Abstract Introduction: Autologous CAR-T therapies targeting B-cell maturation antigen (BCMA) have shown remarkable clinical success in treating multiple myeloma (MM). However, accurate prediction of patient-specific efficacy and adverse events - such as cytokine release syndrome (CRS), immune effector cell-associated neurotoxicity syndrome (ICANS) and Immune effector cell associated hemophagocytic lymphohistiocytosis-like syndrome (IEC-HS) - remains a significant and ongoing challenge. To address the need for personalized screening of CAR-T immunotherapies, we developed and tested a novel humanized mouse model using the NSG-SGM3-IL15-DKO (SDKO) strain, optimized for PBMC engraftment. This model is a new potential preclinical tool to assess individualized CAR-T efficacy and toxicity in vivo. Methods: We evaluated autologous anti-BCMA CAR-T therapy in vivo using five human PBMC donors in the context of an established BCMA+ tumor. For this, SDKO mice were injected intravenously with 1×106 MM.1S-luc cells, a model cell line for multiple myeloma. Ten days post-tumor injection, mice were randomized based on their tumor burden and injected with 3×106 PBMCs. Four hours later, mice received either PBS, 5×106 autologous untransduced T cells (UT), or 5×106 autologous anti-BCMA CAR-T cells. Tumor burden and body weight were monitored over 28 days. Blood samples were collected at SD1, SD2, SD5, SD15, SD21 and SD28 to assess human cell engraftment and plasma cytokine levels and tissues retrieved for histopathology. Results: Autologous CAR-T cells induced rapid and potent tumor regression with low toxicity, whereas UT cells failed to control MM.1S-luc tumors across all donors, with tumor progression even more severe than that observed in the PBS group. However, donor-dependent variability was observed: Donors 0935 and 1295 showed reduced CAR-T expansion, and 0935 failed to achieve complete tumor clearance. Transient tumor resurgence occurred in most CAR-T treated mice, except Donor 5263, where tumors were rapidly and permanently eliminated. In contrast, tumors metastasized to bone marrow in PBMC and UT groups. Cytokine profiling revealed both donor-dependent (IL-9, IL-12p70, IL-12p40) and donor-independent (IL-2, IL-4, IL-5, IFNγ, TNFα, IL-13) secretion patterns. Notably, IL-10 exhibited a unique dynamic: a modest spike at SD1 followed by a CAR-T-dependent decline, while PBS and UT groups showed very high IL-10 levels at SD5, suggesting a link to uncontrolled tumor burden and immune dysregulation. Conclusion: Our results demonstrate that the SDKO PBMC humanized mouse model has the potential to be a valuable tool for preclinical CAR-T testing, enabling patient-specific evaluation of efficacy and safety, and paving the way for safer CAR-T cells in the clinic. Citation Format: Alba Matas-Céspedes, Jiwon Yang, Kushal Prajapati, Destanie Rose, Oanh Pham, Michael Lehmann, Tom McKevitt, Georgina Cornish, Richard Stebbings, James G. Keck, Ilian Radichev. In vivo evaluation of autologous anti-BCMA CAR-T therapy in PBMC-humanized SDKO mice reveals donor-specific immune responses [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 1 (Regular Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(7 Suppl):Abstract nr 4267.

Abstract Introduction Immunotherapies are the largest growing therapeutics applied to cancer and autoimmune disorders, rapidly transforming the drug development landscape. Despite significant improvements in immunotherapy research and available treatments, ample variation in clinical outcomes exposes gaps in our understanding of their mechanisms of action and specific response biomarkers. The development of the NSG-MHC I/II double-knockout (DKO) mice improved significantly the availability of models that can delay graft-versus-host disease (GvHD) while still achieving strong T cells engraftment. Nonetheless, DKO mice perform better with irradiation and engraftment with high doses of PBMCs to achieve these results and there is a challenge on producing multilineage human immune subsets such as natural killer (NK) cells and myeloid populations. Here, we characterize a next-generation DKO strain, SDKO (NSG-SGM3xIL15xDKO) designed for enhanced humanization with lower PBMC doses and improved immune diversity without the need for irradiation. Methods Nonirradiated SDKO (Jax #037320, n=20) and DKO (Jax #025216, n=20) mice between 7-10 weeks of age were intravenously (IV) injected with 10 million PBMC using a total of five different donors. Mice were bled weekly via retro-orbital bleeds to assess engraftment status up for 12 weeks. Half the mice per strain were euthanized at SD21 for spleen engraftment checks. Survival was assessed up to 84 days. Conclusions Survival between the two strains was comparable throughout the entire length of the studies. The newly developed mouse strain, NSG-SGM3xIL15xDKO (SDKO) offers improved overall humanized engraftment over the industry standard NSG-MHC I/II double-knockout (DKO), with higher hCD45+ numbers than DKO strain without the need for irradiation. Also, the SDKO strain present a higher T cell subpopulation (CD4+ and CD8+), higher number of Natural Killer (CD56+) cells and B (CD19+) cells. Noteworthy, SDKO presents high number of differentiated Plasma Cells (CD138+) in the spleen, a population that is not usually observed in engrafted DKO strain. Based on these results, we conclude that the novel SDKO mice better recapitulates the development of human innate and adaptive immunity than its counterpart DKO model. Citation Format: Leandro Salati D'Abronzo, Xiaoqing (Nancy) Zheng, Beau Parry, Guoxiang Yang, Destanie Rose, Li-Chin Yao, James Keck, Jiwon Yang, . Improved humanization of peripheral blood mononuclear cells in NSG-SGM3xIl15xDKO (SDKO) over that of NSG-MHC I/II DKO mice for immuno-oncology modeling [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 1 (Regular Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(7 Suppl):Abstract nr 1600.

Abstract Introduction: Chimeric Antigen Receptor T (CAR-T) cell therapies have revolutionized treatment for hematologic malignancies, particularly B cell cancers, and show promise in autoimmune diseases and solid tumors. Despite this success, the conventional approach to CAR-T manufacturing presents several limitations: it requires leukapheresis, PBMC isolation, ex vivo genetic modification and expansion, and reinfusion back into the patient. These steps are time-consuming, costly, and logistically complex, limiting scalability and patient accessibility. Furthermore, current CAR-T therapies have shown limited efficacy against solid tumors due to poor trafficking, immunosuppressive microenvironments, and antigen heterogeneity. Methods: To improve the cost-inefficient and time-consuming challenges of the standard CAR-T therapy, Syenex developed Vivo-T, a novel T cell-specific lentiviral vector (LVV) platform enabling in vivo delivery of CAR constructs, eliminating the need for ex vivo manipulation and personalized CAR-T manufacturing. This system was evaluated in JAX PBMC-humanized B cell lymphoma mouse model. NSG-MHC I/II double knockout mice were irradiated and injected intravenously with 2.5×105 Raji-Luc cells. Five days later, mice were engrafted with 1×107 human PBMCs. Between 4-24 hours post-engraftment, mice received intravenous doses of T cell-targeted LVVs encoding anti-CD19 CARs. Tumor burden was monitored via IVIS imaging for 3 weeks. Toxicity was assessed through body weight, clinical observations, and serum cytokine analysis. CAR-T engraftment and expansion were evaluated by flow cytometry of peripheral blood. Results: In vivo delivery of the CAR construct resulted in efficient T cell transduction, with rapid and robust CAR-T expansion surpassing that of ex vivo-engineered CAR-T control. Notably, significant anti-tumor activity was observed as early as 7 days post-LVV administration. Initial signs of toxicity were primarily attributed to high viral titers; however, these effects were mitigated by reducing the viral load, without compromising the antitumor efficacy of the treatment. These findings validate the efficacy of the in vivo CAR-T system and support its potential for clinical translation. Conclusion: The Vivo-T system represents a transformative approach to CAR-T therapy by simplifying manufacturing, reducing costs, and enabling scalable, off-the-shelf immunotherapy. Citation Format: Ilian Radichev, Devin Stranford, Hailey Edelstein, Oanh Pham, Destanie Rose, Jiwon Yang, Mahdy Yassine, Henry Schirmer, Teresa Nikolich, Joshua Leonard, James Keck, Matteo Stoppato. In vivo lentiviral CAR-T gene delivery demonstrates high specificity and potent anti-tumor activity in a humanized B cell lymphoma model [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 1 (Regular Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(7 Suppl):Abstract nr 146.

Abstract Humanized mouse models, such as those generated by administering human peripheral blood mononuclear cells (PBMC) into immunodeficient mice, hold great potential for studying immunotherapies and the immunogenicity of biologics and vaccines. Humanized mice are known for their quick engraftment of human cells, strong functional T-cell responses and for their capacity to assess donor-specific responses in vivo. However, these models face several limitations, including partial reconstitution of the human immune system, inadequate human cytokine support, and development of graft-versus-host disease (GvHD). To help address these issues we have developed new immunodeficient mouse strains and here show the potential of hPBMC mice to elicit humoral responses against various antigens. We engrafted human PBMC into NSG-SGM3xIL-15xDKO strain (JAX# 037320) followed by vaccination with formulated vaccines (Tdap, COVID-19 mRNA, or H5N1) or with antigen with or without adjuvant. Elevated levels of human IgG specific for tetanus toxoid, diphtheria, SARS-CoV-2 spike, or H5N1 were found in vaccinated animals compared to unvaccinated controls. Moreover, we also see an increase in total numbers of CD19+ B cells, activated B cells (CD38+) and CD138+ human plasma cells. These findings demonstrate that PBMC humanized mice provide a promising platform for evaluating humoral immunity. Citation Format: Destanie Rose, Ilian Radichev, Beau Parry, Leandro Salati D'Abronzo, Zahid Delwar, Jiwon Yang, Li-Chin Yao, James G. Keck. Inducing memory humoral responses using multiple vaccines in PBMC humanized mice [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 1 (Regular Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(7 Suppl):Abstract nr 4388.

Peripheral Blood Mononuclear Cell (PBMC) humanized mice (hPBMC mice), generated by injecting human PBMCs into immunodeficient mice, are increasingly used to study human immune responses particularly in cancer, infectious diseases, and immunotherapy research. These models are notable for their rapid engraftment of human cells and robust functional T-cell responses and for the ability to evaluate donor-specific responses in vivo. Despite its success in studying toxicity of preclinical Immunotherapeutics the model was believed to have certain limitations, including incomplete reconstitution of the human immune system, insufficient human cytokine support, and development of graft-versus-host disease (GvHD). Here, we demonstrate that hPBMC mice could elicit humoral responses against various antigens. We engrafted human PBMC into newly developed strains based on the NSG-MHC I/II DKO (#025216) from JAX and vaccinated the mice with standard vaccines (Tdap or COVID-19 mRNA) or with a recombinant spike protein from SARS-CoV-2 with or without adjuvant. Circulating tetanus toxoid, diptheria or SARS-CoV-2 spike specific human IgG were found elevated in vaccinated animals compared to PBS controls. Furthermore, these mice had increased numbers of total CD19+ B cells, activated B cells (CD38+) and CD138+ human plasma cells. These results highlight the potential of using hPBMC mice for research studying humoral immune responses. Vaccines and Immunotherapy (VAC)

Chimeric antigen receptor T-cell (CAR T) therapy has revolutionized cancer treatment by providing targeted eradication of cancer cells. However, the efficacy and toxicity of CAR T cells can vary widely between individuals, posing challenges for patient-specific optimization. To address these gaps, we evaluated the efficacy, expansion, and cytokine induction profiles of CAR T using PBMC-humanized mice. Three autologous CAR T products with 4-1BB co-stimulation were manufactured from the PBMCs of three healthy human donors: CD19-specific, CD22-specific, and CD19x22-specific (dual) CAR T cells. The dual CAR T cells were transduced with lentiviral vectors encoding either CD19- or CD22-specific CARs, resulting in three distinct CAR T cell compositions: those targeting CD19 alone, CD22 alone, or both CD19 and CD22. NSG-MHC Class I/II double knock-out (DKO) mice were irradiated and engrafted with 10 million PBMCs from the same donors used for the autologous CAR T cells. Six days post-humanization, the mice were treated with one of the three autologous CAR T cells. We found that all CAR T products effectively eliminated B cells in vivo within three weeks post-treatment, while CD19 CAR Ts achieved clearance significantly faster than CD22 CAR Ts. Expansion levels of CAR T cells differed significantly by donors and constructs. Overall, mice treated with CD19 CAR Ts showed peak expansion earlier than those treated with the other two CAR Ts. Mice treated with CD22 CAR Ts reached the highest expansion across all donors during the 28-day study period. Notably, one donor treated with CD22 CAR Ts displayed more than a 10-fold higher expansion compared to the other two donors, which corresponded to decreased survival and significant body weight loss. In dual CAR T-treated mice, CAR T cells targeting CD19 alone showed more robust expansion than the others (54 to 99% of total CAR T cells, depending on timepoints and donors). Cytokine induction was both donor-dependent and CAR construct-dependent, particularly for IL-1β, IL-2, IL-3, IL-5, IL-6, and IL-10. In summary, PBMC-humanized mice are a valuable platform to assess individual variability in CAR T efficacy and toxicity. These insights highlight the need for personalized preclinical evaluations to optimize CAR T construct selection and predict patient-specific outcomes. Won Lee, Destanie Rose, Beau H. Parry, Joshua Gustafson, Rebecca Gardner, Heather Gustafson, James G. Keck, Jiwon Yang. Using PBMC-humanized mice to identify optimal autologous CAR T products for maximized efficacy and minimized toxicity at an individual level: a study of CD19, CD22, and CD19x22-specific constructs in multiple PBMC donors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 7265.

Abstract Chimeric antigen receptor T-cell (CAR-T) therapy is a significant advancement in treating hematological malignancies, yet it faces challenges due to its variable therapeutic responses and the risk of severe toxicities. This study delves into the effects of tumor burden and CAR-T cell doses on the toxicity profile of the therapy, employing a PBMC-humanized mouse model engrafted with luciferase-labeled Raji B cell lymphoma (Raji-luc). By exploring high vs. low tumor burden and high vs. low CAR-T dose scenarios, we aim to gain insights into the dynamic relationship between these factors and CAR-T-induced toxicity and efficacy. In the first experiment, we established PBMC humanized mouse models with a high tumor burden and a low tumor burden. 10 days (high burden) or 7 days (low burden) after the Raji-luc inoculation, mice were engrafted with 10M human PBMCs and dosed with CD19 autologous CAR-Ts. In mice with low tumor burden, CAR-T treatment demonstrated significant efficacy, as evidenced by IVIS imaging, while the high tumor burden model exhibited reduced efficacy. CAR-T treatment in Raji-bearing mice did not induce weight loss in either high or low burden models. CD19 CAR-T cells effectively reduced the target cell population in peripheral blood, with greater expansion observed in the higher tumor burden model. We also evaluated human cytokine levels post-CAR-T treatment, revealing higher cytokine induction in the high tumor burden model, peaking at 2 days post-treatment. Secondly, in a CAR-T dose-response study, we treated PBMC-humanized Raji-bearing mice with 10M, 15M, and 20M CAR-T cells. Higher CAR-T cell doses (15M and 20M) resulted in observable toxicity measured by body weight loss, while the 10M dose did not. All CAR-T doses effectively slowed tumor growth and induced significant CAR-T cell expansion. Selected human cytokines, such as IL-5 and RANTES, demonstrated a dose-response correlation with CAR-T treatment. Additionally, IL-6 and IL-10 were significantly correlated with tumor burden rather than CAR-T doses. The differential cytokine responses observed in our study provide valuable insights into the utility of the PBMC-humanized mouse model for investigating the biological responses associated with CD19 CAR-T therapy. Our findings underscore the utility of the PBMC-humanized mouse model in assessing variability in toxicity and cytokine responses to CAR-T therapy. This model offers valuable insights into the factors influencing CAR-T treatment outcomes and provides a platform for planning more precise treatment and enhancing the safety and efficacy of CAR-T therapy. Citation Format: Won Lee, Destanie Rose, James G. Keck, Jiwon Yang. Assessing impacts of tumor burden and CAR-T cell dosage on the toxicity and efficacy profile of CD19 CAR-T therapy in a PBMC-humanized mouse model [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 4010.

Background Cancer immunotherapies such as chimeric antigen receptor (CAR) T cell therapies and bispecific antibodies are highly effective treatments for patients with haematological malignancies. However, these immunotherapies are frequently associated with systemic toxicities such as cytokine release syndrome (CRS). In addition to its impact on patient survival and performance status, CRS complicates the administration of the immunotherapies and restricts availability to specialist tertiary centres with access to high-dependency units. CRS is thought to arise from on-target effects of immunotherapy involving undue amplification of specific inflammatory responses. Treatment of CRS includes anti-pyretic or anti-cytokine therapies such as tocilizumab (anti-IL-6R), anakinra (anti-IL-1RA), and corticosteroids. No prophylactic treatments are currently approved. POLB 001 is an oral p38 MAPK inhibitor in development for the prevention of immunotherapy-induced CRS. Inhibition of p38 MAPK is expected to selectively target key inflammatory pathways without causing broad immunosuppressive properties. Adalimumab, an anti-TNF antibody, is a potent inhibitor of CRS in humanized mouse models. The current study was designed to evaluate the effect of POLB 001 on cytokine release compared to adalimumab in a well-characterized animal model of CRS. Methods The Jackson Laboratory provided optimized mouse models, in which CRS was reliably induced in peripheral blood mononuclear cells (PBMC) humanised CD19+ Raji-luc cancer bearing mice. Briefly, to model immunotherapy-induced CRS, female NSG-MHC I/II DKO mice were irradiated at 100 cGy and injected with PBMCs intravenously (15 x 106 cells) which were left to grow for 4 days. On day 4 until study terminus, mice were treated twice daily via subcutaneous injection with either vehicle or POLB 001 at low (2 mg/kg), medium (10 mg/kg), or high (25 mg/kg) doses for 5 days. On day 5, mice were injected intravenously with Raji-luc cells (2 x 106 cells). On day 6, mice were intravenously injected with anti-CD28. The positive control group was immediately dosed intraperitoneally with adalimumab (5 mg/kg). Whole blood was collected from mice by retro-orbital bleeding, samples were used for flow cytometry analysis or processed to serum to measure human cytokines. Mice were euthanised by CO2 asphyxiation for necropsy once they reached the humane endpoint or on the scheduled study terminus. Where mice reached study terminus, whole blood was collected via cardiocentesis for sample analysis. The study objective was to examine the effect of POLB 001 on inflammatory responses following anti-CD28 stimulation, including cytokine analysis and T cell expansion in addition to clinical observations of CRS. A One-Way ANOVA was fitted to the data and comparisons of interest were made using a Bonferroni test to adjust for multiple testing. Results All doses of POLB 001 significantly reduced clinically observed CRS scores (2 mg/kg p<0.05, 10 mg/kg and 25 mg/kg p<0.001). POLB 001 treatment showed statistically significant reductions in IL-6 (at high-dose), IL-4 (low- and high-dose), IL-8 (all doses) and MIP-1α (all doses) and a trend of reduced cytokine production in all other cytokines tested (IFN-γ, TNF, IL-10, IL-2). Adalimumab and POLB 001 (10 mg/kg and 25 mg/kg) effectively prevented CRS symptoms, however POLB 001 produced superior cytokine inhibition (ns). While adalimumab also significantly reduced IFN-γ, TNF, IL-6, and MIP-1α, some cytokines were elevated, including IL-10, IL-4, and IL-2 and there was no significant effect on IL-8. These results indicate that POLB 001 may be effective in preventing CRS. Both test articles were well tolerated. Conclusion In a model of anti-CD28 induced CRS, POLB 001 was shown to dose dependently reduce clinical CRS scores. POLB 001 significantly reduced peak serum levels of IL-4, IL-6, IL-8 and MIP1-α; all other cytokines tested, including IFN-γ, TNF, IL-10 and IL-2, showed trends of reduced peak serum levels. In combination with a previous successful trial in healthy human volunteers, the results of this animal study further support development of POLB 001 in a Phase 2 clinical study as a prophylactic for the prevention of immunotherapy-induced CRS.

Abstract Clear cell renal cell carcinoma (ccRCC), the predominant histological subtype of RCC, is highly correlated with immune cells, an association that is linked to a worse prognosis. Our study aimed to establish an orthotopic ccRCC mouse model reconstituted with human peripheral blood mononuclear cells (PBMCs) and implanted with human ccRCC SKRC-59 cells under the kidney capsule in immunodeficient NSG-MHC I/II double knock-out (DKO) mice. The study was conducted using two experimental designs to optimize humanization and tumor formation. In the first experimental design, the mice were engrafted with SKRC-59 cells under the left kidney capsule 24 hours post-irradiation on study day 0. The PBMCs were then injected intravenously on study day 9. The second experimental design involved irradiating mice on study day -1, followed by engraftment with PBMCs either perfectly (6/6) or partially (3/6) matched human leukocyte antigens (HLA) with the tumors. Subsequently, SKRC-59 cells were placed under the kidney capsule on study day 0. Daily post-operative observations were conducted for six days following surgery to ensure the well-being of the mice. Tumor burden was assessed twice weekly using an in vivo imaging system, along with monitoring body weights and clinical signs. The humanization rates were evaluated 7, 14, and 21 days post-PBMC engraftment, as well as at the end of the study, using peripheral blood. Approximately 5 to 6 weeks post-tumor implantation, the mice were euthanized, and both left and right kidneys were collected and weighed for further analysis. The humanization rates were significantly higher when the PBMCs were engrafted on the same day as irradiation compared to 10 days post-irradiation. The PBMC humanization, and HLA-type PBMC donors did not affect tumor burden. Additionally, we found that distant metastasis in the lungs developed in several mice. Using flow cytometry and immunohistochemistry (IHC) to analyze tumor infiltrating leukocytes (TILs), a predominant number of CD3+ T cells were found in human CD45+ TILs, indicating that this model could be useful for evaluating T cell-mediated immunotherapies. Moreover, lympho-myeloid aggression (LMA) was observed in this peripheral blood leukocytes (PBL) mouse model, showing early stage tertiary lymphoid structures (TLS), which potentially provides a powerful tool to recapitulate TLS formation and study associated mechanisms and therapies. Our study demonstrated the growth of ccRCC SKRC-59 tumors under the renal capsule and observed TILs and metastatic patterns in PBMC humanized DKO mice. This PBMC-humanized orthotopic ccRCC xenograft model can recapitulate tumor microenvironment (TME), and be potentially used to assess the safety and efficacy of various immunotherapeutics. Citation Format: Wonyoung Kang, Yufei Wang, Yasmin N. Laimon, Oanh Pham, Ben Matran, Lauren Bottoms, Sheng Khang, Hsien-Chi Yuan, Nithyassree Murugan, Aseman B. Sheshdeh, Li-chin Yao, Jiwon Yang, Mingshan Cheng, Sabina Signoretti, Wayne A. Marasco, James G. Keck. Establishing orthotopic renal cell carcinoma xenograft in PBMC-humanized immunodeficient NSG-MHC I/II double knock-out mice [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 111.

Chimeric antigen receptor T cell (CART) immunotherapy has been accumulating extraordinary breakthroughs in fighting against hematological malignancies. Particularly, universal, “off-the-shelf”, CARTs (uCARTs) are emerging as an attractive alternative to patient-derived CARTs, offering cost-effectiveness, immediate availability for treatment, and independence from patients' lymphopenic conditions. However, uCARTs share the same safety concerns with autologous CARTs while the efficacy and persistence of uCARTs vary by each product and remain uncertain. Further, T cells sourced from healthy donors for uCARTs may manifest individual-specific immune characteristics upon interaction with cancer cells and the patient's immune system. These characteristics often remain latent when assessed in vitro and complicate the prediction of uCART therapy's toxicity and efficacy on an individual basis. We previously demonstrated that the PBMC-humanized mouse model can capture efficacy and toxicity of autologous and allogeneic CD19 CARTs. We showed that the CART-induced toxicity varied by the combination of PBMC donors and CART cell donors. Here, we extend the use of PBMC-humanized mice to assess the potential toxicity, efficacy, and persistence of uCARTs. Female NSG™-DKO mice (n = 19-20 mice per donor) were irradiated and reconstituted with human PBMCs sourced from four selected donors (Donor A, B, C, and D) out of 58 available in our PBMC bank. The selection criteria were based on their pre-characterized in vivo human cytokine induction profiles, specifically IFN-gamma response levels to OKT3 treatment (Donor A: 268 pg/ml; B: 1936 pg/ml; C: 4862 pg/ml; D: 13538 pg/ml). PBMC-humanized mice were grouped into three different CD19 CART treatments; a) Allogeneic CARTs were manufactured from a healthy donor's T cells containing CD28 costimulatory domain (19-28z) and no genetic modification was made for “off-the-shelf” availability; b) uCART 1 cells (19-28z) were knocked out of TCRab via CRISPR-cas9 (19-28z); c) uCART 2 cells were knocked out of CD3 and TCRab using TALENs and magnetically depleted for remaining CD3/TCRab, and contain 4-1BB costimulatory domain. Body weight change and clinical scores were measured throughout the experiments. We measured the levels of human CD45, human CD19, and CART expansion from blood collected 8 days post-treatment using flow cytometry. We measured 54 human cytokine levels from serum collected 48 hours post-treatment using a multiplex assay. CART-induced toxicity, measured by body weight loss, dramatically varied by PBMC donors and the treatments (Figure 1). uCART 2 induced significant body weight loss in Donor B and D reaching the lowest 6 days post-treatment. Notably, all mice humanized with Donor C and treated with uCART 2 reached a humane endpoint within 12 days post-treatment. We only observed noticeable body weight loss from uCART 1 in Donor C. Allogeneic CART did not induce body weight loss across the four donors. Efficacy, measured by the % of elimination of human CD19 cells in peripheral blood compared to the PBS-treated group, also varied by PBMC donor and the treatments (Figure 2). Overall, Allogeneic CART showed the best elimination of CD19 cells. Mice humanized with Donor D responded with the poorest efficacy of the three CART treatments. CART expansion was observed only in uCART 2 treated mice that reached a humane endpoint within 6 days post-treatment. CART persistence measured 16 days post-treatment was not observed across three CARTs and this corresponds to the clinical data of uCARTs. Principal component analysis of human cytokine data revealed that uCART 2 induced the most distinct cytokine responses compared to the other CARTs, driven by IFN-gamma, IL-10, IL-5, IL-3, and GM-CSF. In Donor-A humanized mice, the same set of human cytokines (IFN-gamma, IL-10, IL-5, IL-3, GM-CSF, IL-13) was induced in response to the three CARTs, while the degree of induction varied by treatment. In contrast, Donor D-humanized mice showed vastly different responses to each treatment; uCART 2 induced significant levels of IL-6, TNF-beta, MIG/CXCL9, MDC, MCP-3, IL-13, and IL-2. In sum, we show that PBMC-humanized mice capture individual variations in the toxicity and efficacy of uCARTs. This platform can be used to preclinically assess efficacy and persistence of uCARTs as well as used to predict toxicity from the interaction between the host and healthy T cell donor of uCARTs.

Background The development of new bispecific antibodies such as bispecific T cell engagers (BiTE) is a hot avenue in cancer immunotherapy. BiTE antibodies interact simultaneously with epitopes located on T cells and target cells, so can direct T cells to cancer. Despite significant success, adverse effects are often reported with cancer immunotherapies. In recent years, PBMC-humanized mice have emerged as a valuable pre-clinical model that recapitulates patient responses observed in clinics. Therefore, such models can be used for personalized screening of a patient ’ s immune response, and an evaluation of immunotherapy ’ s efficacy and safety before starting treat-ment. Here, we used JAX ’ s established protocol for cancer therapy in PBMC-humanized mice to compare the efficacy and cytokine responses of two BiTE products – Blinatumo-mab, an FDA-approved drug