Purpose:Wet age-related macular degeneration is a leading cause of irreversible vision loss, primarily due to choroidal neovascularization (CNV) and subsequent fibrosis. Although current anti-vascular endothelial growth factor A (anti-VEGF) therapies offer significant benefits, many patients exhibit limited or no response and develop drug resistance over time, necessitating the exploration of complementary or alternative therapeutics. This study aimed to identify and characterize a platelet-derived growth factor-C (PDGF-C)-targeting DNA aptamer and to evaluate its therapeutic potential for suppressing CNV and fibrosis, including in an anti-VEGF-refractory setting. Methods:A DNA aptamer against PDGF-C (α-PC aptamer) was identified using systematic evolution of ligands by exponential enrichment. Its binding to PDGF-C and inhibition of PDGF-C/platelet-derived growth factor receptor alpha (PDGFRα) interaction were assessed using surface plasmon resonance. The effects of the α-PC aptamer on PDGF-C-induced proliferation, migration, and PDGFRα, Akt, and extracellular-regulated kinase (ERK) signaling were examined in fibroblasts and human umbilical vein smooth muscle cells (HUVSMCs). In vivo efficacy was evaluated in a laser-induced CNV mouse model, including anti-VEGF refractory aged mice. Results:The α-PC aptamer specifically bound to PDGF-C and effectively blocked its binding to PDGFRα. The α-PC aptamer significantly inhibited PDGFRα, Akt, and ERK activation and suppressed PDGF-C-induced proliferation and migration of both fibroblasts and HUVSMCs. Importantly, in a laser-induced CNV mouse model, the α-PC aptamer markedly reduced neovascularization and fibrosis; it particularly retained efficacy in suppressing CNV in anti-VEGF refractory aged mice, where anti-VEGF treatment failed to do so. Conclusions:These findings suggest that the α-PC aptamer represents a promising therapeutic agent for treating neovascular diseases, especially in patients refractory to anti-VEGF treatment.

1 Abstract Adoptive cell therapy using tumor antigen-targeting T cell receptors (TCRs) offers a compelling approach to treat both hematological cancers and solid tumors due to broad antigen accessibility and the ability to target cancer-specific neoantigens. However, unlike clinically validated second generation CAR-T cells bearing built-in co-stimulatory signaling modules (i.e. 41BB or CD28), TCR-T cells receive little to no co-stimulation within most tumor microenvironments leading to attenuated cellular responses. Additionally, CD4+ TCR-T cells engineered to express HLA-Class I restricted TCRs possess minimal T-helper cell activity and thus do not effectively mobilize CD8+ TCR-T cells or host anti-tumor immune responses. To address these limitations, we used CRISPR-Cas9 to engineer TCR-T cells with targeted integration of chimeric CD8 constructs containing intracellular co-stimulatory domains. We found that expression of wild-type CD8αβ, but not CD8αα, could promote CD4+ T cell activities in HLA-Class I restricted TCR-T cells. However, this was insufficient to drive durable anti-tumor responses in challenging tumor mouse models when using a high-affinity WT1-directed TCR. To address this, several CD8 co-stimulatory fusion constructs containing CD28 or 41BB intracellular domains were designed and screened, identifying two CD8-41BB based chimeras that substantially increased TCR-T cell activity relative to wild-type CD8αβ. WT1-TCR-T cells co-expressing the CD8-41BB fusions demonstrated not only enhanced CD4+ activity including strong and polarized Th1-type cytokine secretion, but also enhanced the proliferation, cytokine release, and cytotoxicity of CD8+ CTLs. Remarkably, when combined with TGFBR2 gene disruption, WT1-TCR-T cells co-expressing CD8-41BB receptors were able to completely regress established cell line-derived ovarian tumors, showed robust in vivo expansion and persistence, and provided long-term protection from tumor rechallenge. Importantly, the specificity profile of the WT1-TCR including its HLA-A*02:01 restriction and WT1 peptide recognition motif was preserved upon expression of CD8-41BB. To simplify cell engineering processes for clinical applications, we configured a homology directed repair (HDR) cassette to allow for efficient CRISPR-Cas9-based insertion of both the TCR and CD8-41BB transgenes in the TRAC locus in a single step with >80% efficiency. Lastly, the enhanced activity conferred by CD8-41BB expression was validated with a second clinically relevant TCR targeting PRAME, suggesting this platform can be a universal approach for enhancing the therapeutic potential of TCR-based cell therapies.

The limited clinical benefits of immunotherapy in epithelial ovarian cancer (EOC) highlight the urgent need for innovative treatment strategies to improve outcomes. This study employs a dual-pronged strategy to promote EOC immunotherapy, focusing on tumor-specific T-cell receptor (TCR)-redirected T cells. Cancer cells derived from the ascites of EOC patients were analyzed to determine the expression of selected tumor-associated antigens (Mesothelin, HER2, NY-ESO-1, and WT1) and membrane-bound inhibitory receptor (IR) ligands. These antigens, critical for tumor progression, were selected as targets for our T cell products. Using TCR gene editing techniques, T cells with HLA-A*02:01-restricted receptors specific for these antigens were generated, drawing from both our laboratory’s library and relevant literature. In clinically relevant models using patient ascites-derived primary cultures (PC), T cells engineered with WT1-, MSLN-, and NY-ESO-1-specific TCRs exhibited potent and specific elimination of target cells, with WT1-TCR emerging as the most promising candidate for further development. We hypothesized that disrupting IR pathways could shield engineered T cells from immunosuppression within the tumor microenvironment (TME), thereby enhancing their therapeutic efficacy. To explore this, T-cell products were engineered to express WT1-TCR while incorporating gene disruptions of specific IRs (TIM3 or LAG3) (IRKO WT1-TCR). To assess the impact of IR disruption on anti-tumor efficacy, WT1-TCR and IRKO WT1-TCR T cells were tested against WT1-positive, HLA-A*02:01-positive PC and tumor organoids (PDOs). Evaluations included cytokine secretion, T cell activation using flow cytometry, and apoptosis measured via Incucyte live cell imaging. IRKO WT1-TCR T cells demonstrated superior cytotoxicity compared to WT1-TCR T cells, with TIM3KO WT1-TCR showing heightened effectiveness against tumor cells expressing TIM3 ligands. Co-culture experiments with PDOs further confirmed the increased induction of apoptosis mediated by TIM3KO and LAG3KO WT1-TCR T cells compared to their IR-competent counterparts. Finally, in vivo experiments were conducted to monitor tumor growth using IVIS live imaging and ultrasound. Considering the results of in vitro experiments, our primary focus was to analyze the efficacy of TIM3KO WT1-TCR T cells in comparison to IRCOMPETENT WT1-TCR T cells. Both T cell products significantly inhibited tumor growth compared to control groups that received no T cell therapy. Ultrasound imaging revealed a reduction in the size of abdominal masses, with TIM3KO WT1-TCR treatment achieving the most pronounced decrease in mass diameter, particularly in the intermediate size range (4-8 mm). These findings support the hypothesis that TIM3 knockout enhances the anti-tumor efficacy of WT1-TCR-engineered T cells for EOC. Maria Chiara Maffia, Eliana Ruggiero, Elena Tassi, Anna Simioni, Zulma Magnani, Alice Bergamini, Giorgia Mangili, Giorgio Candotti, Luca Bocciolone, Giacomo Pavone, Massimo Candiani, Miriam Sant’Angelo, Claudio Doglioni, Birgit Schultes, Aaron Prodeus, Chiara Bonini. Adoptive cell therapy with genetically engineered T cells for epithelial ovarian cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_2):Abstract nr LB027.

BACKGROUND AIMS:Autologous T-cell therapies have shown profound clinical responses; however, their widespread use has been limited primarily as the result of their individualized manufacturing requirements. METHODS:To develop a persistent "off-the-shelf" allogeneic (Allo) approach, a multiplex Nme2Cas9-based cytosine base editor was deployed to knockout select human leukocyte antigens (HLA) class I and II alleles (HLA-A, HLA-B and the class II transactivator [CIITA]) while retaining HLA-C to protect from natural killer (NK) cell rejection. RESULTS AND CONCLUSION:Matching the residual HLA-C allele from homozygous donors to the host prevented rejection of the donor T cells by allogeneic host T and NK cells. Site-specific integration of a tumor-specific CAR or TCR into the TRAC locus using SpyCas9 nuclease and an adeno-associated virus template allowed for a high localized insertion rate while simultaneously removing the endogenous TCR and preventing graft-versus-host disease. Using an optimized T-cell engineering process involving orthogonal CRISPR/Cas9 cleavage and base editors coupled with lipid nanoparticle delivery, we achieved efficient production of Allo-CAR T cells with high editing rates and cell expansion in a scalable manner. These allogeneic T cells demonstrated comparable functional activity to their autologous counterparts in preclinical assays. Moreover, this gene-editing approach significantly minimized the occurrence of chromosomal aberrations. This promising allogeneic approach also has been applied to induced pluripotent stem cells (iPSCs) with triple edits targeting HLA-A, HLA-B and CIITA (TKO). Pancreatic progenitor cells or cardiomyocytes derived from TKO iPSCs were protected from host peripheral blood mononuclear cell-mediated rejection when matched for HLA-C, suggesting potential applications in regenerative medicine applications.

Autologous T cell therapies have shown profound clinical responses; however, their widespread use has been limited primarily due to their individualized manufacturing requirements. To develop a persistent "off-the-shelf" allogeneic (Allo) approach, a multiplex Nme2Cas9-based cytosine base editor was deployed to knockout select HLA Class I and II alleles ( HLA-A , HLA-B , and the class II transactivator ( CIITA )), while retaining HLA-C to protect from NK cell rejection. Matching the residual HLA-C allele from homozygous donors to the host prevented rejection of the donor T cells by allogeneic host T and NK cells. Site-specific integration of a tumor-specific CAR or TCR into the TRAC locus using SpyCas9 nuclease and an adeno-associated virus (AAV) template allowed for high localized insertion rate while simultaneously removing the endogenous TCR and preventing GvHD. Using a lipid nanoparticle (LNP)-based delivery system of the editing components enabled a robust cell engineering process, achieving high editing rates and cell expansion. These allogeneic T cells demonstrated comparable functional activity to their autologous counterparts in preclinical assays. Moreover, this gene editing approach generated cells with minimal chromosomal aberrations. The Allo strategy has also been applied to induced pluripotent stem cells (iPSCs), suggesting potential applications in regenerative medicine applications.

Objective. To find the relationship between the quantitative indicators of DNA TREC and KREC with three groups of pathological conditions: sudden death syndrome, frequent diseases of the bronchopulmonary system and allergic diseases. Materials and methods. The study was conducted retrospectively. The survey group included infants and young children with various diseases, in which, based on the anamnesis, the presence of a pathology of the immune system was assumed. A total of 43 children were examined, of which group I - 5 children with sudden death syndrome, group II - 25 patients with frequent diseases of the bronchopulmonary system who received long-term antibiotic therapy, group III - 13 children with allergic diseases. Quantitative determination of TREC and KREC was carried out from samples of "dry spots" of blood obtained during neonatal screening. Results. Low TREC values were recorded more frequently than low KREC values, found in 79.1% and 48.8%, respectively. In the group of patients with sudden death syndrome (I group), who died in the first - fourth months of life, there were high rates of intrauterine growth retardation, dysembryogenesis stigma, congenital malformations, intrauterine infections. This group of children is characterized by thymomegaly and hypoplasia of the adrenal glands, TREC and KREC values are reduced in all children. The group of children with a high frequency of bronchopulmonary diseases (Group II) is characterized by a persistent course of herpes virus infection, prolonged subfebrile condition, neutropenia, transient hypoimmunoglobulinemia A, and thymomegaly. Low TREC values were found in 88% of children of group II, KREC - in 32%. In the III group of patients with allergic diseases (bronchial asthma, atopic dermatitis, allergic rhinitis, food allergy), TREC values are reduced in 53.8% of children, KREC – in 61.5% of children. Children of this group often suffered ARI, laryngotracheitis, obstructive bronchitis. During the first months of life, 75% of children manifested atopic dermatitis. In 62.5% of children with allergic diseases, a low level of serum immunoglobulin A was detected. Conclusions. Low values of TREC and KREC in a significant part of the examined patients make it possible to suspect the pathology of the immune system. The presence of diseases belonging to the group of warnings for the presence of primary immunodeficiency may also indicate the pathology of the immune system, primary or transient, due to infections, prematurity, intrauterine growth retardation, toxic effects. Comparison of the results of the survey in the neonatal period and diseases formed in infancy and early childhood was carried out retrospectively. The introduction in the Russian Federation of neonatal screening of primary immunodeficiencies based on the quantitative determination of TREC and KREC makes it possible to timely diagnose immunity errors and prescribe modern methods of treatment. However, the cut-off of the TREC and KREC values needs to be revised in the future.

The excisional rings of the T cell receptor rearrangement (TREC) and the κ-deletion element (KREC) are extrachromosomal DNA structures formed during V(D)J-recombination. A decrease in the number of TREC and KREC below age-related values may be a manifestation of immunodeficiency conditions, which can be caused by oncological and hematological diseases. Objective: To study the change in the amount of TREC and KREC in breast cancer. Methods. For the study, blood was taken from patients with malignant neoplasms in the main group and group of healthy women of various ages. Of these 35 healthy individuals and 77 patients with breast cancer. Median age 54 years (Q1-Q3: 36-81 years). Quantification of TREC and KREC was performed by real-time PCR using the IMMUNO-BIT reagent kit (ABV-test LLC) in accordance with the instructions for the kit. DNA extraction from whole blood was performed using the AmpliPrime RIBO-prep reagent kit (NextBio LLC). According to our study, in healthy individuals, the TREC level is 75.6/105 PBMC (Q1-Q3: 19.2-135.3), the KREC level is 317.3/105 (Q1-Q3: 118.1-565.9). Whereas in patients with breast cancer, the level of TREC is 4.4/105 PBMC (Q1-Q3: 0.9-17.3), the level of KREC is 101.3/105 (Q1-Q3: 29.3-339.28). When comparing the TREC and KREC indicators in different groups, statistically significant differences were established (p<0.001, p=0.006). The levels of TREC and KREC in the healthy population were significantly higher than among patients with cancer (median TREC were 75.6 and 4.4, median KREC 317.3 and 101.3, respectively). The data obtained demonstrate significant changes in T- and B-cell lymphopoiesis in patients with breast cancer. Quantitative determination of TREC and KREC makes it possible to assess the state of the T- and B-cell link of the immune system in patients with malignant neoplasms.

Background The success of T cell therapies for the treatment of solid tumors has been limited. Factors limiting efficacy in solid tumors are poor infiltration, exhaustion, and an immunosuppressive tumor microenvironment (TME). Methods To address these issues, we developed multiple mouse syngeneic tumor models to conduct in vivo CRISPR screening to identify Immune Enhancing Edits (IEEs) that augment CD8+ T cell function across TMEs. Several IEEs were validated in multiple mouse syngeneic solid tumor models, displaying significant in vivo tumor control. To better understand the therapeutic potential in human T cells, we engineered Wilms Tumor 1 (WT1)-specific TCR-T cells with these IEEs and tested them against WT1-expressing human solid tumor xenografts. Results WT1-specific TCR-T cells with either single IEE or a combination of IEEs induced tumor regression across multiple human tumor models. These IEE targets were also able to enhance CAR-based T cell therapies against solid tumors, adding to the broad applicability of this platform. Conclusions Coupling CRISPR-engineered immune enhancements with our allogeneic platform, Intellia is creating next-generation cell therapies for the treatment of solid tumors. Ethics Approval The mouse studies described here have been performed in compliance with protocols approved by Intellia Therapeutics' IACUC (Institutional Animal Care and Use Committee). (Mainly protocol number IT008)

Although VEGF-B was discovered as a VEGF-A homolog a long time ago, the angiogenic effect of VEGF-B remains poorly understood with limited and diverse findings from different groups. Notwithstanding, drugs that inhibit VEGF-B together with other VEGF family members are being used to treat patients with various neovascular diseases. It is therefore critical to have a better understanding of the angiogenic effect of VEGF-B and the underlying mechanisms. Using comprehensive in vitro and in vivo methods and models, we reveal here for the first time an unexpected and surprising function of VEGF-B as an endogenous inhibitor of angiogenesis by inhibiting the FGF2/FGFR1 pathway when the latter is abundantly expressed. Mechanistically, we unveil that VEGF-B binds to FGFR1, induces FGFR1/VEGFR1 complex formation, and suppresses FGF2-induced Erk activation, and inhibits FGF2-driven angiogenesis and tumor growth. Our work uncovers a previously unrecognized novel function of VEGF-B in tethering the FGF2/FGFR1 pathway. Given the anti-angiogenic nature of VEGF-B under conditions of high FGF2/FGFR1 levels, caution is warranted when modulating VEGF-B activity to treat neovascular diseases.

T cell receptor (TCR)-based therapy has the potential to induce durable clinical responses in patients with cancer by targeting intracellular tumor antigens with high sensitivity and by promoting T cell survival. However, the need for TCRs specific for shared oncogenic antigens and the need for manufacturing protocols able to redirect T cell specificity while preserving T cell fitness remain limiting factors. By longitudinal monitoring of T cell functionality and dynamics in 15 healthy donors, we isolated 19 TCRs specific for Wilms' tumor antigen 1 (WT1), which is overexpressed by several tumor types. TCRs recognized several peptides restricted by common human leukocyte antigen (HLA) alleles and displayed a wide range of functional avidities. We selected five high-avidity HLA-A*02:01-restricted TCRs, three that were specific to the less explored immunodominant WT137-45 and two that were specific to the noncanonical WT1-78-64 epitopes, both naturally processed by primary acute myeloid leukemia (AML) blasts. With CRISPR-Cas9 genome editing tools, we combined TCR-targeted integration into the TCR α constant (TRAC) locus with TCR β constant (TRBC) knockout, thus avoiding TCRαβ mispairing and maximizing TCR expression and function. The engineered lymphocytes were enriched in memory stem T cells. A unique WT137-45-specific TCR showed antigen-specific responses and efficiently killed AML blasts, acute lymphoblastic leukemia blasts, and glioblastoma cells in vitro and in vivo in the absence of off-tumor toxicity. T cells engineered to express this receptor are being advanced into clinical development for AML immunotherapy and represent a candidate therapy for other WT1-expressing tumors.

Post-traumatic knee osteoarthritis is characterized by cartilage degeneration, subchondral bone remodeling, osteophyte formation, and synovial changes. Therapeutic targeting of inflammatory activity in the knee immediately post injury may alter the course of osteoarthritis development. This study aimed to determine whether CD200R1 agonists, namely the protein therapeutic CD200Fc or the synthetic DNA aptamer CCS13, both known to act as anti-inflammatory agents, are able to delay the pathogenesis of injury-associated knee osteoarthritis in a murine model. Ten week old male C57BL/6 mice were randomized and surgical destabilization of the medial meniscus (DMM) to induce knee arthritis or sham surgery as a control were performed. CCS13 was evaluated as a therapeutic treatment along with CD200Fc and a phosphate-buffered saline vehicle control. Oligonucleotides were injected intra-articularly beginning one week after surgery, with a total of six injections administered prior to sacrifice at 12 weeks post-surgery. Histopathological assessment was used as the primary outcome measure to assess cartilage and synovial changes, while µCT imaging was used to compare the changes to the subchondral bone between untreated and treated arthritic groups. We did not find any attenuation of cartilage degeneration or synovitis in DMM mice with CD200Fc or CCS13 at 12 weeks post-surgery, nor stereological differences in the properties of subchondral bone. The use of CD200R1 agonists to blunt the inflammatory response in the knee are insufficient to prevent disease progression in the mouse DMM model of OA without anatomical restoration of the normal joint biomechanics.

Introduction. Despite the success of autologous chimeric antigen receptor (CAR)-T cells, barriers to a more widespread use of this potentially curative therapy include manufacturing failures and the high cost of individualized production. There is a strong desire for an immediately available cell therapy option; however, development of “off-the-shelf” T cells is challenging. Alloreactive T cells from unrelated donors can cause graft versus host disease (GvHD) for which researchers have successfully used nucleases to reduce expression of the endogenous T cell receptor (TCR) in the allogeneic product. The recognition of allogeneic cells by the host is a complex issue that has not been fully solved to date. Some approaches utilize prolonged immune suppression to avoid immune rejection and increase persistence. Although showing responses in the clinic, this approach carries the risk of infections and the durability of the adoptive T cells is uncertain. Other strategies include deletion of the B2M gene to remove HLA class I molecules and avoid recognition by host CD8 T cells. However, loss of HLA class I sends a “missing-self” signal to natural killer (NK) cells, which readily eliminate B2Mnull T cells. To overcome this, researchers are exploring insertion of the non-polymorphic HLA-E gene, which can provide partial but not full protection from NK cell-mediated lysis. Because activated T cells upregulate HLA class II, rejection by alloreactive CD4 T cells should also be addressed.