4-1BB Signaling Research Articles

322.6: Delineating the role of biological sex in organ transplantation: Identifying estrogen receptor α enhanced 4-1BB signaling as a critical component of T cell-mediated alloimmunity.

322.6: Delineating the role of biological sex in organ transplantation: Identifying estrogen receptor α enhanced 4-1BB signaling as a critical component of T cell-mediated alloimmunity.

Functional Activity of Cytokine-Induced Killer Cells Enhanced by CAR-CD19 Modification or by Soluble Bispecific Antibody Blinatumomab.

Strategies to increase the anti-tumor efficacy of cytokine-induced killer cells (CIKs) include genetic modification with chimeric antigen receptors (CARs) or the addition of soluble T-cell engaging bispecific antibodies (BsAbs). Here, CIKs were modified using a transposon system integrating two distinct anti-CD19 CARs (CAR-MNZ and CAR-BG2) or combined with soluble CD3xCD19 BsAb blinatumomab (CIK + Blina). CAR-MNZ bearing the CD28-OX40-CD3ζ signaling modules, and CAR-BG2, designed on the Tisagenlecleucel CAR sequence (Kymriah®), carrying the 4-1BB and CD3ζ signaling elements, were employed. After transfection and CIK expansion, cells expressed CAR-CD19 to a similar extent (35.9% CAR-MNZ and 17.7% CAR-BG2). In vitro evaluations demonstrated robust proliferation and cytotoxicity (~50% cytotoxicity) of CARCIK-MNZ, CARCIK-BG2, and CIK + Blina against CD19+ target cells, suggesting similar efficacy. All effectors formed an increased number of synapses, activated NFAT and NFkB, and secreted IL-2 and IFN-ɣ upon encountering targets. CIK + Blina displayed strongest NFAT and IFN-ɣ induction, whereas CARCIK-BG2 demonstrated superior synapse formation. All the effectors have shown therapeutic activity in vivo against the CD19+ Daudi tumor model, with CARCIK cells showing a more durable response compared to CIK + Blina, likely due to the short half-life of Blina in this model.

IMT030122, A novel engineered EpCAM/CD3/4-1BB tri-specific antibody, enhances T-cell recruitment and demonstrates anti-tumor activity in mouse models of colorectal cancer

Colorectal cancer is a major global health burden, with limited efficacy of traditional treatment modalities in improving survival rates. However, recently advances in immunotherapy has improved treatment outcomes for patients with this cancer. To address the continuing need for improved treatment efficacy, this study introduced a novel tri-specific antibody, IMT030122, that targets EpCAM, 4-1BB, and CD3. We evaluated the pharmacological efficacy and mechanism of action of IMT030122 in vitro and in vivo. In in vitro studies, IMT030122 exhibited differential binding to antigens and cells expressing EpCAM, 4-1BB, and CD3. Moreover, IMT030122 relied on EpCAM-targeted activation of intracellular CD3 and 4-1BB signaling and mediated T cell cytotoxicity specific to HCT116 colorectal cancer cells. In vivo, IMT030122 demonstrated potent anti-tumor activity, significantly inhibiting the growth of colon cancer HCT116 and MC38-hEpCAM subcutaneous grafts. Further pharmacological analysis revealed that IMT030122 recruited lymphocytes from peripheral blood into colorectal cancer tissue and exerted durable anti-tumor activity, predominantly by promoting the activation, proliferation, and differentiation of CD8T cells. Notably, IMT030122 still exhibited anti-tumor efficacy even in the presence of significantly depleted lymphocytes in colorectal cancer tissue. The potent pharmacological activity and anti-tumor effects of IMT030122 suggest it may enhance treatment efficacy and substantially extend the survival of patients with colorectal cancer in the future.

Phase 1 trial safety and efficacy of ragistomig, a bispecific antibody targeting PD-L1 and 4-1BB, in advanced solid tumors.

2529 Background: Activation of T cells in the tumor microenvironment by 4-1BB agonist antibodies is a promising approach to augment PD-(L)1 inhibitor efficacy. Ragistomig(ABL503/TJ-L14B) is a bispecific antibody combining PD-L1 antagonist with 4-1BB agonistic activity, designed to induce 4-1BB signaling only when bound to the PD-L1 tumor antigen on cancer cells, which may overcome resistance to PD-(L)1 inhibition and avoid hepatoxicity seen with traditional 4-1BB mAbs. Methods: ABL503 was investigated at doses ranging from 0.7 mg (flat dose) to 10 mg/kg (weight-based dose) IV every 2 weeks (Q2W) in patients with advanced or relapsed/refractory solid tumors, to assess safety, preliminary anti-tumor effect and pharmacokinetic (PK)/pharmacodynamic (Pd) activity. The BOIN design was utilized during dose escalation (regardless of CPS/TPS score). Additional patients were enrolled in dose expansion cohorts at 3 and 5 mg/kg (CPS/TPS>=1 required). Results: As of Jan 2024, the study enrolled 49 patients (30 dose escalation, 19 dose expansion). At least 1 treatment related adverse event (TRAE) occurred in 37 patients (75.5%); most common TRAE (≥ 10%, any grade/grade 3-4) were elevated AST (30.6%/18.4%), elevated ALT (26.5%/18.4%), rash (14.3%/4.1%), nausea (12.2%/0%), pyrexia (12.2%/2.0%) and fatigue (10.2%/0%). Dose limiting toxicities occurred in 5 patients and were observed at dose levels of 1, 5, and 10 mg/kg. All DLTs were recovered/recovering. MTD was not reached. Based on the safety, efficacy, PK and Pd analysis, the optimal dose was determined to be 5 mg/kg Q2W. Objective responses were observed in 6 out of 39 efficacy-evaluable patients, and all responses were observed at 3 and 5 mg/kg, including 1 complete response (CR) in a patient with ovarian cancer who received 6 prior lines of treatment and 5 partial responses (PRs) in patients with ovarian (n=1), melanoma (n=1), gastric (n=1), head and neck squamous cell (n=1), and esophageal cancer (n=1). Overall response rate (ORR) for all dose levels was 15.3%, and ORR at 5 mg/kg was 30% (3/10). Clinical benefit rate (CBR) for all dose levels was 61.5%, and CBR at 5 mg/kg was 80% (8/10). 66.7% of responders received prior PD-(L)1 inhibitors. One patient achieved sustained tumor regression over 6 months, even after treatment discontinuation. PK was dose proportional, and half-life was ~5 days. Dose-dependent increase of Pd marker s4-1BB was observed, demonstrating target engagement. Conclusions: ABL503 had a manageable safety profile and demonstrated promising anti-tumor activity, with objective responses in 6 out of 39 efficacy-evaluable patients across multiple tumor types in heavily pre-treated patients, including patients previously treated with checkpoint inhibitors. The data support continued development of ABL503 alone and in combination with other compounds, as a potential therapeutic option for patients with solid tumor cancers. Clinical trial information: NCT04762641 .

Incorporating IL7 receptor alpha signaling in the endodomain of B7H3-targeting chimeric antigen receptor T cells mediates antitumor activity in glioblastoma.

CAR-T-cell therapy has shown promise in treating hematological malignancies but faces challenges in treating solid tumors due to impaired T-cell function in the tumor microenvironment. To provide optimal T-cell activation, we developed a B7 homolog 3 protein (B7H3)-targeting CAR construct consisting of three activation signals: CD3ζ (signal 1), 41BB (signal 2), and the interleukin 7 receptor alpha (IL7Rα) cytoplasmic domain (signal 3). We generated B7H3 CAR-T cells with different lengths of the IL7Rα cytoplasmic domain, including the full length (IL7R-L), intermediate length (IL7R-M), and short length (IL7R-S) domains, and evaluated their functionality in vitro and in vivo. All the B7H3-IL7Rα CAR-T cells exhibited a less differentiated phenotype and effectively eliminated B7H3-positive glioblastoma in vitro. Superiority was found in B7H3 CAR-T cells contained the short length of the IL7Rα cytoplasmic domain. Integration of the IL7R-S cytoplasmic domain maintained pSTAT5 activation and increased T-cell proliferation while reducing activation-induced cell death. Moreover, RNA-sequencing analysis of B7H3-IL7R-S CAR-T cells after coculture with a glioblastoma cell line revealed downregulation of proapoptotic genes and upregulation of genes associated with T-cell proliferation compared with those in 2nd generation B7H3 CAR-T cells. In animal models, compared with conventional CAR-T cells, B7H3-IL7R-S CAR-T cells suppressed tumor growth and prolonged overall survival. Our study demonstrated the therapeutic potential of IL7Rα-incorporating CAR-T cells for glioblastoma treatment, suggesting a promising strategy for augmenting the effectiveness of CAR-T cell therapy.

Abstract 6705: A novel tri-specific T cell engager targeting the intracellular oncoprotein WT1

Abstract Background: Wilms' tumor gene 1 (WT1) is considered an oncogenic factor, with high expression observed in several hematological malignancies and solid tumors. However, its expression is rarely found in normal adult tissue. WT1 can be degraded by the proteasome, processed and presented on the cell surface as major histocompatibility complex (MHC) I epitopes and recognized by T cell receptors (TCR). Here, we described the development of WT1-TOPAbody, a novel tri-specific T cell engager (TCE), that activated T cells by both CD3 and 4-1BB signaling upon recognition of WT1 in the context of HLA-A02, leading to superior anti-tumor efficacy. Materials and Methods: WT1-TOPAbody was evaluated for its antigen binding activity and target specificity by a series protein/peptide and cell-based assays. In vitro cytotoxicity and T cell activation mediated by WT1-TOPAbody was further assessed in a panel of WT1+ tumor cell lines in coculture with effector cells. Anti-tumor activity was investigated in a humanized syngeneic mice model bearing WT1/HLA-A02 expressing tumors. Safety assessment including cytokine release assay (CRA) and preliminary CMC developability was also performed. Results: WT1-TOPAbody exhibited superior binding affinity to the WT1/HLA-A02 complex while displaying lower binding to off-target peptides and CD34+ HSC from healthy donor than clinical benchmark WT1-CD3 bispecific antibody, RG-6007. WT1-TOPAbody effectively induced tumor lysis in multiple WT1+ tumors including leukemia, ovarian, breast, and colon cancer cells. Moreover, serial killing of tumor cells were observed in WT1-TOPAbody treated T cells, accompanied by lower exhaustion markers expressed, suggesting a prolonged anti-tumor inhibition effect of WT1-TOPAbody. In a humanized CD3e/4-1BB/HLA-A2.1 mouse model, WT1-TOPAbody significantly inhibited tumor growth in a dose-dependent manner and the efficacy was superior than that of the benchmark. Additionally, IL-6 release was minimal in PBMC upon treatment of the antibody, indicating a reduced risk of inducing cytokine release syndrome (CRS). Drug developability assessment indicated that the leading WT1-TOPAbody was a reasonably developable molecule to be taken forward. Conclusion: WT1-TOPAbody is a differentiated WT1/HLA-A02 targeted T cell engagers that displayed potent and durable anti-tumor activity. These results collectively support the potential of WT1-TOPAbody as a novel therapeutic agent against WT1/HLA-A02+ cancers and warrant its further development. Citation Format: Shan Gao, Liu Yang, Jun Du, Wenqing Jiang, Lei Fang. A novel tri-specific T cell engager targeting the intracellular oncoprotein WT1 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6705.

Abstract 6362: The bispecific antibody KA-3002, targeting PSMA and 4-1BB, specifically enhances T cell activity within prostate tumors

Abstract Cancer immunotherapy with 4-1BB agonists has faced limitations in further clinical development due to dose-limiting toxicity. PSMA, highly expressed in normal prostate tissue and prostate cancer, is a clinically validated target for prostate cancer treatment. In this study, we developed a bispecific antibody (BsAb), KA-3002, targeting PSMA and human 4-1BB, with the aim of restricting 4-1BB stimulation to the interior of tumors. KA-3002 was developed based on our nanobody antibody discovery platform. KA-3002, in IgG1 format, features mutations in the Fc domain to eliminate ADCC, ADCP, and CDC activity. It contains two identical nanobodies at the C-terminal against 4-1BB to stimulate 4-1BB signaling in T cells and binds to PSMA with two Fabs at the N-terminal. KA-3002 exhibits high avidity binding to prostate tumor cells expressing PSMA (EC50<1nM) and also binds to 4-1BB expressed on 293T cells and activated human T cells. In the presence of a low concentration of CD3 antibody to stimulate T cell activation, co-culture of tumor cells expressing PSMA with primary T cells isolated from human PBMC enhances T cell activation and proliferation. In PBMC humanized immunodeficient mouse tumor models, KA-3002 effectively suppresses the growth of 22Rv1 tumors, which highly express PSMA. The antibody also demonstrates a half-life of more than five days in the blood of Balb/c mice. Finally, we assessed the developability of this antibody with different treatments such as high concentration, low pH, repeated freezing and thawing, and high temperature. KA-3002 shows neither aggregation nor degradation. In conclusion, the BsAb KA-3002, developed with our nanobody antibody discovery platform, emerges as a promising pre-clinical candidate drug for the treatment of prostate cancer. Citation Format: Guojin Wu, Jiabei Liang, Hao Peng, Feng Hao, Tongtong Liu, Jinying Ning. The bispecific antibody KA-3002, targeting PSMA and 4-1BB, specifically enhances T cell activity within prostate tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6362.

Abstract 6361: KY-HC-mouse: A low-cost and rapid nanobody discovery platform

Abstract Nanobodies, primarily derived from camelid animals, have gained prominence in antibody-drug development due to their small molecular weight and ease of modification. However, the high cost associated with camelid animals for nanobody discovery led us to develop a strain of heavy-chain mice known as KY-HC-mouse. This genetically modified mouse expresses IgG without the CH1 domain, resulting in the production of nanobodies in IgG format. Utilizing hybridoma techniques, we developed a variety of nanobodies against multiple targets, such as PSMA, CD38, PD-L1, CD28, and 4-1BB, from KY-HC-mouse. These nanobodies all demonstrate high affinity, with KD ranging from 0.01 nM to 10 nM. We also selected antagonist nanobodies that block the interaction of PD1 and PDL1, as well as agonist nanobodies that stimulate 4-1BB signaling in T cells. After humanization, most nanobodies have a half-life of more than five days in the blood of both mice and monkeys. Subjected to various treatments, such as high concentration, low pH, repeated freezing and thawing, and high temperature, most humanized nanobodies show neither aggregation nor degradation, suggesting good developability of these nanobodies. Finally, we have developed multiple bispecific antibodies (BsAbs) targeting both PDL1 and 4-1BB, PSMA and 4-1BB, or B7H3 and 4-1BB. These BsAbs exhibit good developability and efficacy. In conclusion, our KY-HC-mouse is a powerful nanobody discovery platform. Citation Format: Guojin Wu, Feng Hao, Feng He, Hao Peng, Yi Gu, Jinying Ning. KY-HC-mouse: A low-cost and rapid nanobody discovery platform [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6361.

Abstract 2380: The bispecific antibody KA-3004, targeting PD-L1 and 4-1BB, is designed to enhance anti-tumor activity

Abstract Checkpoint inhibitors (CPI) have transformed the treatment landscape for advanced solid tumors; however, a majority of cancers still do not respond to immune checkpoint therapy. In this study, we developed a bispecific antibody (BsAb) named KA-3004, targeting PD-L1 and human 4-1BB, with the aim of enhancing the functionality and survival of stimulated CD8+ T cells in the tumor microenvironment. This antibody was designed using our nanobody antibody discovery platform. KA-3004, in IgG1 format, incorporates mutations in the Fc domain to eliminate ADCC, ADCP, and CDC activity. It features two identical nanobodies at the C-terminal against 4-1BB to stimulate 4-1BB signaling in T cells and binds to PD-L1 with two Fabs at the N-terminal. KA-3004 binds to PD-L1 with high affinity, blocking the interaction of PD-1 and PD-L1. It also binds to 4-1BB expressed on 293T cells and activated human T cells. In the presence of a low concentration of CD3 antibody to stimulate T cell activation, co-culture of tumor cells expressing PD-L1 with primary T cells isolated from human PBMC enhances T cell activation and proliferation. In PBMC humanized immunodeficient mouse tumor models, KA-3004 effectively suppresses the growth of NCI-H292 tumors expressing PD-L1. Additionally, KA-3004 has a half-life of more than five days in the blood of Balb/c mice. Finally, we assessed the developability of this antibody with different treatments such as high concentration, low pH, repeated freezing and thawing, and high temperature, and KA-3004 shows neither aggregation nor degradation. In conclusion, the BsAb KA-3004, developed with our nanobody antibody discovery platform, stands out as a promising pre-clinical candidate drug for the treatment of solid cancers. Citation Format: Jiabei Liang, Zhengcheng Guo, Hao Peng, Feng Hao, Tongtong Liu, Jinying Ning, Guojin Wu. The bispecific antibody KA-3004, targeting PD-L1 and 4-1BB, is designed to enhance anti-tumor activity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2380.

Abstract 5299: A novel anti-4-1BB antibody with no liver toxicity and its application in a bi-specific antibody

Abstract 4-1BB (CD137, TNFRSF9) is a co-stimulatory receptor initially identified on T cells, belonging to the tumor necrosis factor receptor superfamily. Primarily expressed on activated CD8+ T cells, it plays a crucial role in immune activation. Moreover, the intracellular domain of 4-1BB is leveraged to enhance the proliferation and cytotoxicity of Chimeric Antigen Receptor-T (CAR-T) cells. However, the development of 4-1BB agonist antibodies has encountered challenges, with limited clinical efficacy (e.g., utomilumab) or dose-dependent liver toxicity (e.g., urelumab). Consequently, there is a significant demand for the development of next-generation 4-1BB agonist antibodies, leading to numerous ongoing preclinical R&D efforts and clinical trials. Here, we introduce a novel fully humanized anti-4-1BB antibody, HLX25, and discuss the development of HLX34, a bispecific antibody targeting both 4-1BB and Her2. HLX25 boasts a unique binding epitope distinct from first-generation 4-1BB antibodies and demonstrates multiple cross-species reactivities. Additionally, HLX25 incorporates an engineered Fc region to enhance intratumoral clustering while reducing toxicity. In vitro studies reveal that HLX25 exhibits activity similar to natural 4-1BB ligands. In contrast, utolimumab shows inadequate activity, and urelumab displays superior activity, suggesting a correlation between efficacy and safety. As reported previously, (As been profiled in our Lab,) HLX25 exhibits robust dose-dependent anti-tumor activity in the MC38/h4-1BB KI mouse model. Noteworthy is the superior safety profile of HLX25 in the h4-1BB KI model, where both HLX25 and utolimumab induce no significant AST and ALT upregulation, unlike urelumab, which demonstrates such upregulation in vivo. A comparative analysis of HLX25 with other second-generation anti-4-1BB antibodies in clinical trials in vivo reveals that HLX25 provides superior tumor inhibition without observed toxicity within the effective dose range. Furthermore, we've developed HLX34, a Her2x4-1BB bispecific antibody, derived from Trastuzumab and HLX25. Its format underwent optimization using a reporter assay, indicating superior in vitro activity. Validation through a cytokine release assay with primary PBMCs in vitro and confirmed in vivo activity in animal models, without observed toxicity, strongly supports the efficacy and safety of HLX34 in Her2-positive tumors. These results provide evidence that our novel anti-4-1BB antibody activates proper 4-1BB signaling through tumor-enriched FcgRIIB or tumor-associated antigen-mediated clustering, inhibiting tumor growth with good safety in both in vitro and in vivo settings. This underscores HLX34 as a promising alternative therapeutic strategy for next-generation cancer immunotherapy. Citation Format: Jen-Kuan Chang, Jie Xue, Chen Dong, An Ju, Toya Baral, Julie Yoo, Yanling Wang, Marco Muda, Qiang Liu, Wenfeng Xu, Weidong Jiang, Lixin Feng. A novel anti-4-1BB antibody with no liver toxicity and its application in a bi-specific antibody [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5299.

Comparison of antibody-based immunotherapeutics for malignant hematological disease in an experimental murine model

AbstractAntibody-based immunotherapies have revolutionized leukemia and lymphoma treatment, with animal studies being crucial in evaluating effectiveness and side effects. By targeting the evolutionary conserved Slamf7 immune receptor, which is naturally expressed by the murine multiple myeloma cell line MPC-11, we have developed a syngeneic mouse model for direct comparison of 3 immunotherapies: monoclonal antibodies (mAb), bispecific T-cell engagers (BiTE), and chimeric antigen receptor (CAR) T cells (CART), all targeting Slamf7. Slamf7-BiTE is a bispecific single-chain antibody consisting of α-Slamf7 and α-CD3 Fv fragments joined through a Gly-Ser linker, and Slamf7-CART comprises the α-Slamf7 Fv fragment fused to the msCD8α transmembrane and msCD28, 4-1BB, and CD3ζ intracellular signaling domains. Slamf7-BiTE and Slamf7-CART effectively killed MPC-11 cells in vitro, independently of Slamf7-mediated inhibitory signaling by self-ligation. After chimerizing the constant region of the rat–anti-mouse Slamf7 antibody to mouse Fc-immunoglobulin G2a for enhanced effector functions, Slamf7-mAb triggered antigen-specific antibody-dependent cellular cytotoxicity by binding to Fcγ receptor IV. In vivo, all 3 immunotherapies showed antitumor effects against Slamf7-expressing targets. Unlike Slamf7-mAb, Slamf7-BiTE led to considerable side effects in test animals, including weight loss and general malaise, which were also observed to a lesser extent after Slamf7-CART infusion. In allogeneic transplant, Slamf7-BiTE and Slamf7-CART maintained activity compared with the nontransplant setting, whereas Slamf7-mAb displayed enhanced antimyeloma activity. In summary, our model faithfully replicates treatment efficacy and side effects detected after human immunotherapy. It aids in developing and improving immunotherapies and may help devise novel approaches to mitigate undesired effects in steady state and allogeneic stem cell transplantation.

Abstract A042: Quantitative multiplex immunoprecipitation reveals distinct protein interaction networks responsible for discrepant in vivo activity exhibited by two second generation CD22-targeted chimeric antigen receptors

Abstract The Pediatric Leukemia Adoptive Therapy (PLAT) trials at Seattle Children’s Hospital have pioneered the use of chimeric antigen receptor (CAR) T cell (CAR-T) therapies in pediatric patients with B cell Acute Lymphoblastic Leukemia. The landmark PLAT-02 trial (n=45), utilizing a second-generation CD19-directed CAR, observed a 93% remission rate following CAR-T infusion. However, approximately half of these subjects relapsed within one year and nearly a third of those tumors no longer expressed the CD19 target. The PLAT-04 trial (n=4) was subsequently conceived with a second-generation CAR (SCRI-C22v1) engineered to target CD22, an alternative antigen expressed on the B cell surface. Despite promising preclinical data, SCRI-C22v1 failed to generate anti-leukemic activity in all four subjects and the trial was halted. In response, a different second-generation CD22-directed CAR (SCRI-C22v2) was engineered, replacing the CD28 transmembrane domain and IgG4 hinge domain in SCRI-C22v1 with a continuous CD8 hinge and transmembrane domain while retaining identical 4-1BB and CD3ζ intracellular signaling domains. The modified SCRI-C22v2 displayed improved anti-tumor activity in preclinical models and was ultimately tested in patients in the PLAT-07 trial (n=4), where it exhibited strong in vivo expansion and persistence while inducing complete remission after 1 month in all four subjects. To investigate the molecular signaling responsible for clinical differences observed between SCRI-C22v1 and SCRI-C22v2, we applied an emerging proteomic technology, Quantitative Multiplex Immunoprecipitation (QMI), that captures medium-throughput quantitative data about fold changes in protein interaction networks downstream of the CAR. CAR-T products from healthy donors expressing either SCRI-C22v1 or SCRI-C22v2 were stimulated with fixed K562 cells expressing CD22 and protein interaction networks were profiled with QMI, revealing distinct differences in signal transduction downstream of the CAR. Contrary to expectation, it was the ineffective SCRI-C22v1 that exhibited significantly enhanced signalosome formation, engaging more strongly than SCRI-C22v2 with both the classical CD3ζ-ZAP70-LAT-SLP76 pathway as well as the non-classical TRAF pathway. Interestingly, the clinically successful SCRI-C22v2 demonstrated stronger baseline engagement with inhibitory molecules that attenuate canonical TCR signaling such as SHP2 and UBASH3A, suggesting regulatory control of CAR signaling is crucial for optimal clinical responses. Since the CAR is a synthetic receptor, it should be possible to bioengineer optimized signal transduction to some extent, and our group continues to work towards identifying an optimized QMI signature that can be used as a CAR-tune network for the rational design of more effective CAR-T therapies. Citation Format: Eric Bueter, Corinne Summers, Isabella Draper, Joshua Gustafson, Stephen EP Smith, Kamila Gwiazda, Julie Park, Colleen Annesley, Rebecca Gardner, Michael Jensen. Quantitative multiplex immunoprecipitation reveals distinct protein interaction networks responsible for discrepant in vivo activity exhibited by two second generation CD22-targeted chimeric antigen receptors [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 A042.

Gp350-Targeted CAR-T Therapy in EBV-Positive NK/T-Cell Lymphoma: Pre-Clinical Development of gp350CAR-T

Introduction: Epstein-Barr virus (EBV) positive NK/T cell lymphoma is a subtype of non-Hodgkin's lymphoma strongly associated with EBV infection. EBV infection has been reported in 85-100% of cases. It has been suggested to be central in pathogenesis. The incidence of EBV+ NK/T cell lymphoma is increasing worldwide, particularly in Asia and Latin America, and it has a poor prognosis with few effective treatment options. Therefore, novel approaches for treating EBV-associated NK/T cell lymphoma are urgently needed. Developing chimeric antigen receptor (CAR)-T cells therapy targeting EBV specific protein in the context of this EBV-positive aggressive lymphoma will provide a novel therapeutic strategy for patients with limited treatment options. Targeting EBV lytic protein for control EBV in vivo replication and tumorigenesis have been explored lately. The gp350 protein is a major glycoprotein on the surface of EBV during its lytic phase, after fusion between virus and host cell membrane, gp350 is left on the host cell surface. The distinct surface distribution pattern of gp350 in both freshly infected host cells and the lytic phase host cells renders it an attractive target for immunotherapy. Method and Result: We developed EBV specific CAR-T cells targeting gp350. We utilized single-chain variable fragments (scFvs) derived from four novel, gp350-binding, neutralizing monoclonal antibodies (mAbs) (12C10, 7D11, 6A12, 7A8) to generate the CAR constructs. We demonstrated that these scFvs, when fused to a second-generation CAR backbone consisting of 4-1BB and CD3ζ signal domains, are efficiently expressed on the surface of transduced T cells. These gp350 CAR-T cells exhibit specific activation and cytotoxic effects in vitro. Of note, the clone 6A12 scFv of CAR displayed the most potent cytotoxicity effects in gp350 positive cancer coculture toxicity assays. To optimize the CAR structure further, we designed various linker and transmembrane domains based on the clone 6A12 scFv, including CD8 linker, IgG4 hinge, IgG1 Fc linker, and CD28 linker and transmembrane domains. Our cytotoxicity assay screening indicated that the CD8 linker and transmembrane domain resulted in the most advanced cytotoxicity and target specificity (Figure A, B). To assess gp350 CAR-T efficacy in vivo, we established a T-cell leukaemia animal model in NOD immune-deficient mice through intravenous injection of Jurkat-gp350-luc cells. On post xenograft day 7, the Jurkat-gp350-luc xenografted mice were separated into three groups and each group with 5 mice (except PBS group with 2 mice). CAR-T and T cell were infused by tail vein injection with mock T 2x10^6/mouse, 6A12CAR-T 2x10^6/mouse and PBS control. The tumor growth in mice was monitored by in vivo luminescence with IVIS imaging system. Upon engraftment, Jurkat-gp350-luc cells established disseminated leukaemia expanding preferentially in the spine, femur, head, and pelvis, with few cells in peripheral blood. Mice receiving control mock T cells or without T cell PBS control developed rapid disease progression and were euthanized by day 35 after Jurkat-gp350-luc engraftment. In contrast, mice receiving gp350 CART cells were significantly protected from rapid progression (Figure C, D). Conclusion: In pre-clinical development, we designed, constructed, and screened gp350 CAR-T cells to demonstrate its efficacy in vitro and in vivo against and kill gp350 positive leukaemia cells and prevent formation and development leukaemia in xenograft mice model.

CD38 KO/CD38-CAR Human Primary Natural Killer Cells Enhance Cytotoxicity Against CD38-Expressing Primary Lymphoma, Leukemia, and Myeloma

Natural killer (NK) cells have strong cancer immunotherapeutic potential that can be further augmented by engineering with a chimeric antigen receptor (CAR). CD38, a transmembrane glycoprotein involved in signal transduction and cellular adhesion, is highly expressed on the surface of the hematologic malignancies multiple myeloma (MM), acute myeloid leukemia (AML), Burkitt Lymphoma (BL) and T-cell acute lymphoblastic leukemia (T-ALL). FDA approved CD38 monoclonal antibodies (mAb) daratumumab and isatuximab are used to treat MM in patients, and have been tested to treat other CD38 expressing malignancies. NK cells play an imperative role in the efficacy of these antibodies and have natural antitumor activity against these cancers. However, because NK cells also highly express CD38, anti-CD38 mAb can cause NK cell fratricide, limiting their efficacy. Here, we generated multiple CD38 scFv based on isatuximab sequences and generated fratricide-resistant primary CD38-CAR NK cells using CRISPR/Cas9 genome editing and AAV6 gene delivery. Using this approach, we inserted the CD38-CAR gene into the CD38 locus to efficiently generate simultaneous CD38 KO and CD38-CAR KI with a mean expression level of %60. The CAR-expressing NK cells could be expanded on mbIL21-expressing feeder cells to a clinically relevant number of cells with no decline in CAR-expression level during two weeks of expansion confirming the lack of CAR mediated fratricide. We evaluated transduction efficiency and expression of CD38-CAR, and compared the cytotoxic function of two constructs with CD8 transmembrane (TM)/stalk and 41BB signaling domains and reverse heavy and light chains against AML, MM, T-ALL and BL immortalized cell lines and primary patient samples. We enhanced CD38 expression in target cancer cells by pre-treatment with all-trans retinoic acid (ATRA), a metabolite of vitamin A that pushes cells to maturation, consequently upregulating CD38. The sequential treatment of target cells with 10nM ATRA for 48 hours enhanced the cytotoxic activity of the CD38 KO/CD38-CAR-NK cells against primary MM, BL and AML samples. This would be particularly important for patients with low expression of CD38 on their cancers. The enhanced anti-tumor activity of the CAR was also studied and observed by CyTOF. Additionally, to better understand the therapeutic potential of CD38-CAR NK cells compared to CD38-CAR T cells we generated CD38 KO/CD38-CAR T cells using the same engineering approach and comparing their cytotoxic function. We showed that both CD38-CAR-T and CD38-CAR-NK cells have comparable anti-tumor activity compare to their paired non-edited wild type cells. Finally, we investigated the need for a CD38 KO to develop a fratricide-resistant therapy by comparing the cytolytic and metabolic functions of CD38 +/AAVS1 KO and CD38 KO CD38-CAR NK cells. We conclusively report a CD38-CAR NK cell therapy with enhanced metabolism and cytotoxicity toward a variety of hematologic malignancies, which can be further augmented by combination treatment with ATRA to target CD38-low malignancies.

Anti-TIM3 chimeric antigen receptor-natural killer cells from engineered induced pluripotent stem cells effectively target acute myeloid leukemia cells

BackgroundAcute myeloid leukemia (AML) is a clonal malignant disorder which originates from a small number of leukemia-initiating cells or leukemic stem cells (LSCs)—the subpopulation that is also the root cause of relapsed/refractory AML. Chimeric antigen receptor (CAR)-T cell therapy has proved successful at combating certain hematologic malignancies, but has several hurdles that limit its widespread applications. CAR-natural killer (NK) cells do not carry the risk of inducing graft-versus-host disease (GvHD) frequently associated with allogeneic T cells, thereby overcoming time-consuming, autologous cell manufacturing, and have relatively safer clinical profiles than CAR-T cells. The present study aimed to generate anti-TIM3 CAR-NK cells targeting LSCs from a clonal master induced pluripotent stem cells engineered with the third-generation anti-TIM3 CAR.MethodsA clonal master umbilical cord blood NK-derived induced pluripotent stem cell (iPSC) line, MUSIi013-A, was used as a starting cells for engineering of an anti-TIM3 CAR harboring TIM3 scFv fragment (clone TSR-022), CD28, 4-1BB, and CD3ζ signaling (CAR-TIM3). The established CAR-TIM3 iPSCs were further differentiated under serum- and feeder-free conditions into functional CAR-TIM3 NK cells and tested for its anti-tumor activity against various TIM3-positive AML cells.ResultsWe successfully established a single-cell clone of CAR-TIM3 iPSCs, as validated by genomic DNA sequencing as well as antibody and antigen-specific detection. We performed thorough iPSC characterization to confirm its retained pluripotency and differentiation capacity. The established CAR-TIM3 iPSCs can be differentiated into CAR-TIM3 NK-like cells, which were further proven to have enhanced anti-tumor activity against TIM3-positive AML cells with minimal effect on TIM3-negative cells when compared with wild-type (WT) NK-like cells from parental iPSCs.ConclusionsiPSCs engineered with CARs, including the established single-cell clone CAR-TIM3 iPSCs herein, are potential alternative cell source for generating off-the-shelf CAR-NK cells as well as other CAR-immune cells. The feasibility of differentiation of functional CAR-TIM3 NK cells under serum- and feeder-free conditions support that Good Manufacturing Practice (GMP)-compliant protocols can be further established for future clinical applications.

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.

A novel CAR-T cell product targeting CD74 is an effective therapeutic approach in preclinical mantle cell lymphoma models

BackgroundMantle cell lymphoma (MCL) is a rare B-cell non-Hodgkin lymphoma subtype which remains incurable despite multimodal approach including chemoimmunotherapy followed by stem cell transplant, targeted approaches such as the BTK inhibitor ibrutinib, and CD19 chimeric antigen receptor (CAR) T cells. CD74 is a nonpolymorphic type II integral membrane glycoprotein identified as an MHC class II chaperone and a receptor for macrophage migration inhibitory factor. Our group previously reported on CD74's abundant expression in MCL and its ability to increase via pharmacological inhibition of autophagosomal degradation. Milatuzumab, a fully humanized anti-CD74 monoclonal antibody, demonstrated significant activity in preclinical lymphoma models but failed to provide meaningful benefits in clinical trials mainly due to its short half-life. We hypothesized that targeting CD74 using a CAR-T cell would provide potent and durable anti-MCL activity.MethodsWe engineered a second generation anti-CD74 CAR with 4-1BB and CD3ζ signaling domains (74bbz). Through in silico and rational mutagenesis on the scFV domain, the 74bbz CAR was functionally optimized for superior antigen binding affinity, proliferative signaling, and cytotoxic activity against MCL cells in vitro and in vivo.ResultsFunctionally optimized 74bbz CAR-T cells (clone 42105) induced significant killing of MCL cell lines, and primary MCL patient samples including one relapse after commercial CD19 CAR-T cell therapy with direct correlation between antigen density and cytotoxicity. It significantly prolonged the survival of an animal model established in NOD-SCIDγc−/− (NSG) mice engrafted with a human MCL cell line Mino subcutaneously compared to controls. Finally, while CD74 is also expressed on normal immune cell subsets, treatment with 74bbz CAR-T cells resulted in minimal cytotoxicity against these cells both in vitro and in vivo.ConclusionsTargeting CD74 with 74bbz CAR-T cells represents a new cell therapy to provide a potent and durable and anti-MCL activity.

Use of locally produced anti-CD19 CAR-T cells in the treatment of relapsed/refractory B-cell lymphomas in adults

Background. Patients with B-cell lymphoma have an extremely unfavorable prognosis after relapse or in case of refractoriness to the first and consecutive lines of immunochemotherapy with the anti-CD19 CAR-T cells being the only therapeutic option to such patients. the manual preparation of anti-CD19 CAR-T lymphocytes was reproduced in the N. N. Alexandrov republican research and practical center for oncology and medical radiology (Minsk). Their safety was demonstrated. Aim. To estimate safety, tolerability and efficacy of the in-house CAR-T cells, including objective response rate, progression-free and overall survival. Materials and methods. The second generation anti-CD19 chimeric antigen receptor contained an anti-CD19 antibody scFv fragment, CD28 transmembrane domain, 4-1BB and CD3z signaling domains. the coding sequence was cloned into the lentiviral vector S4. The cell product was obtained by expansion of CD4- and CD8-positive lymphocytes populations with IL-7 and IL-15 after initial activation and lentiviral transduction with vector S4. CAR-T cells were infused into 8 patients with refractory forms of B-cell lymphoma after the preliminary lymphodepleting chemotherapy. Persistence of CAR-T cells was assessed by flow cytometry. therapeutic efficiency was assessed by positron emission tomography-computed tomography with 18F-fluorodeoxyglucose. Results. Expansion of CAR-T cells with resulting b-cell aplasia was observed in all patients. the median of observation was 113 days (range 22–529 days). objective response rate was 100 %, complete remission was observed in 6 patients, partial response – in 1 patient. One patient died because of complications before the clinical response. Overall survival was 88 ± 12 %. cytokine release syndrome and neurotoxicity were not observed in 6 out of 8 patients despite a high tumor burden. Conclusion. Our study demonstrated efficiency and safety of the in-house CAR-T cells for the treatment of patients with refractory B-cell lymphomas.

Ligand-based adoptive T cell targeting CA125 in ovarian cancer

BackgroundOvarian cancer (OC) is a highly aggressive gynecological malignancy prevalent worldwide. Most OC cases are typically diagnosed at advanced stages, which has led to a 5-year overall survival rate of less than 35% following conventional treatment. Furthermore, immune checkpoint inhibitor therapy has shown limited efficacy in the treatment of patients with OC, and CAR-T therapy has also demonstrated modest results owing to inadequate T cell infiltration. Therefore, novel strategies must be developed to enhance T cell persistence and trafficking within the OC tumor microenvironment.MethodsIn this study, we developed a novel adoptive T-cell therapy for ovarian cancer based on a chimeric antigen receptor structure. We used a ligand-receptor binding motif to enhance the therapeutic effect of targeting CA125. Since mesothelin can naturally bind to CA125 with high affinity, we concatenated the core-binding fragment of mesothelin with the 4-1BB and CD3ζ signal fragments to assemble a novel CA125-targeting chimeric receptor (CR). The CAR structure targeting CA125 derived from the 4H11 antibody was also constructed. CR- and CAR-encoding RNA were electroporated into T cells to evaluate their antitumor activity both in vitro and in vivo.ResultsWhile CR-T or CAR-T cells exhibited moderate activity against two ovarian cancer cell lines, T cells co-expressing CR and CAR exhibited a superior killing effect compared to T cells expressing either CR or CAR alone. Furthermore, upon interaction with ovarian tumors, the ability of CR and CAR T cells to release activation markers and functional cytokines increased significantly. Similarly, CR and CAR co-expressing T cells persistently controlled the growth of transplanted ovarian cancer tumors in NSG mice and significantly prolonged the overall survival of tumor-challenged mice. Transcriptome sequencing revealed that the survival and cytotoxicity of T cells co-expressing CR and CAR were significantly altered compared with those of T cells expressing either CR or CAR.ConclusionOur findings demonstrate that CA125 targeting CR and CAR can synergistically kill ovarian cancer cells, indicating that CA125 targeting by the two binding motifs simultaneously in tumors may improve the therapeutic outcomes of ovarian cancer treatment.

A NOVEL MSLN×4-1BB BISPECIFIC ANTIBODY FOR SOLID TUMOR

Abstract Background Mesothelin (MSLN) is a 70 KD glycosylphosphatidylinositol (GPI)-anchored cell surface glycoprotein that is rarely expressed in normal tissues but overexpressed in a variety of cancers, including mesothelioma, ovarian cancer, pancreatic cancer and breast cancer et.al. 4-1BB is a member of the tumor necrosis factor receptor superfamily that functions as a co-stimulatory molecule. Agonistic antibodies bind to 4-1BB, triggering a signaling cascade leading to T-cell activation and expansion of cytotoxic CD8+ T lymphocytes. Here, we developed two bispecific antibodies (bsAbs) targeting both MSLN and 4-1BB with an intact Fc fragment from human IgG1 or IgG4, named HK013-G1 and HK013-G4 respectively. We suspected that HK013-G1 can simultaneously exert the cytotoxic effect of CD8+T cells and NK cells on tumor cells expressing MSLN to achieve better antitumor efficacy. Methods Both HK013-G1 and HK013-G4 were constructed by fused a single-chain variable fragment (scFv) targeting hu4-1BB to the C terminus of an anti-MSLA nanobody. And their affinity was optimized to making it highly effective in tumor localization. Next, we tested the killing ability of bsAbs-mediated PBMC or NK92 against tumor cells with different expression levels of MSLN in vitro. And the IFN-γ secretion was detected when CD8+T cells co-cultured with MSLN+ or MSLN- cells in the presence of antibodies. Also, the 4-1BB agonist activity of bsAbs was measured in a luciferase report gene assay. To confirm the safety of HK013-G1, non-specific activation of 4-1BB signal mediated by Fc receptor and CRS was evaluated in vitro. Finally, we compared the antitumor activity of two bispecific antibodies in both MC38/hMSLN and CT26/hMSLN tumor model and hepatotoxicity as well as cardiotoxicity was evaluated. Results Affinity-optimized HK013-G1 has an order of magnitude greater affinity for MSLN(KD≈10−9M) than 4-1BB (KD≈10−8M). HK013-G1 induced stronger PBMC against tumor cells than MOARb009 while HK013-G4 does not. Also, HK013-G1 could only mediate the killing of NK92 on MSLN-positive tumor cells. In co-cultured assay, HK013-G1 had superior ability to stimulate CD8+T cell secretion of IFN-γ than urelumab in the presence of MSLN. In luciferase reporter assay, the bsAbs-induced 4-1BB activation is dependent on expression level of MSLN. In addition, HK013-G1 was shown no stronger ability to inducing non-specific activation of 4-1BB signal mediated by Fc receptor and CRS in vitro. Compared with HK013-G4, HK013-G1 showed a more significant anti-tumor effect in both MC38/hMSLN and CT26/hMSLN tumor model. And, HK013-G4 showed significant hepatotoxicity in mice while HK013-G1 not. Moreover, HK013-G1 can protect mice against tumor re-challenge. Conclusions HK013-G1, an MSLN×4-1BB bsAb with human IgG1 Fc fragment, prevents tumor development by killing tumor cells directly via effector functions mediated by NK and cytotoxic T cells. More importantly, HK013-G1 showed no stronger toxic side effects both in vitro and in vivo. These results show that HK013-G1 has the potential to develop into a new clinical therapy for cancer types with MSLN expression.