Targeting CD47 Research Articles

Targeting CD73 to Overcomes Resistance to First-Generation EGFR Tyrosine Kinase Inhibitors in Non-Small Cell Lung Cancer.

In patients with epidermal growth factor receptor (EGFR)-mutant non-small cell lung cancer (NSCLC), EGFR tyrosine kinase inhibitors (TKIs) improve response rate and survival. However, most patients eventually develop resistance. This study aimed to identify the role of CD73 in EGFR-mutant NSCLC and explore whether CD73 inhibition may serve as a therapeutic strategy in NSCLC patients with acquired resistance to EGFR-TKIs. We evaluated the prognostic role of CD73 expression in EGFR-mutant NSCLC using tumor samples from a single institution. We silenced CD73 in EGFR-TKI-resistant cell lines using short hairpin RNA (shRNA) targeting CD73 and also transfected a vector alone as a negative control. Using these cell lines, cell proliferation and viability assays, immunoblot assays, cell cycle analysis, colony-forming assays, flow cytometry, and apoptosis analysis were performed. High expression of CD73 was associated with shorter survival in patients with metastatic EGFR-mutant NSCLC treated with first-generation EGFR-TKI. CD73 inhibition synergistically inhibited cell viability with first-generation EGFR-TKI treatment compared with the negative control. When CD73 inhibition and EGFR-TKI treatment were combined, G0/G1 cell cycle arrest was induced through the regulation of p21 and cyclin D1. In addition, the apoptosis rate was increased in CD73 shRNA-transfected cells treated with EGFR-TKI. High expression of CD73 adversely affects the survival of patients with EGFR-mutant NSCLC. The study demonstrated that inhibiting CD73 in EGFR-TKI-resistant cell lines resulted in increased apoptosis and cell cycle arrest, which overcame the acquired resistance to first-generation EGFR-TKIs. Further research is needed to determine whether blocking CD73 plays a therapeutic role in EGFR-TKI-resistant patients with EGFR-mutant NSCLC.

Bicistronic CAR-T cells targeting CD123 and CLL1 for AML to reduce the risk of antigen escape

PurposeAcute myeloid leukemia (AML) is a highly heterogeneous neoplastic disease with a poor prognosis that relapses even after its treatment with chimeric antigen receptor (CAR)-T cells targeting a single antigen. CD123 and CLL1 are expressed in most AML blasts and leukemia stem cells, and their low expression in normal hematopoietic stem cells makes them ideal targets for CAR-T. In this study, we tested the hypothesis that a new bicistronic CAR targeting CD123 and CLL1 can enhance antigenic coverage and prevent antigen escape and subsequent recurrence of AML. MethodsCD123 and CLL1 expressions were evaluated on AML cell lines and blasts. Then, in addition to concentrating on CD123 and CLL1, we introduced the marker/suicide gene RQR8 with a bicistronic CAR. Xenograft models of disseminated AML and in vitro coculture models were used to assess the anti-leukemia efficacy of CAR-T cells. The hematopoietic toxicity of CAR-T cells was evaluated in vitro by colony cell formation assays. It was demonstrated in vitro that the combination of rituximab and NK cells caused RQR8-mediated clearance of 123CL CAR-T cells. ResultsWe have successfully established bicistronic 123CL CAR-T cells that can target CD123 and CLL1. 123CL CAR-T cells effectively cleared AML cell lines and blasts. They also demonstrated appreciable anti-AML activity in animal transplant models. Moreover, 123CL CAR-T cells can be eliminated in an emergency by a natural safety switch and don't target hematopoietic stem cells. ConclusionsThe bicistronic CAR-T cells targeting CD123 and CLL1 may be a useful and secure method for treating AML.

Targeting CD70 using CAR NK cells to enhance NK cells cytolytic effect against osteosarcoma

Abstract Background: Cure rates for osteosarcoma (OS) have not improved in >30 years and no new effectivetherapies have been identified for metastatic disease. Natural killer (NK) cells have become an attractive tool for cancer immunotherapy. Efficay is improved by direct tumor targeting introducing a specific chimeric antigen receptor (CAR). Emerging knowledge suggests CD70 to be a viable candidate as expression is higher in lung metastases and it has been correlated with tumor escape from immune surveillance. Objective: To assess whether using CD70 CAR-NK cells we can enhance trafficking, homing and proliferation of NK cells and therefore therapeutic efficacy against OS. Methods: We analyzed the cBioportal database to verify CD70 expression in OS. Surface expression of CD70 on human OS cell lines and normal osteoblasts was determined by flow cytometry. CD70 CAR constructs were sequenced, verified and transduced into NK cells. CD70 CAR NK and control NK cells cytolytic activity against OS cells was evaluated by IncuCyte. In vitrocytokine release was measured upon CD70 CAR NK/NK cells exposure to OS cells (IsoPlexis). Results: Database analysis confirmed OS CD70 gene amplification. Flow cytometry showed variable expression of CD70 on OS cells and no expression on human osteoblasts. There was an increase in CD70 CAR NK percent lysis against OS cells at the highest effector:target ratios compared to control NK cells. We found a predominant release in the chemoattractive/stimulatory cytokines upon OS cells exposure to CD70 CAR NK cells. Conclusion: CD70 has immunotherapy potential against OS. CD70 CAR NK cells have increased cytolytic activity against OS cells in vitro. Cytolytic activity may be influenced by the release of specific cytokines.

CD40-dependent mechanism of virus control in a mouse model of HBV infection

Abstract An estimated 296 million people worldwide currently live with chronic hepatitis B virus (HBV) infection. Up to 90% of newborns and 30% of children with acute hepatitis B develop chronic infections, causing lifelong complications including liver cirrhosis and cancer. HBV persistence is a consequence of T and B cell dysfunction, so immunotherapy has been proposed as a means of HBV control. TNF receptor superfamily member CD40 has shown therapeutic value in overriding T cell exhaustion and inducing lymphocyte responses in solid tumors and viral infections. To determine the efficacy of CD40 targeting in generating HBV-resolving immune responses, mice were simultaneously transduced with adeno-associated virus encoding the HBV genome (AAV-HBV) and treated with the agonistic CD40 antibody FGK4.5 to activate CD40 signaling. AAV-HBV mice receiving FGK4.5 saw a transient reduction in HBV as determined by serum antigen analysis, while antigen levels in CD4-depleted mice treated with FGK4.5 became undetectable over seven weeks. HBV control was strongly associated with the generation of HBV-specific CD8 +T cells, increased serum ALT, and elevated expression of intrahepatic CD8 +T cell-related genes. Histological analysis of the liver revealed loss of hepatitis B core antigen-positive hepatocytes and increased inflammatory foci. Neither Treg cell depletion nor checkpoint inhibition in CD4-replete mice affected HBV control. Lastly, CD40 activation in CD4-depleted chronically infected mice also reduced both serum antigenemia and liver HBV RNA. Taken together, these results implicate a critical role of CD4 +T cells in maintaining HBV immune tolerance and further suggest CD40 as a potential therapeutic target for chronic HBV infection. Supported by the National Institute of Allergy and Infectious Diseases of the National Institutes of Health under award numbers R01AI124006 and R01AI148354. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.

Plasmacytoid dendritic cell neoplasms.

Plasmacytoid dendritic cells (pDCs) are type I interferon-producing cells that modulate immune responses. There are two types of pDC neoplasms: 1) mature pDC proliferation (MPDCP) associated with myeloid neoplasm and 2) blastic pDC neoplasm (BPDCN). MPDCP is a clonal expansion of mature pDCs that is predominantly associated with chronic myelomonocytic leukemia. In contrast, BPDCN is a clinically aggressive myeloid malignancy involving the skin, bone marrow, lymphatic organs, and central nervous system. There are various types of skin lesions, ranging from solitary brown or violaceous to disseminated cutaneous lesions, which often spread throughout the body. The expression of CD4, CD56, CD123, and pDC markers (TCL-1, TCF4, CD303, and CD304, etc.) are typical immunophenotype of BPDCN. Historically, BPDCN treatment has been based on acute leukemia regimens and allogeneic hematopoietic cell transplantation in selected patients. Recent advances in molecular biology and genetics have led to the development of targeted agents, such as tagraxofusp (a recombinant fusion protein targeting CD123), anti-CD123 CAR-T cells, XmAb14045, and IMGN632. Lastly, this review provides a comprehensive overview of pDC neoplasms.

Site-specific 68Ga-labeled nanobody for PET imaging of CD70 expression in preclinical tumor models

BackgroundCD70-CD27 is a costimulatory ligand-receptor pair in the tumor necrosis factor receptor family. With only limited expression in normal tissues, CD70 is constitutively expressed in a variety of solid tumors and hematologic malignancies, facilitating immunosuppression through CD27 signaling in the tumor microenvironment by enhanced survival of regulatory T cells, induction of T cell apoptosis, and T cell exhaustion. Consequently, CD70 is an increasingly recognized target for developing antibody-based therapies, but its expression patterns vary among different tumor types in spatial distribution, magnitude of expression and percentage of positive cells. In that regard, individual confirmation of CD70 expression at screening and during treatment could enhance the successful implementation of anti-CD70 therapies. Here, we developed a gallium-68 (68Ga) radiolabeled single-domain antibody-fragment targeting CD70 for in vivo positron emission tomography (PET) imaging.ResultsAn anti-CD70 VHH construct containing a C-direct-tag with a free thiol was developed to enable site-specific conjugation to a NOTA bifunctional chelator for 68Ga radiolabeling. [68Ga]Ga-NOTA-anti-CD70 VHH was obtained in good radiochemical yield of 30.4 ± 1.7% and high radiochemical purity (> 94%). The radiolabeled VHH showed excellent in vitro and in vivo stability. Specific binding of [68Ga]Ga-NOTA-anti-CD70 VHH was observed on CD70high 786-O cells, showing significantly higher cell-associated activity when compared to the blocking condition (p < 0.0001) and CD70low NCl-H1975 cells (p < 0.0001). PET imaging showed specific radiotracer accumulation in CD70 expressing human tumor xenografts, which was efficiently blocked by prior injection of unlabeled anti-CD70 VHH (p = 0.0029). In addition, radiotracer uptake in CD70high tumors was significantly higher when compared with CD70low tumors (p < 0.0001). The distribution of the radioactivity in the tumors using autoradiography was spatially matched with immunohistochemistry analysis of CD70 expression.Conclusion[68Ga]Ga-NOTA-anti-CD70 VHH showed excellent in vivo targeting of CD70 in human cancer xenografts. PET imaging using this radioimmunoconjugate holds promise as a non-invasive method to identify and longitudinally follow-up patients who will benefit most from anti-CD70 therapies.

Design of a novel chimeric peptide via dual blockade of CD47/SIRPα and PD-1/PD-L1 for cancer immunotherapy.

Although immune checkpoint inhibition has been shown to effectively activate antitumor immunity in various tumor types, only a small subset of patients can benefit from PD-1/PD-L1 blockade. CD47 expressed on tumor cells protects them from phagocytosis through interaction with SIRPα on macrophages, while PD-L1 dampens T cell-mediated tumor killing. Therefore, dual targeting PD-L1 and CD47 may improve the efficacy of cancer immunotherapy. A chimeric peptide Pal-DMPOP was designed by conjugating the double mutation of CD47/SIRPα blocking peptide (DMP) with the truncation of PD-1/PD-L1 blocking peptide OPBP-1(8-12) and was modified by a palmitic acid tail. Pal-DMPOP can significantly enhance macrophage-mediated phagocytosis of tumor cells and activate primary T cells to secret IFN-γ in vitro. Due to its superior hydrolysis-resistant activity as well as tumor tissue and lymph node targeting properties, Pal-DMPOP elicited stronger anti-tumor potency than Pal-DMP or OPBP-1(8-12) in immune-competent MC38 tumor-bearing mice. The in vivo anti-tumor activity was further validated in the colorectal CT26 tumor model. Furthermore, Pal-DMPOP mobilized macrophage and T-cell anti-tumor responses with minimal toxicity. Overall, the first bispecific CD47/SIRPα and PD-1/PD-L1 dual-blockade chimeric peptide was designed and exhibited synergistic anti-tumor efficacy via CD8+ T cell activation and macrophage-mediated immune response. The strategy could pave the way for designing effective therapeutic agents for cancer immunotherapy.

G protein-coupled receptor 183 mediates the sensitization of Burkitt lymphoma tumors to CD47 immune checkpoint blockade by anti-CD20/PI3Kδi dual therapy.

Immunotherapy-based regimens have considerably improved the survival rate of B-cell non-Hodgkin lymphoma (B-NHL) patients in the last decades; however, most disease subtypes remain almost incurable. TG-1801, a bispecific antibody that targets CD47 selectively on CD19+ B-cells, is under clinical evaluation in relapsed/refractory (R/R) B-NHL patients either as a single-agent or in combination with ublituximab, a new generation CD20 antibody. A set of eight B-NHL cell lines and primary samples were cultured in vitro in the presence of bone marrow-derived stromal cells, M2-polarized primary macrophages, and primary circulating PBMCs as a source of effector cells. Cell response to TG-1801 alone or combined with the U2 regimen associating ublituximab to the PI3Kδ inhibitor umbralisib, was analyzed by proliferation assay, western blot, transcriptomic analysis (qPCR array and RNA sequencing followed by gene set enrichment analysis) and/or quantification of antibody-dependent cell death (ADCC) and antibody-dependent cell phagocytosis (ADCP). CRISPR-Cas9 gene edition was used to selectively abrogate GPR183 gene expression in B-NHL cells. In vivo, drug efficacy was determined in immunodeficient (NSG mice) or immune-competent (chicken embryo chorioallantoic membrane (CAM)) B-NHL xenograft models. Using a panel of B-NHL co-cultures, we show that TG-1801, by disrupting the CD47-SIRPα axis, potentiates anti-CD20-mediated ADCC and ADCP. This led to a remarkable and durable antitumor effect of the triplet therapy composed by TG-1801 and U2 regimen, in vitro, as well as in mice and CAM xenograft models of B-NHL. Transcriptomic analysis also uncovered the upregulation of the G protein-coupled and inflammatory receptor, GPR183, as a crucial event associated with the efficacy of the triplet combination. Genetic depletion and pharmacological inhibition of GPR183 impaired ADCP initiation, cytoskeleton remodeling and cell migration in 2D and 3D spheroid B-NHL co-cultures, and disrupted macrophage-mediated control of tumor growth in B-NHL CAM xenografts. Altogether, our results support a crucial role for GPR183 in the recognition and elimination of malignant B cells upon concomitant targeting of CD20, CD47 and PI3Kδ, and warrant further clinical evaluation of this triplet regimen in B-NHL.

Abstract 1873: D3L-001, a novel bispecific antibody targeting HER2 and CD47, demonstrates potent preclinical efficacy in solid tumors

Abstract Background: HER2 is overexpressed in different solid tumors, including 10-20% breast and gastric cancers. Many HER2-targeted drugs, including trastuzumab, pertuzumab, lapatinib, trastuzumab emtansine (T-DM1), and trastuzumab deruxtecan (T-DXd), have been approved for treatment of HER2-positive early and metastatic breast cancer (BC). However, trials involving pertuzumab, lapatinib and T-DM1 have failed to provide significant clinical improvements in patients with HER2-positive gastric cancer (GC), suggesting differences in HER2 mediated disease biology between HER2-positive BC and GC. Recent studies have suggested efficacy of HER2 antibodies could be further enhanced by antibody-dependent cellular phagocytosis (ADCP) principally regulated by “don’t-eat-me” CD47 signals, which is overexpressed in many HER2-positive cancers and suppresses phagocytosis through binding to SIRPα. Methods: BC and GC cell lines were used. Monocytes were isolated from human PBMC and differentiated into macrophages, which were used for in vitro ADCP assays. In in vivo animal experiments, D3L-001 and comparator antibodies were examined for efficacy in solid tumor xenograft models. Results: D3L-001 was designed to have higher HER2 affinity (KD &amp;lt; 1 nM) than that of CD47 (KD &amp;gt;10 nM). With this unique design, D3L-001 showed preferential binding to HER2/CD47 double positive tumor cells as compared to CD47 single positive cells. D3L-001 showed better anti-tumor effect than trastuzumab in a panel of solid tumor models, including but not limited to BC and GC cell lines or patient derived xenografts. Immunohistochemistry (IHC) analysis was performed in tumor FFPE slides to evaluate expression of HER2 and CD47 in the efficacy tested models, and we observed that D3L-001 showed broad tumor growth inhibition in HER2 or CD47 high/low expression models. In both in vitro and in vivo studies using breast cancer HCC1954, JIMT-1 and gastric cancer N87 models, we observed a synergistic effect in the combination of Trastuzumab with Magrolimab. Moreover, D3L-001 showed a trend of better efficacy than the combination of Trastuzumab and Magrolimab. These results confirm the synergistic effect of dual-targeting HER2 and CD47 simultaneously and suggest advantage of using bispecific antibodies to confer such effect. D3L-001 also displayed potent and comparable in vivo efficacy as Enhertu in gastric N87 model, and it showed synergistic in vivo efficacy, when combining with Pertuzumab or Paclitaxel, in breast HCC1954, JIMT-1 and gastric cancer N87 models. Conclusion: D3L-001, a novel HER2×CD47 bsAb, demonstrated potent anti-tumor effect in a variety of solid tumor models via HER2 guided CD47 co-blocking. It also showed synergistic efficacy when combining with other anti-tumor agents. D3L-001 will be further evaluated in clinic for its potential as a novel treatment option in HER2-positive BC, GC and other solid tumors. Citation Format: Haopeng Rui, Xiaofeng Yang, Jiahao Chen, Jia Wang, Zhiqiang Zheng, Zhiqiang Chen, Jingtao Lu, Peggy Wu, Janet Chen, Allison Wang, George Chen. D3L-001, a novel bispecific antibody targeting HER2 and CD47, demonstrates potent preclinical efficacy in solid tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1873.

Abstract 6341: Preclinical development of a monoclonal antibody targeting CD70 as cancer immunotherapy

Abstract CD70 is a novel immune checkpoint highly expressed on cancers of kidney, blood and lymphoid tissues, mesothelium and gastrointestinal system. High expression of CD70 is a prognostic marker for poor survival in patients with ovarian carcinoma, pancreatic cancer and mesothelioma, etc. Several CD70 antibodies are under early stages of clinical trials for oncology indications. We have developed IMM40H, a humanized IgG1 antibody that differentially induces phagocytosis and cytotoxicity in CD70-expressing cancer cells. Results of in vitro study have demonstrated that, in comparison with cusatuzumab, IMM40H possesses better CD70 binding (kD= 3.22E-11M) and functional inhibition ability, as well as more potent activities of ADCC (antibody-dependent cellular cytotoxicity, EC50 = 0.5 -2.0ng/ml) and CDC (complement-dependent cytotoxicity, EC50 = 170 ng/mL) against certain cancer cells. IMM40H also induces strong ADCP (antibody-dependent cellular phagocytosis) activity (EC50 = 0.02nM). At doses between 0.3 mg/kg to 10mg/kg, IMM40H significantly reduced tumor volume in mouse models of multiple myeloma, lymphoma and kidney cancer. Notably, in a U266 multiple myeloma model, IMM40H, at the dose of 0.3 mg/kg, completely inhibited tumor growth in a way that therapeutic effects appeared significantly earlier than that of cusatuzumab at a higher dose of 1 mg/kg. Therapeutic effects of IMM40H at the dose of 3 mg/kg also appeared earlier than that of bortezomib at the dose of 0.5 mg/kg. Interestingly, combination of IMM40H and IMM01 (a SIRPα-IgG1 Fc fusion protein targeting CD47) generated significant therapeutic synergy in models of A498 kidney cancer and Raji lymphoma. Importantly, maximum tolerated dose of IMM40H exceeds 100 mg/kg (IV, single dose) in cynomolgus monkeys and no significant adverse reaction was observed at a dose range of 3 to 30 mg/kg (IV, QW, 5 times) in monkeys. Taken together, our data show that targeting CD70 on tumor cells using IMM40H antibody may serve as a potent immunotherapy for multiple cancer indications. IND of IMM40H has been approved in China and US. This antibody is now in a phase 1 study in patients with advanced malignancies expressing CD70. Citation Format: Wenzhi Tian, Song Li, Dianze Chen, Dandan Liu, Huiqin Guo, Chunmei Yang, Li Zhang, Wei Zhang, Xiaoping Tu, Liang Peng, Gui Zhao, Ruliang Zhang, Fan Zhang, Frank X. Gan. Preclinical development of a monoclonal antibody targeting CD70 as cancer immunotherapy. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6341.

Abstract 6345: Non-clinical pharmacology of HX009, a novel FIC PD1xCD47 BsAb

Abstract Immune checkpoint inhibitors (ICIs) have been widely tested as cancer therapeutics but with respective limitations such as marginal activity and/or poor safety profiles. It is possible that some of these limitations could be overcome by specially engineered molecules, e.g. bi-functional agents. Here we report a novel rationally designed bi-specific antibody (BsAb) targeting both PD1 and CD47, HX009, in order to improve both efficacy and safety over the two respective single-targeting ICIs. HX009 features IgG4 and 2 × 2 symmetric format, but with specifically weakened CD47 binding affinity. We performed a comprehensive preclinical pharmacology characterization of HX009 to test our hypothesis by confirming its candidacy as a novel first-in-class biologic for cancer therapy. The affinity constant (KD) of the HX009 binding to PD1 was determined to 3.96 × 10−11 M, while 5.06 × 10−9 M for binding to CD47, significantly reduced affinity as compared to the binding to PD1 and the binding between native SIRPα and CD47. HX009 has little binding to red blood cell (RBC) and platelets so to minimize hematological toxicity that usually associated with CD47 targeting. The antibody injectable formulation caused little cytokine release from PBMC (CRS or cytokine storm). HX009 cross-species binds to cynomolgus monkey PD1 and CD47 receptors, with the similar affinity as with the counterparts of human, but not to those of rodents. Cynomolgus monkey was thus chosen to investigate non-clinical pharmacokinetic (PK) and toxicology profiles of HX009. The intravenous (IV) infusion PK study revealed that HX009 is largely distributed within vasculature, with terminal half-life (T1/2) around 50hr and dose proportional exposure. Repeat dosing caused no accumulation. The production of antidrug antibody (ADA) were observed in the dosed animals, as expected for administration into different species, which impacted PK parameters of repeated dosing. The IV single dose toxicology study revealed the maximal tolerated dose (MTD) of 150mg/kg with 14-day observation, while the maximum non-severely toxic dose (HNSTD) was determined to be 15 mg/kg in the repeated dose study after once weekly dose for 4 consecutive weeks (5 times total). The main drug related toxicities observed seemed to be related to CD47 targeting as expected. Strong antitumor activity was also confirmed in in several preclinical cancer models, including humanized syngeneic models. In summary, the desired PK and safety profiles of HX009, along with anti-tumor activity, supporting further clinical development. Currently, HX009 is under clinical investigation (ClinicalTrials.gov Identifier: NCT05189093). Citation Format: Faming Zhang, Hang Ke, Feiyu Peng, Cen Chen, Lei Zhang, Henry Qixiang Li. Non-clinical pharmacology of HX009, a novel FIC PD1xCD47 BsAb [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6345.

Abstract 2980: HX009, a first-in-class PD1xCD47 BsAb, demonstrated anti-AML activity in PDX models

Abstract Acute myeloid leukemia (AML) is an aggressive leukemia of myeloid lineage with different subtypes (global annual mortality ~150,000). The first-line treatment is usually induction chemotherapies, followed by further chemo-/radiation therapies or stem cell transplant, etc. Immune checkpoint inhibitor (ICIs), e.g. PD1 blocker, although a great success for treating many solid tumors, has yet to show convincing activity in AML. CD47, a “don’t eat me” receptor frequently found on the surface of tumor cells, including hematological malignances such as AML, prevents phagocytosis by interacting to its ligand, SIRPα, on the surface of macrophages. CD47 is also now shown as a key immune checkpoint for both innate and adoptive immunity (Liu et al., 2018). CD47 targeting, actively being tested for MSD and AML in clinics, encounters challenges for the reported toxicities (e.g. anemia/thrombocytopenia) as well as inadequate efficacy (Feng et al., 2019). Majority of investigational CD47 targeting agents in the clinics are combination therapies with other modality in order to increase the likelihood of success. To this end, it is prudent to develop agents with dual targeting agents, particularly for both the innate and adoptive immune checkpoints. We describe a novel specially designed/constructed bi-specific antibody (BsAb), HX009, targeting CD47 and PD1 for the treatment of cancers. We tested HX009 in several preclinical AML-PDXs (patient-derived xenograft models) via systemic engraftment: AM8096 (CD47lo, H-score of 55 by IHC), AM7577 (CD47hi, H-score of 237)(An et al., 2017) and AM5512 (CD47hi, H-score of 198). All three PDXs responded to 10mg/kg twice weekly dose of HX009 (IP) as measured either by leukemic burden in peripheral blood at different timepoints, and in both spleen and bone marrow at the termination, or by survival. Since there is no T-cell function in xenograft models, all anti-leukemic activities observed should be attributed to the CD47 blockade, not PD1 blockade. AM7577 model responded significantly better than AM8096, likely due to the significantly higher expression of CD47 and also better than Ara-C standard of care (SOC) treatment at 3mg/kg. In contrast, AM8096 responded even less to HX009 than Ara-C. In contrast, in the subcutaneous transplanted AML cell line-derived model, MV4-11, there is little anti-tumor activity observed, although there is significant expression of CD47 on the tumor cells. In conclusion, our data seems to suggest that HX009 could be a candidate immunotherapy for CD47hi AML, with CD47 expression being a positive predictive biomarker, warranting further evaluation. Citation Format: Xiaoyu An, Jinping Liu, Hang Ke, Lingxin Xiong, Xiaolong Tu, Lei Zhang, Faming Zhang, Henry Li. HX009, a first-in-class PD1xCD47 BsAb, demonstrated anti-AML activity in PDX models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2980.

Abstract 1751: Prognostic value of CD20/CD73 ratio in sensitive and relapsed/refractory diffuse large B-cell lymphoma cell lines

Abstract Purpose: Developing novel therapy strategies to treat relapsed/refractory diffuse large B-cell lymphoma (r/r DLBCL) is critically needed. Previous studies in our group found despite dysregulation of apoptotic pathway in resistant DLBCL, upregulation in glycose metabolism and energetic ATP production were also found in r/r DLBCL. Therefore, decreasing or blockage ATP production may provide a novel method to overcome resistance in lymphoma. ATP/ADP is hydrolyzed into AMP by NTPDases, and AMP into adenosine and phosphate by ecto-5’-nucleotidase, known as CD73. CD73 is the rate limiting enzyme anchored the surface of tumor and immune cell. The expression level of CD73 on the tumor was reported significantly associated with inferior prognosis in variety of cancers. Moreover, expression level of CD73 on immune cell has been shown immunosuppression in multiple cancers. Targeting CD73 by monoclonal antibody or small inhibitor currently underwent multiple clinical trials. However, the role of CD73 in r/r DLBCL and the relationship between CD73 and CD20 have not been explored. Methods: Germinal center B-cell like, activated B-cell like lymphoma, Burkitt’s lymphoma and rituximab resistant cell lines generated in our lab were used. Surface expression of CD73 and CD20 were determined by staining anti-CD73 FITC and anti-CD20 Per-cy5.5 antibodies and running flow cytometry. The expression levels of CD73 and CD20 were detected by media fluorescence index (MFI). The CD20/CD73 ratio was calculated by CD73 MFI/CD20 MFI. Result: Previously our group found surface CD20 levels exhibited a linear correlation with rituximab induced cellular mediated cytotoxicity. The CD20 MFI of germinal center B-cell like, activated B-cell like lymphoma, Burkitt’s lymphoma and rituximab resistant cell lines were: DHL4 8652, DOHH2 6472, TMD8 3981, RL 2849, Raji 2011, U2932 604, RL 4RH 599 and Raji 4RH 576. The higher MFI of CD20, the more sensitive to rituximab treatment. Here we discovered that the surface level of CD73 had an inverse correlation with CD20 level in these cell lines. In detail, CD73 MFI were: DHL4 464, DOHH2 387, TMD8 363, RL 299, Raji 611, U2932 486, RL 4RH 631 and Raji 4RH 702. The CD20/CD73 ratio of cell lines from high to low were DOHH2, DHL4, TMD8, RL, Raji, U2932, Raji 4RH and RL 4RH, respectively. This CD20/CD73 ratio in each of cell lines was found more straight forward to represent the sensitivity of rituximab or chemo drug treatment in vitro condition. Conclusion: Our study was first time to reveal the association between CD20/CD73 ratio and immune-chemo response in lymphoma cell lines, especially in resistant lymphoma in vitro condition. This may give us prognostic value of CD20/CD73 ratio in predicting lymphoma response to immune/chemotherapy in clinical setting. Further study to find CD20 and CD73 association in the clinical setting and mechanism of action need to be warranted. Citation Format: Juan J. Gu, Cory Mavis, Jessica Wang, Taylor Mandeville, Francisco Hernandez-Ilizaliturri. Prognostic value of CD20/CD73 ratio in sensitive and relapsed/refractory diffuse large B-cell lymphoma cell lines [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1751.

Abstract 2923: JAB-X1800: A potent immunostimulatory antibody-drug conjugate (iADC) targeting CD73

Abstract Background: STING emerges as a key immune sensor and activated STING pathway plays a key role in innate and adaptive immune response. STING agonist has shown promising potency to stimulate anti-tumor immunity. Considering immune-related toxicity due to systemic administration, as well as difficulty in intra-tumor administration, targeted delivery of STING agonist by iADC is warranted. We developed CD73-STING iADC JAB-X1800, which uses anti-CD73 mAb for targeted delivery of payload JAB-27670, a highly potent non-cyclic dinucleotide STING agonist. Method: Binding assay and HEK293-STING-luciferase reporter assay were performed to test affinity of JAB-27670. Tumor cells with CD73 expression and THP-1 cells were co-cultured in the presences of iADC JAB-X1800 to assess the IFN-β production. The cell killing activity of iADC JAB-X1800 was evaluated when cancer cells co-cultured with human PBMCs. Furthermore, human and mouse plasma stability of iADC JAB-X1800 was assessed. Finally, the anti-tumor effects of iADC JAB-X1800 alone or in combination with anti-PD-1 were evaluated in animal models. Results: JAB-27670 is a highly potent STING agonist with sub-nanomolar EC50 in binding and luciferase reporter assays. In CD73-expressing cancer cells and THP-1 co-culture experiments, iADC JAB-X1800 induced IFN-β production with significant improvement compared with the free payload. JAB-X1800 also induced potent killing in vitro. JAB-X1800 is stable in human and mouse plasma. A single dose of JAB-X1800 induced tumor regression in MDA-MB-231 xenograft model and hCD73-MC38 syngeneic model. JAB-X1800 conferred immunologic memory when tumor free mice were re-challenged. JAB-X1800 showed synergistic anti-tumor activity with anti-PD-1 in hCD73-MC38 syngeneic model. Conclusions: The CD73-targeted STING agonist iADC JAB-X1800 demonstrated high stability in plasma, robust activity, and good tolerability in animal models, supporting clinical development of JAB-X1800 alone and in combination with anti-PD-1 for patients with CD73-expressing tumors. Citation Format: Yang Ye, Guiqun Yang, Hongling Wang, Xin Sun, Yanping Wang, Fashun Yan, Lei Wang, Wei Long. JAB-X1800: A potent immunostimulatory antibody-drug conjugate (iADC) targeting CD73 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2923.

Abstract 2715: Utilizing a pH sensitive peptide (pHLIP) for tumor targeted delivery of an immunogenic peptide motif

Abstract Chimeric antigen receptor (CAR) T cell and immune-cell therapies are revolutionizing how patients are treated in the clinic by targeting tumor-specific antigens. Specifically, advances in antigen targeting of CD20 and CD19 with CAR T cell therapy has increased overall survival of this patient population and has influenced to expand T cell repertoire to novel antigen targets. However, limitations to these cell-based therapies are the lack of development against solid tumor antigens, partly due to the paucity of antigen candidates that are sufficiently specific to epithelial cancers. Therefore, technologies that can mark tumor cells for immune recognition could prove beneficial in the treatment of these solid cancers. This project aims to artificially deliver an antigen to tumor cells by harnessing a universal property of tumors: acidic microenvironment. The pH low insertion peptide (pHLIP) is a technology that targets solid tumors based on the low pH produced by the Warburg effect. pHLIP-conjugates have undergone extensive development for drug delivery, with one notable candidate CBX12 entering phase I clinical trials in 2021. Our system uses a pHLIP-conjugated SIINFEKL peptide (derived from ovalbumin) to serve as our model antigen system, with SIINFEKL-targeting T cells (OT1) that can be used as a tool to investigate antigen delivery, recognition, and functionality. We have observed through immunofluorescence that B16 F10 mouse melanoma cells treated in vitro with the 1uM pHLIP-SIINFEKL construct under acidic conditions can recruit OT1 cells and induce a T cell synapse. Preliminary in vivo experiments in C57BL/6-Tg OT1 mice bearing B16 F10 flank tumors showed that treatment with pHLIP-SIINFEKL by intravenous administration led to a suppression of tumor growth compared to controls, with approximately 50% smaller fold change in tumor volume. Cumulatively, these initial results suggest that pHLIP-SIINFEKL can be delivered to tumors to recruit OT1 cells to engage in antigen recognition and lead to efficacy in tumor growth delay in vivo. This technology has the capability to provide a novel approach to decorate solid tumors with engineered antigens for enhanced immune recognition and anti-tumor efficacy. Citation Format: Annali M. Yurkevicz, Yanfeng Liu, Peter M. Glazer. Utilizing a pH sensitive peptide (pHLIP) for tumor targeted delivery of an immunogenic peptide motif [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2715.

Abstract 5087: Combination therapy of immune checkpoint inhibitor and HIF1α/c-MET peptide vaccine suppresses metastasis by modulating tumor microenvironment in triple-negative breast cancer

Abstract Triple-negative breast cancer (TNBC) accounts for 20% of all breast cancer and has limited therapeutic option except for immunotherapy because ER, PR, and HER2 targeted for treatment are absent. It also tends to be more aggressive meaning more likely to metastasize faster to other organs such as lungs, liver, and bone, and its recurrence rate is high which leads to a poor prognosis. Immunotherapy is an option to treat TNBC such as programmed death ligand 1 (PD-L1) antibody. However, tumor cells use various immune evasion mechanisms in addition to PD-1/PD-L1 pathway to interrupt the effect of immunotherapy. Moreover, tumor microenvironment (TME) is one of the important factors for the effective anti-tumor immune responses from immunotherapy. Hypoxia is a typical event in TNBC. Hypoxia inducible factor 1-alpha (HIF1α) is a transcription factor that regulates angiogenesis by expressing VEGF, and in addition, upregulates the c-MET which increases the proliferation, migration, and survival of tumor cells. Moreover, HIF1α directly regulates the expression of CD47. CD47 is a transmembrane protein that binds to SIRPα of macrophages to inhibit the phagocytosis of macrophages. Therefore, the efficacy of immune checkpoint inhibitors decreases even if there is a higher degree of tumor-infiltrating lymphocytes (TILs). Combination therapy with immune checkpoint and tumor associated antigens (TAAs) derived peptide vaccine could inhibit metastasis. In present study, we found that HIF1α/c-MET peptide vaccination delayed metastasis in C3(1)Tag mouse. We also investigated whether combination therapy with immune checkpoint inhibitors and peptide vaccine inhibited metastasis. Next, we observed that combination strategy of HIF1α/c-MET peptide vaccine and treated anti-PD-L1 antibody or/and anti-CD47 antibody was more effective in systemically inoculated M6 cells growth in C3(1)Tag mice. Immunohistochemical analysis of the tumors showed that the protein expression of CD47, HIF1α, and c-MET were significantly decreased. On the other hand, CD4+ T cells, CD8+ T cells and M1 macrophages increased, but M2 macrophages decreased, resulting in delayed metastasis in combination therapy group compared to single therapy and control group. In addition, IFNγ ELISPOT showed that peptide vaccination significantly increased the HIF1α/c-MET specific IFNγ-secreting T cell response in splenocytes. We also assessed osteoclastogenesis in bones, which showed reduction of osteoclast in combination therapy group compared to single therapy and control group.Taken together, combination therapy with immune checkpoint inhibitor targeting PD-L1 and CD47 and peptide vaccine appears to be a promising strategy that inhibit metastasis by modulating tumor microenvironment in triple negative breast cancer. Citation Format: JinHwa Hong, Jimin Lee, Soon Young Lim, Ju Won Kim, Ah Reum Lim, Kyong Hwa Park. Combination therapy of immune checkpoint inhibitor and HIF1α/c-MET peptide vaccine suppresses metastasis by modulating tumor microenvironment in triple-negative breast cancer. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5087.

Abstract 6307: A novel pegylated bispecific antibody-drug conjugate (P-BsADCpb-adc) targeting cancers co-expressing PD-L1 and CD47

Abstract ADCs have demonstrated improved efficacy and target selectivity comparing to the non-specific small molecule cytotoxicity drugs in cancer treatment. Yet, in solid tumor therapies, effort for further improvement of efficacy and safety has been hindered by the poor tumor penetration of conventional IgG based ADC, low internalization efficiency, undesired efflux of ADC from tumor cells, narrow therapeutic window due to the on-target/off tumor and Fc induced toxicity, etc. To address those issues, we previously reported that the compound JY201, a Polyethylene Glycol (PEG)-based bispecific ADC (P-BsADC) targeting two epitopes of Her2, demonstrated advantages in tumor penetration, internalization efficiency, lysosome trafficking, no Fc related toxicity, and better efficacy in tumor inhibition than transtuzumab deruxtecan (Ds-8201). In this study, we reveal another novel pegylated P-BsADC (JY207) formed by site-specific conjugation of a bispecific single chain fusion protein targeting PD-L1 and CD47 with PEG-MMAE (a pegylated cytotoxic payload MMAE). As expected, the compound JY207 retains all the advantages that JY201 possesses. Furthermore, JY207 does not bind to human red blood cells, but preferentially binds to CD47/PD-L1 double positive tumor cells, thus reduces the possibility of on-target toxicities. In vitro cytotoxicity studies showed that JY207 has strong potencies in CD47/PD-L1 double positive tumor cells, while showing almost no killing effect to CD47 or PD-L1 single positive tumor cells. In CDX models and a PDX model of transplanted tumor tissues from lung cancer patients, the compound demonstrated excellent tumor inhibition at low doses. In an in vitro plasma stability test, JY207 displays high stability in cynomolgus monkey and human serums. Preliminary repeated-dosing toxicological study has found the maximum tolerated dose of JY207 in CD47/PD-L1 double transgenic mice is 50mg/kg. In vivo pharmacokinetics and toxicological studies of JY207 are being conducted in cynomolgus monkeys and are expected to show desirable results. All those findings in this study warrant JY207 as a promising candidate for the clinical development for patients with CD47/PD-L1 double positive cancers. Citation Format: Shumin Liu, Weidong Lyu, Shuqiang Yin, Yang Lei, Qiudong Zhuo, Liling Zheng, Bin Sun, Shuangyu Tan, Lidong Jiang, Teng Zhang, Bo Gao, Rui Xu, Dechang Huang, Yong Li, Zibin Wu, David Wu, Yvonne (Yu) Wen. A novel pegylated bispecific antibody-drug conjugate (P-BsADCpb-adc) targeting cancers co-expressing PD-L1 and CD47. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6307.

Abstract 2958: BSI-508, a novel bispecific fusion molecule targeting PD1 and CD47 for cancer immunotherapy

Abstract Introduction: While anti-PD1/PDL1 mAbs have shown success in the market only a small fraction of patients actually benefit from the therapies. Novel approaches to improve outcomes for patients who are resistant or refractory to current checkpoint inhibitors (CPIs) are needed. CD47 expressed on tumor cells can provide a “don’t eat me” signal to antigen-presenting cells (APCs) via the binding to SIRPα. Blocking this interaction, enhancing the phagocytosis of tumor cells, has emerged as an effective alternative to current CPIs. Here we report on the generation and characterization of a novel bispecific fusion molecule BSI-508, composed of an anti-PD1 antibody fused with a SIRPα extracellular domain (ECD). The SIRPα-ECD binds CD47 and blocks the CD47/SIRPα-mediated cell signaling pathway. BSI-508 is designed to target PD1 on T cells and CD47 on tumor cells, providing tumor killing by T cells and macrophages synergistically. Methods: An anti-PD1 mAb was identified from PD1 KO mice immunized with PD1-ECD-Fc and screened by our proprietary H3 (High-throughput, High-content and High-efficiency) platform. Human SIRPα-V2-ECD1 was fused to the N-terminus of the heavy chain of the anti-PD1 antibody via a flexible linker. The binding activities and affinities to PD1 and CD47 were evaluated by ELISA, FACS and SPR. Ligand blocking activity was measured by ELISA and FACS. A cell-based reporter assay was used to evaluate the function of the anti-PD1 portion and a macrophage mediated phagocytosis assay was used to evaluate the function of the SIRPα-V2-ECD1 portion of the bispecific. B-hPD-1/hPD-L1/hSIRPα/hCD47 mice bearing B-hPDL1/hCD47 MC38 tumors were used to evaluate the tumor inhibitory activity of the bispecific fusion molecule. Results: BSI-508 demonstrated comparable activity to the parental anti-PD1 monoclonal antibody regarding PD1 binding and PD1/PDL1 blocking. It also exhibited comparable activity to the SIRPα-V2-ECD1-Fc fusion protein in CD47 binding and CD47/SIRPα blocking. BSI-508 was able to reverse PD1 mediated T-cell suppression and exhibited comparable bioactivity to the parental anti-PD1 antibody. The bispecific molecule showed comparable bioactivity to the SIRPα-V2-ECD1-Fc fusion protein in inducing macrophage mediated phagocytosis of Jurkat cells. In addition, BSI-508 showed superior antitumor efficacy in vivo. Summary: BSI-508 is a novel anti-PD1xanti-CD47 bispecific fusion molecule combining both T-cell activation and macrophage mediated phagocytosis together for superior tumor killing. BSI-508 demonstrates favorable biophysical and functional characteristics, supporting the initiation of development activities including manufacturing and IND-enabling studies. Citation Format: Zeyu Peng, Xiaodong F. Liu, Hongyan Li, Wenwen Dai, Jinyu Liu, Xiaoyao Hao, Shukai Xia, Qun Lv, Hugh M. Davis, Mingjiu Chen, Mark Z. Ma. BSI-508, a novel bispecific fusion molecule targeting PD1 and CD47 for cancer immunotherapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2958.

Abstract 1151: CD6 isoforms improve CAR T cell fitness

Abstract The ability of chimeric antigen receptor (CAR) T cells to initiate and sustain an effective immune response entails a delicate balance between T-cell activation and deactivation and is key for complete tumor elimination. CD6 is a co-receptor that fine-tunes T cell receptor signal strength through a unique dual function intracellular domain. CD6 is alternatively spliced, creating isoforms that lack distinct regions which interact with activating or inhibitory adapter proteins, thereby delivering inhibitory or activating signals, respectively. We hypothesized that CAR T cell fitness, namely their ability to sustain their effector function, can be achieved by using alternatively spliced CD6 isoforms which lack exons responsible for inhibitory signaling. First, we discovered that overexpression of the canonical full-length CD6 molecule, isoform CD6A, resulted in marginal improvement of CAR T cell function, while its deletion using CRISPR/Cas9 depressed the antitumor CAR T cell functionality. This supported a net stimulatory role for CD6 in CAR T cell function. To study their effect on cellular fitness, we then synthesized and overexpressed known alternatively spliced cytoplasmic isoforms CD6B, CD6C, CD6D, CD6E, and CD6F on CAR T cells and, noted a significant tendency towards preserving the CAR T cell naïve and central memory compartments, upon tumor encounter. Overexpression of isoform CD6F, which lacks exon 9, and to a lesser extent isoforms CD6C and CD6E enhanced the short- and long-term cytotoxicity of HER2 and CD19 targeting CAR T cells when compared to CAR T cells alone or CAR T cells overexpressing, CD6A. Despite exhibiting a lower proliferation index, and lower CAR CD3ζ phosphorylation at rest, CD6F CAR T cells secreted significantly higher concentrations of the Th1 cytokine, IFN-γ, and mediated more sustained in vitro cytotoxicity long-term, with more complete elimination of large tumor cell loads. Lastly, in an orthotopic model of human glioblastoma, the adoptive transfer of CD6F HER2 CAR T cells induced a more significant but also more sustained anti-glioma effect that translated into a significant survival advantage for experimental animals. Our data support that the overexpression of the alternatively spliced CD6 isoforms improves CAR T cell fitness resulting in superior antitumor activity both in vitro and in vivo. Citation Format: Jessica S. Morris, Ahmed Z. Gad, Lea M. Godret-Miertschin, Ryan Fleischmann, Vita Salsman, Sujith K. Joseph, Nabil Ahmed. CD6 isoforms improve CAR T cell fitness [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1151.

Abstract 913: Engineering allogeneic off-the-shelf CD19-directed CAR-iNKT cells without additional genetic manipulations for the treatment of hematological malignancies

Abstract Despite the approval of three autologous CAR-T cell products, B-cell malignancies still represent an unmet need as several of the relapsed/refractory patients are ineligible for autologous CART cell therapies. Invariant natural killer T (iNKT) cells are a unique subset of immune cells that display properties of both T cells and natural killer cells and bridge the innate and adaptive immune responses upon antigen recognition. Unlike conventional β T cells, iNKT cells express an invariant T cell receptor (TCR) that recognize glycolipids presented in the context of the monomorphic, MHC-class I related molecule, CD1d. iNKT cells are ideal cell types for allogeneic, off the shelf approach as they can target cancers without the risk of graft-versus-host disease (GvHD), circumventing the need to delete or knock out the endogenous TCR. Here, we report the engineering of ‘off-the-shelf’ iNKT cells directed against CD19 and their robust in vitro and in vivo functional profile. Briefly, peripheral blood derived iNKT cells were isolated from healthy donors and were engineered to express a CD19 CAR using a 3rd generation lentiviral vector. To demonstrate the CAR-dependent and independent anti-tumor activity, CAR19-iNKT cells were compared in vitro against non-transduced (NT) iNKT cells in cytotoxicity assays and intracellular cytokine staining assays. Allogeneic CAR19-iNKT cells upregulated activation markers, secreted cytokines, and lysed cancer cell lines and primary tumor cells from chronic lymphocytic leukemia and marginal zone lymphoma patients in an antigen-specific manner. Excellent post thaw recovery and viability was noted in cryopreserved CAR19-iNKT cells. Finally, the anti-tumor activity of cryopreserved CAR19-iNKT cells were evaluated in an established aggressive NSG mice model of SEM-luc, a B cell lymphoblastic leukemia cell line expressing luciferase. Robust antitumor activity by CAR19-iNKT cells was observed without any obvious adverse effects compared to controls (untreated and NT iNKT cells). The persistence of CAR19-iNKT cells in this model is currently being evaluated. These data demonstrate a unique, off the shelf CAR19-iNKT cell platform for targeting hematological malignancies that combines the specificity of CD19 targeting CAR with the innate features of iNKT cells. The data indicates that iNKT cells are a potent cell type for generating allogeneic off the shelf products for treating a broad spectrum of hematological malignancies and solid tumors by targeting different receptors on the respective tumors. Citation Format: Kanagaraju Ponnusamy, Janani Kanagaraju, Yingxin Wang, Bryan Lye, Hongwei Ren, Sandhya Buchanan, Michael J. Baker, Mini Bharathan, Anastasios Karadimitris. Engineering allogeneic off-the-shelf CD19-directed CAR-iNKT cells without additional genetic manipulations for the treatment of hematological malignancies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 913.