcription. In MTCL specimens, a significant increase in BCL-xL transcription and sensitization to Bcl-xL antagonism were observed upon TCR/CD28 stimulation. A TCR-dependent GEM model was utilized, demonstrating that these MTCL are Bcl-xL dependent and sensitive to navitoclax. Conclusions: These findings suggest that BCL-xL is a therapeutic vulnerability for MTCL subsets, particularly non-ALCL subtypes that are TCR dependent, and provide a robust pre-clinical rationale for future studies investigating navitoclax in these lymphomas.

High Efficacy and Safety of Nanobody Based Anti-BCMA CAR-T Cell Therapy in Treating Patients with Relapsed or Refractory Multiple Myeloma

Introduction Refractory or relapsed (R/R) multiple myeloma (MM) is often associated with a poor prognosis following chemotherapy or even transplantation. BCMA-targeted CAR-T therapy has demonstrated notable efficacy in R/R MM. In the past, the overall response rate (ORR) was relatively low at around 60%, but in recent years, advancements in the manufacturing of BCMA-CAR-T products have led to a substantial improvement in efficacy. Here we explore the efficacy and safety of nanobody-based anti-BCMA CAR-T cell therapy in R/R MM from a clinical trial (https://clinicaltrials.gov NCT04447573). Methods Autologous peripheral blood lymphocytes were collected from patients, who subsequently underwent intravenous fludarabine (30mg/m 2/d) and cyclophosphamide (300mg/m 2/d) lymphodepletion chemotherapy from day-5 to day -3. The SL-BCMA CAR molecule, an anti-BCMA CAR-T cell product generated by Senlang Biotechnology, features a structure that includes dual nanobody VHHs targeting BCMA (dVHHs), CD8α hinge region, CD8α transmembrane domain (TMD), co-stimulatory domain (4-1BB), and CD3 zeta domain (CD3ζ). Among them, dVHHs consist of two different VHHs connected by short peptides. The CD8α hinge region provides an effective spatial architecture for the binding of dVHHs and BCMA protein. The first signal of CAR-T cell activation is completed along with the intracellular CD3ζ domain. The 4-1BB intracellular co-stimulatory domain provides co-stimulatory signals for the activation of CAR-T cells. Under the dual signal stimulation, CAR-T cells are activated and expanded. After the BCMA CAR-T cells infusion on day 0, patients underwent evaluations, including immunoglobulin fixed electrophoresis, free light chains in blood and urine, bone marrow penetration, and whole-body PET CT to evaluate the efficacy at the first month, second and third month, and every three months thereafter. Results From April 2021 to May 2023, 21 R/R MM patients in our hospital were treated with SL-BCMA CAR-T, including 10 males and 11 females. The median age is 57 years (30-73 years). Among them, there were 2 cases with Plasma cell leukemia, 10 had extramedullary multiple lesions, and 2 had paralysis caused by central nervous system infiltration. Pre-CAR-T, 11 patients had a prior transplant history including 10 who relapsed after autologous transplantation and 1 who relapsed after allogeneic transplantation. One patient had a rare type of anaplastic Plasma cell tumor. Seven patients presented high-risk factors characterized by FISH abnormalities such as t (4; 14) (p16; q32), t (14; 16) (q32; q23), and 3 had TP53 mutations. The median BCMA expression rate among 19 patients was 53.3% (range: 18.5% -100%), while the remaining 2 patients displayed non-BCMA expression. The median BCMA CAR-T cell dose was 1*10 6/Kg (0.3-2*10 6/Kg). One month following BCMA-CAR-T cell infusion, the ORR was 95%(20/21), and the CR rate was 38% (8/21). The patient who initially showed no response achieved a partial response following a second infusion of BCMA-CAR-T cells. One died at about 2.5 months due to lung infection. Evaluations conducted at the three-month mark showed an ORR of 95% (19/20) and a CR rate of 80% (16/20). One patient relapsed at the second month and underwent salvage allogeneic hematopoietic stem cell transplantation. For the long follow-up data, the median duration of remission was 11 months. Of the 7 deaths, 2 were attributed to infection during disease control, and 5 were due to disease progression. (Figure 1) In terms of side effects, 6 patients developed grade 2 cytokine release syndrome (CRS), and only 1 patient developed grade 3 CRS. Only one patient had grade 1 neurotoxicity. Conclusions The clinical trial demonstrated that BCMA CAR-T therapy, composed of dual nanobody VHHs targeting BCMA (dVHHs), exhibits a high ORR with a manageable safety profile in treating R/R MM patients. This extends even to high-risk patients, such as those with extramedullary lesions, cytogenetics high-risk groups, and patients with plasma cell leukemia or anaplastic plasma cell tumor.

A Prospective Randomized Clinical Trial Comparing a Calcineurin-Free Gvhd Prophylaxis Regimen with Post-Transplant Cyclophosphamide and Abatacept to Methotrexate and Tacrolimus

Background: Chronic Graft Versus Host Disease (GVHD) is one of the major causes of mortality and morbidity after an allogeneic stem cell transplantation. We hypothesized that we could induce post-transplant tolerance by using the combination of post-transplant cyclophosphamide (PTCy) and abatacept (CTLA4Ig) for GVHD prophylaxis. PTCy can eliminate most host-reactive donor T cells. Abatacept (CTLA4Ig) blocks the costimulatory signals given to naïve T cells during immune reconstitution, thus favoring an anergic phenotype and promoting tolerance towards recipient derived antigens. CTLA4Ig gives an activating signal to NK cells and has therefore the ability to preserve the graft-versus-tumor effect. Methods: We conducted a prospective randomized clinical trial. Patients with hematologic malignancies in need of an allogeneic transplant and with an 8/8 matched related or unrelated donor were randomized 1:1 to tacrolimus and methotrexate for GVHD prophylaxis (SOC arm) or PTCy on days +3 and +4 followed by abatacept on an extended schedule: days +5, +14,+28, +56, +84, +112, +140 and +168 (Aba arm). Patients were stratified by conditioning regimen (myeloablative vs reduced intensity) and donor type (matched sibling vs matched unrelated donor). They received peripherally collected stem cells. The primary endpoint was moderate and severe chronic GVHD at 1 year and the secondary endpoints overall chronic GVHD, Grade II-IV and III-IV acute GVHD rate, overall (OS), disease-free (DFS), and GVHD-relapse-free-survival (GRFS) at on year, transplant related mortality and infection rate. The study stopped enrolling on the SOC arm when abatacept was approved for GVHD prophylaxis and the institutional SOC changed to include abatacept. Results: 40 patients were treated on this study, 25 of which on the aba arm. There were two cases of secondary graft failure (one on each arm) that were excluded from further analysis other than OS. The trial met its primary end point: Kaplan-Meier estimates of moderate/severe cGVHD were 0% on the aba and 65.8% on the SOC arm (p < 0.0001). GRFS was 58.3% in the aba and 21.4% in the SOC arm (p=0.043). There were no treatment related deaths on the aba and two on the SOC arm. There was no statistically significant difference in the rates of OS (92% aba, 80% SOC, p=0.28), DFS (62.5% aba, 85.7% SOC, p=0.103) and infection. Grade III-IV acute GVHD rate was 4.1% on the aba and 20% on the SOC arm (p=0.132), whereas Grade II-IV 12.5% on aba and 35.7% on SOC (p=0.117). There were no cases of acute GVHD of the gut or liver on the aba arm. Overall chronic GVHD rate (including mild) was 9.1% on the aba and 74% on the SOC arm (p<0.0001). Conclusions: The calcineurin inhibitor-free combination of PTCy and abatacept for GVHD prophylaxis is feasible, well tolerated and associated with a very low rate of chronic GVHD and a favorable GRFS rate.

Unraveling the transcriptomic signatures of Parkinson's disease and major depression using single-cell and bulk data.

Motor symptoms are well-characterized in Parkinson's disease (PD). However, non-motor symptoms, such as depression, are commonly observed and can appear up to 10 years before motor features, resulting in one-third of individuals being misdiagnosed with a neuropsychiatric disorder. Thus, identifying diagnostic biomarkers is crucial for accurate PD diagnosis during its prodromal or early stages. We employed an integrative approach, combining single nucleus RNA and bulk mRNA transcriptomics to perform comparative molecular signatures analysis between PD and major depressive disorder (MDD). We examined 39,834 nuclei from PD (GSE202210) and 32,707 nuclei from MDD (GSE144136) in the dorsolateral prefrontal cortex (dlPFC) of Brodmann area 9. Additionally, we analyzed bulk mRNA peripheral blood samples from PD compared to controls (GSE49126, GSE72267), as well as MDD compared to controls (GSE39653). Our findings show a higher proportion of astrocytes, and oligodendrocyte cells in the dlPFC of individuals with PD vs. MDD. The excitatory to inhibitory neurons (E/I) ratio analysis indicates that MDD has a ratio close to normal 80/20, while PD has a ratio of 62/38, indicating increased inhibition in the dlPFC. Microglia displayed the most pronounced differences in gene expression profiles between the two conditions. In PD, microglia display a pro-inflammatory phenotype, while in MDD, they regulate synaptic transmission through oligodendrocyte-microglia crosstalk. Analysis of bulk mRNA blood samples revealed that the COL5A, MID1, ZNF148, and CD22 genes were highly expressed in PD, whereas the DENR and RNU1G2 genes were highly expressed in MDD. CD22 is involved in B-cell activation and the negative regulation of B-cell receptor signaling. Additionally, CD86, which provides co-stimulatory signals for T-cell activation and survival, was found to be a commonly differentially expressed gene in both conditions. Pathway analysis revealed several immune-related pathways common in both conditions, including the complement and coagulation cascade, and B-cell receptor signaling. This study demonstrates that bulk peripheral immune cells play a role in both conditions, but neuroinflammation in the dlPFC specifically manifests in PD as evidenced by the analysis of single nucleus dlPFC datasets. Integrating these two omics levels offers a better understanding of the shared and distinct molecular pathophysiology of PD and MDD in both the periphery and the brain. These findings could lead to potential diagnostic biomarkers, improving accuracy and guiding pharmacological treatments.

Generation of Bi-Specific Chimeric Antigen Receptor (CAR) T Cell Therapy for the Treatment of AIDS-Related B Cell Malignancies from Healthy Donors and HIV Patients

Introduction: Individuals with human immunodeficiency virus (HIV) face a substantially greater likelihood of developing cancers compared to the general population. The prevalence of cancer diagnosis among HIV-positive individuals ranges from 25% to 40%. Non-Hodgkin lymphoma (NHL) stands out as the most prevalent cancer type associated with people living with HIV/AIDS (PLWHA). NHL primarily manifests in two forms in PLWHA: Burkitt lymphoma, which poses an 18-fold higher risk, and diffuse large B cell lymphoma (DLBCL), accounting for 75% of cases. The advent of chimeric antigen receptor (CAR) T cell therapies, notably CD19-targeting CAR T cells, has emerged as a groundbreaking treatment for B cell NHL. Unfortunately, PLWHA has been excluded from participating in all of these clinical trials for years due to safety concerns and the challenges associated with manufacturing CAR T cell products from HIV-positive donors. Recent researches explored the feasibility of developing CD19-targeting CAR T cell products utilizing cells from HIV-positive donors with lymphoma. Although these studies have provided promising evidence that alleviates concerns regarding the safety profile and limited effectiveness of CAR T cell therapy in this particular population, they do not address the underlying HIV condition. We previously generated CAR T cells targeting either lymphoma (CD19-CAR) or HIVgp120 (N6-CAR) that show efficacy against their respective diseases in clinical and preclinical testing, respectively. We hypothesized that a dual construct capable of targeting both antigens could simultaneously target lymphoma cells and HIV-infected cells in a same individual. We report our experience developing a bi-specific N6-huCD19 CAR T cell platform that can target both antigens in a single therapeutic product. Methods: We engineered 3 bi-specific CAR candidate designs including 2 tandem and 1 loop constructs. These constructs were developed by integrating a humanized (hu) CD19 single-chain variable fragment (scFv) and an N6 scFv from an anti-HIV broadly neutralizing antibody (bNAb) into a backbone of a 2 nd-generation CAR construct. The bi-specific tandem CAR constructs consisted of N6 and huCD19 scFvs fused with a G4S linker in either huCD19-N6 or N6-huCD19 orientation. The loop-CAR was generated by fusing huCD19(VL):N6(VH):N6(VL):huCD19(VH) with a whitlow linker. All constructs included the CD4 transmembrane domain, a double-mutated IgG4 Fc spacer, 4-1BB co-stimulatory and CD3-zetta signaling domains, and EGFRt separated by a T2A ribosomal skip sequence (Fig.1). We tested the 3 bi-specific constructs in healthy donor-derived T cells using cytotoxicity co-culture assay against either Raji (CD19+) or 8E5 (gp120+) target cells. We generated N6-huCD19 CAR T cells from HIV-positive patients and confirmed the functionality of these CAR T cells against tumor cells. Results: The functional assessment of all 3 bi-specific CAR constructs revealed their efficacy against CD19 and HIVgp120 antigens. However, the N6-huCD19 tandem CAR T cells demonstrated comparable and better efficacy against both antigens. Notably, the N6-huCD19 tandem CAR T cells exhibited the capability to sequentially target either a single antigen or alternate between the 2 antigens (Fig.2). We successfully generated N6-huCD19 tandem CAR T cells using HIV-positive donors, signifying the practicality of this approach within the context of PLWHA. Remarkably these HIV donor-derived N6-huCD19 CAR T cells effectively killed tumor cells expressing CD19 or HIVgp120 antigens, thus confirming their dual functionality against these 2 antigens. Conclusion: Here we demonstrate the development of the novel N6-huCD19 bi-specific CAR T cells with functionality against both CD19 and HIVgp120 antigens. We also showed the feasibility of generating these CAR T cells from HIV-donors. This strategy could potentially provide life-saving approach tailored for treatment of HIV-patients who develop CD19+ lymphomas.

Dual Split-Signaling TIM3+CLEC12a Targeting CAR T-Cells with Optimized Signaling As a Safe Potential Therapy for Acute Myeloid Leukemia

Background: The success of chimeric antigen receptor (CAR) T-cell therapies targeting B cell maturation antigen (BCMA) in multiple myeloma and CD19 in B-cell malignancies has demonstrated the immense potential of this immunotherapeutic strategy. However, the translation of CAR T-cell therapy to acute myeloid leukemia (AML) patients has encountered challenges due to the absence of surface antigens specifically or preferentially expressed on AML cells. To overcome this hurdle, we explored the feasibility and safety of simultaneously targeting the AML-associated antigens CLEC12a and TIM3 using an AND-gate strategy in which the essential stimulatory and co-stimulatory T-cell activation signals are segregated into separate CARs. To improve the safety of our strategy we also attenuated signaling through the stimulatory anti-CLEC12a CAR via affinity-optimization or through targeted mutations in its immunoreceptor tyrosine-based activation motif (ITAM) regions. Methods: To identify the most optimal dual-split CAR T-cell configuration, we designed several chimeric antigen receptor constructs in which we targeted CLEC12a with stimulatory CARs (sCARs) with high (C hi) or low (C low) affinities. In addition, the high affinity C hi stimulatory CAR was mutated in the second and third ITAM regions (denoted as C hixx) to attenuate the CD3ζ stimulatory signal. TIM3 was targeted by a costimulatory CAR (cCAR) containing both CD28 and 41BB signaling domains. The dual-split CARs were generated using two separate retroviral constructs. After the assessment of the CAR expression and specific surface markers associated with memory phenotype and exhaustion, dual-split CAR T-cells were functionally tested using an isogenic cell line model for specific lysis against dual CLEC12a/TIM3 positive or single CLEC12a or TIM3 positive isogenic target cells as well as for dual antigen-specific cytokine secretion. The dual-split CAR T-cells were also tested for their efficiency to specifically eliminate leukemic stem/progenitor cells by the colony formatting unit (CFU) assay. Moreover, anti-AML activity and persistence of the dual-split CAR T-cells was assessed using an AML PDX mouse model in which human primary AML cells expressing TIM3 and CLEC12a were injected. Results: Using the isogenic cell line model, we revealed that the dual-split CAR T-cells with the combination of the high affinity CLEC12a sCAR and TIM3 cCAR (C hi+T) show antigen-specific cytokine secretion and cytotoxicity, and that this specificity was enhanced when the CLEC12a sCAR was attenuated through ITAM mutations (C hixx+T). In CFU assays, all tested CLEC12a/TIM3 dual-split CAR T-cell combinations exhibited potent inhibition (up to 95%) of leukemia blast viability and leukemia stem/progenitor self-renewal. In vivo, although AML did not engraft in all mice, the best-performing dual-split C hixx+T1 CAR T-cells displayed potent anti-leukemic activity, as they prevented engraftment in two additional mice compared to control groups (n=6 each group). Furthermore, in contrast to control groups dual-split CAR T-cells exhibited prolonged persistence in the blood up to 20 weeks after injection. Conclusion: We successfully generated highly specific dual-targeting CAR T-cells by splitting the T-cell activation signals into two simultaneously expressed CARs, and using a novel approach of attenuating the sCAR through mutations in ITAM regions. Overall, we show that dual-split CAR T cells targeting CLEC12a and TIM3, particularly the combination C hixx+T CAR T-cells, have specific and promising anti-AML activity. Our findings underscore the potential of innovative dual-split CAR T-cell therapies as a novel and effective immunotherapeutic approach for combating AML.

Human Single Domain Antibody-Based CAR-T Cells Targeting BAFF-R Demonstrate Promising Preclinical Activity in B Cell Malignancies

Background: B-cell activating factor-receptor (BAFF-R) is a potential B-cell specific target, highly expressed in B cell malignancies and regulates B cell proliferation and survival. Single-chain antibody-based CAR-T cells targeting BAFF-R (PMB101) has demonstrated antitumor effects against human B cell malignancies and can overcome CD19 antigen loss (Qin et al. Sci Transl Med. 2019;11(511)). However, CARs built on antigen-specific single chain antibody variable fragment (scFv) may have some immunological risks. Recently, single variable domain of heavy chain antibodies are becoming an alternative to scFv to construct CARs. To improve the function of BAFF-R CAR-T cells, we designed CARs with only a fully human heavy-chain variable domain. Methods: A fully human antibody phage display library (IMARS, IASO Biotherapeutics) was used to select anti-BAFF-R clones using optimal protein/cell alternate panning. The enzyme-linked immunosorbent assay (ELISA) and flow cytometry were applied to evaluate the specificity of clones. The screened clones were grafted into a second-generation CAR with CD8α hinge, 4-1BB costimulatory and CD3ζ intracellular signaling domains to generate CAR-T cells. PMB101 and CAR-T cells constructed with VAY736 were used as positive controls. Non-transduced T cells from the same donor were used as a negative control. Results: Four candidate clones including clone 1, 5, 77 and 80 were finally screened by in vitro functional evaluation, among which clone 5, 77 and 80 were variable domain heavy chain antibodies. The expression levels of the degranulation marker CD107a on CAR-T cells constructed with the four candidate clones were significantly upregulated after coculture with BAFF-R positive tumor cells, while BAFF-R negative tumor cells could not effectively activate CAR-T cells (data not shown). The four clones exhibited similar cytotoxicity in CAR-T cells compared with the PMB101 and VAY736 CAR-T cells using the luciferase-based cytotoxicity assay (data not shown). Xenografts were established in NPG mice following intravenous injection of 1 x 10 6 jeko-1 cells expressing the firefly luciferase gene on day - 5, and a single dose of 4 x 10 6 CAR T cells were infused IV on day 0. Compared to the other groups, no progressive tumor growth was discovered in mice treated with clone 5 CAR-T cells until day 49. Clone 5 CAR-T cells showed excellent in vivo antitumor activity to PMB101 and conferred long-term survival (data not shown). Conclusion: We developed a novel BAFF-R CAR-T cell product structured on single-domain antibody. This product demonstrated promising preclinical activity, and it may provide an alternative choice for patients with B cell malignancies.

Cooperative Super-Enhancer Inactivation through Loss of Crebbp and KMT2D Skews B Cell Fate Decisions and Yields T Cell-Depleted Lymphomas

Mutations in chromatin modifiers are a hallmark of many tumors, especially lymphomas arising from germinal center (GC) B cells. Given that most of these lymphoma mutations induce aberrant gene repression, it is surprising that they often co-occur in individual patients. The most common pairing are mutations affecting CREBBP and KMT2D, even though regulating overlapping gene enhancers and pathways. Hence their co-occurrence is especially puzzling. Herein, we report that combined haploinsufficiency of Crebbp and Kmt2d (C+K) do indeed induce a more severe lymphoma phenotype (vs either allele alone) and unexpectedly conferred an immune evasive phenotype manifesting as reduced CD8+ T cell infiltration. This was linked to profound repression of immune synapse genes that provide costimulatory effects to T-cells and others. Repression of immune synapse genes was reflected in impaired response to T-cell directed cell fate decisions and weakening of costimulatory signals. From the epigenetic perspective we observed interaction and mutually dependent cooperative binding of C+K to chromatin. Notably loss of C+K cooperativity was selectively severe at superenhancers (vs enhancers), especially those driving expression of immune synapse signaling genes and pointing to a particular dependency for both co-activators at these specialized regulatory elements. Therefore, we show that critical features of the lymphoma microenvironment are shaped by specific combinations of chromatin modifier somatic mutations, induced by the dependency of immune synapse superenhancers on cooperative actions of CREBBP and KMT2D.

CD58 Genetic Alterations and Its Contribution to Upregulation of PD-L1 and IDO Via LYN/CD22/SHP1 Axis in DLBCL

Abstract Background: Recurrent abnormalities of immune surveillance-related genes play a crucial role in DLBCL progression. Prior studies have shown that CD58, a key adhesion molecule that acts as a ligand for the T-cell costimulatory molecule CD2, is frequently mutated or deleted in certain hematological malignancies. Aberrant expression of CD58 may result in tumor cells evading immune surveillance mechanisms mediated by T/NK cells. Downregulation or loss of CD58 is linked to resistance to ICB therapy in melanoma and CAR-T therapy in B-cell malignancies. Nevertheless, the role of CD58 in cancer is not yet well understood. Thus, the aim of this study was to comprehensively characterize CD58 genetic alterations in DLBCL, unveil the multiple roles of CD58 in anti-tumor immunity, and provide insights for potential therapeutic strategies. Methods: Comprehensive analysis of the genetic characteristics of CD58 were performed through targeted deep sequencing (n=176), whole exome sequencing (n=38), and RNA-sequencing (n=162) in patients with de novo DLBCL. To investigate the mechanistic impacts of CD58 alterations on co-inhibitory molecules expression and immune cell function, we performed bulk and single-cell RNA-sequencing analysis of tumor samples and conducted co-IP, flow cytometry and co-culture assays in vitro. Results: We identified that CD58 mutagenesis rate was 9.1%, with 88.9% of the identified mutations resulting in truncated protein or impairment of its normal function. 61.1% mutations occurred in exon 2, which may disrupt CD58-CD2 binding. Co-mutation analysis revealed higher frequencies of KMT2D, CD79B and MYD88 mutations in patients with CD58 mutations(p < 0.05) (Fig 1A). The CD58 copy number loss rate was 44.7% (Fig 1B). Both mutations and copy number loss led to a mild decrease in mRNA expression of CD58. Patients with CD58 mutation, copy number loss or low expression exhibited lower complete response rates following first-line R-CHOP therapy, as well as significantly poorer PFS and OS. To reveal the profound impact of CD58 on the immune microenvironment of DLBCL, we performed bulk and single-cell RNA sequencing and revealed that increased NK cells, activated CD4 +/CD8 + T cells, as well as the weaker exhaustion status of CD8 + T cells, correlated with CD58 high expression. By co-culturing assays, we identified that CD58 downregulation or mutation in DLBCL cells suppressed T cell proliferation and CAR-T cell-mediated killing. Interestingly, blocking CD58-CD2 signaling with a CD2 antibody did not fully eliminate the inhibitory effect, indicating that CD58 alterations could trigger additional immunosuppressive mechanisms. We further revealed that CD58 downregulation or mutation in DLBCL cells resulted in elevated expression of PD-L1 and IDO. This suggested that CD58 may have a dual effect on T cells, whereby its downregulation or mutation may lead to a lack of CD2 co-stimulatory signaling, as well as an increase in PD-L1 and IDO inhibitory signaling. We found that CD58 downregulation or mutation caused JAK2/STAT1 phosphorylation, which in turn promoted PD-L1 and IDO expression. Co-IP identified that CD58 can interact with Lyn, which phosphorylated ITIM of the inhibitory receptor CD22, leading to recruitment and activation of the inhibitory phosphatase SHP1. CD58 downregulation suppressed the negative regulation of the LYN/CD22/SHP1 axis, thereby promoting the activation of JAK2/STAT1 and upregulating PD-L1 and IDO expression. To overcome the dual effects induced by CD58 alterations, anti-PD-L1 antibody or IDO inhibitor and recombinant CD58-Fc chimera protein were added alone or in combination to the co-culture cells. We showed that the combination regimens reversed the inhibitory effect of CD58-downregulated or mutated DLBCL cells on T cell proliferation and restored sensitivity to CAR-T cell-mediated killing (Fig 1C, D). Conclusions: Our study comprehensively characterized CD58 genetic alterations in DLBCL. We demonstrated that CD58 downregulation or mutation led to upregulation of PD-L1 and IDO expression mainly by regulating the LYN/CD22/SHP1 axis. Moreover, we explored strategies to directly activate CD2 co-stimulatory signaling or in combination with immune checkpoint inhibitors to address the diverse effects of CD58 alterations. Our findings provide novel insights for individualized therapy for DLBCL patients with CD58 mutation or deletion.

Dual Co-Stimulation with CD28 and ICOS Enhances T Cell Activation and Optimizes CAR-T Cell Antitumor Activity

Background: The Chimeric antigen receptor (CAR) T cell therapy for hematological malignancies has revolutionized anticancer therapy. CAR-T cell expansion and persistence are strikingly associated with the intrinsic programmes of T cells. However, the current process for CAR-T cells manufacturing resulted in high frequencies of exhausted and senescent CAR-T cells with reduced mitochondrial mass. T cell receptor (TCR) signaling cooperates with co-stimulatory signaling to regulate T cells activation and differentiation. Considering that ICOS has a well-defined role in T cell differentiation, especially with regard to the incorporation of ICOS into CAR to boost CAR-T cells antitumor activity, we therefore hypothesize that TCR activation in synergy with dual CD28 and ICOS co-stimulation rapidly triggers T cell activation and may be a potential approach to optimize CAR-T cells antitumor activity. Methods: In this study, we established a culture system using plate-coated CD3 in combination with CD28 and ICOS mAbs to simultaneously stimulate T cells, and further studied whether dual CD28 and ICOS co-stimulation could reprogram the transcriptional, epigenetic and proteomic states of T cells and optimize CAR-T cells antitumor activity. In vitro CAR-T cell phenotype and cytotoxicity were detected via flow cytometry, co-culture killing assay and degranulation assay. NSG mice were inoculated with Raji cells to establish xenograft mouse model, and the tumor load and survival time were dynamically monitored after treatment with CAR-T cells. Results: As expected, dual co-stimulation potently enhanced T cells activation and expansion via reprogramming their transcriptome, epigenome and proteome. Mechanistically, dual CD28 and ICOS co-stimulation rapidly upregulated the pathways such as ribosome, citrate cycle (TCA cycle) and oxidative phosphorylation (OXPHOS) to drive T cells exiting from quiescence. Moreover, metabolic pathways are rewired in CD28 and ICOS-stimulated T cells to guarantee sufficient energy for cell proliferation. Meaningfully, dual co-stimulation confers CAR-T cells with high lentiviral transfection efficiency, possibly correlating with highly open chromatin accessibility in CD28 and ICOS-stimulated T cells. CAR-T cells manufactured with dual CD28 and ICOS co-stimulation demonstrated significantly enhanced OXPHOS and mitochondrial biogenesis, resulting in a superior expansion and being more effective at killing tumor cells in immunodeficient animals. Conclusions: Collectively, our data indicated that activating CD28 and ICOS signaling during CAR-T cells preparation is a potential strategy for optimizing CAR-T cells antitumor activity.

Signaling Role of Viral Protein Motif and Its Application in CAR T Cell Therapy

Adoptive cell therapy using Chimeric Antigen Receptor (CAR) T cells, such as Tisagenlecleucel and Axicabtagene Ciloleucel, has revolutionized cancer immunotherapy and become a game-changer in cancer care. However, several challenges, including durable remission and limited expansion after infusion, persist. Recent work from the Riddell lab (PMID: 34429382) demonstrated that T Cell Receptors (TCRs) are more sensitive than CARs due to the presence of T cell signaling proteins like CD3-, CD3ε, and CD3-, which facilitate the recruitment of Lck to phosphorylate immunoreceptor tyrosine-based activation motifs (ITAMs). Although CARs were designed to mimic TCR signaling, they still lack the full sensitivity. In this study, we hypothesized that enhancing tyrosine phosphorylation of the immunoreceptor tyrosine-based activation motifs (ITAMs) presented on CD3 in CAR T cells amplifies the CAR-mediated signaling as well as improves sensitivity of CAR co-stimulatory signaling. To achieve this, we developed a novel co-stimulation system where anti-CD19 CAR T cells co-expressed Tip37, a 37-amino acid segment of the Herpesvirus saimiri (HVS) tyrosine-protein kinase-interacting protein (Tip). This specific segment interacts exclusively with the major T cell kinase, Lck, effectively regulating its kinase activation. The critical region for this interaction lies within 37 amino acids (146-182) in the central part of Tip, which contains two motifs: the SH3 binding motif (SH3B; 174-182) and the C-terminal Src-related kinase homology motif (CSKH; 146-155). The 37 amino acid peptide fragments alone form a stable complex with Lck. Lck is crucial in the initial stages of T cell activation, not only when triggered by the native TCR stimulation but also in response to CAR-antigen stimulation. Following antigen-induced T cell activation, Lck initiates the phosphorylation of tyrosines in the ITAMs present in CD3ζ domains. This leads to the recruitment of ZAP70 kinases to the ITAMs of CD3ζ, facilitating the formation of signaling protein complexes. The results of our in vitro experiments using the immortalized T cell line, Jurkat cells, demonstrate that the inclusion of the Tip37 component in the CAR leads to the formation of the Lck-CAR complex. This, in turn, significantly enhances the phosphorylation of ITAMs in CARs and triggers downstream signaling. Importantly, the CAR T cells with the Tip37 co-stimulatory domain do not secrete cytokines when in an unstimulated state. This suggests that the heightened Lck phosphorylation does not lead to the auto-activation of T cells. As a result, CAR T cells engineered with Tip37 can be considered safe and hold promise for future applications. We engineered CAR19 T cells co-expressing Tip (Tip-CART19 cells) with a co-stimulatory domain of 4-1BB and CD3 (Tip-CART19 cells). As a control, we generated CART19 cells lacking the TIP domain (WT CART19 cells). We confirmed that Tip expression induced enhanced CD3 phosphorylation in CART19 T cells. Additionally, we observed that a significantly augmented outgrowth of CD8+ cells engineered with the TIP-containing CAR compared with canonical CAR T cells. Further, TIP-CAR T cells displayed increased expression of CD38 and Ki67. Interestingly, we also noted an expansion of CCR7+CD45RA- central memory subsets in CD4+ CART19 cells and CCR7-CD45RA- effector memory subsets in CD8+ CART19 cells. Finally, as evidenced by apparent changes in phenotype, Tip-CART19 cells exhibited improved cytotoxic ability and significant outgrowth of CART19 cells with in vitro repeated CAR stimulation using CD19-expressing tumor cells, as previously described in our study (Fraietta et al., Nature 2018). This study highlights that amplification of the constitutive Lck recruitment and amplified CD3 phosphorylation in CART19 cells via the introduction of Tip37 domain results in enhanced anti-leukemia cytotoxic ability and preservation of proliferative function in CART19 cells. Our discovery introduces a novel approach for amplifying CAR signaling to improve CAR T cell therapy.