Background Chimeric antigen receptor (CAR) T-cell therapies are highly efficacious for several different hematologic cancers. However, for most CAR T targets it is observed that low surface antigen density on tumors can significantly reduce therapeutic efficacy. In this study, we explore this dynamic in the context of CD72, a surface antigen we recently found as a promising target for refractory B-cell cancers, but for which CD72 low antigen density can lead to therapeutic resistance in preclinical models.Methods Primary samples were accessed via institutional review board-approved protocols. Affinity-matured and humanized nanobody clones were previously described in Temple et al. (2023). CAR T cells were generated via lentiviral transduction. In vitro cytotoxicity assays were performed using luciferase-labeled cell lines. In vivo studies were performed using cell line-derived or patient-derived xenografts implanted in NOD scid gamma mice.Results We first confirmed ubiquitous CD72 expression across a range of primary B-cell non-Hodgkin lymphomas. We further found that after resistance to CD19-directed therapies, across both B-cell acute lymphoblastic leukemia (B-ALL) models and primary tumor samples, surface CD72 expression was largely preserved while CD22 expression was significantly diminished. Affinity maturation of a nanobody targeting CD72, when incorporated into CAR T cells, led to more effective elimination in vitro of isogenic models of CD72 low-expressing tumors. These results suggested that nanobody-based CAR T cells (nanoCARs) may exhibit a similar relationship between binder affinity, antigen expression, and efficacy as previously demonstrated only for single chain variable fragment-based CAR T cells. Surprisingly, however, this significantly improved in vitro efficacy only translated to modest in vivo survival benefit. As a parallel strategy to enhance CAR T function, we found that the small molecule bryostatin could also significantly increase CD72 surface antigen density on B-cell malignancy models. Structural modeling and biochemical analysis identified critical residues improving CD72 antigen recognition of our lead affinity-matured nanobody.Conclusions Together, these findings support affinity-matured CD72 nanoCARs as a potential immunotherapy product for CD19-refractory B-cell cancers. Our results also suggest that for B-ALL in particular, CD72 may be a preferable second-line immunotherapy target over CD22.

There remains an unmet clinical need for improved treatment strategies in Acute Myeloid Leukemia (AML). Although radiopharmaceutical therapies targeting non-cancer-selective antigens have shown promise in AML, their clinical utility is often limited by prolonged bone marrow suppression. Using a unique proteomics-based strategy, we recently identified the active conformation of integrin-β2 (aITGB2) as a novel, tumor-selective target for AML. Importantly, this conformational epitope is expressed widely on AML cells but minimally on normal marrow progenitors/healthy tissues. Here we first confirmed widespread aITGB2 expression on AML tumors that was largely independent of tumor genotype or prior therapeutic regimen. We developed diagnostic and therapeutic radiopharmaceuticals targeting aITGB2 utilizing a conformation-specific antibody (clone 7065). PET/CT imaging with 89Zr and 134Ce-labeled 7065 in AML models revealed high target-mediated uptake, greater than that compared to standard of care [18F]-FDG. PET/CT imaging with [89Zr]DFO*-7065 showed reduced binding to normal bone marrow and immune cells in humanized immune system mice compared to [89Zr]DFO*-anti-CD33. For therapy, we developed [225Ac]Macropa-PEG4-7065 using an optimized chelator-linker combination. Treatment with [225Ac]Macropa-PEG4-7065 in Nomo-1 and PDX AML disseminated models delayed tumor growth and improved overall survival compared to controls, including [225Ac]DOTA-anti-CD33, a clinical stage-radioimmunotherapy under evaluation in AML. Relapsed tumors demonstrated persistent aITGB2 expression, supporting continued development of fractionated dosing schemes, and proteomics analysis indicated activation of TCA cycle and carbon metabolism pathways, consistent with therapy-induced stress responses. These findings highlight [89Zr]DFO*-7065 and [225Ac]Macropa-7065 as a promising aITGB2-targeted theranostic pair with potential for imaging and treatment in future clinical translation.

CD19 CAR T-cells have markedly improved the survival of patients with B-cell malignancies. However, over half of patients relapse after this therapy, many due to CD19 antigen loss. Our laboratory discovered CD72, a B cell specific surface antigen widely expressed on B-cell malignancies. We developed an effective humanized nanobody-based CAR-T (“H24 nanoCAR”) against this target (Nix et al. Cancer Discov 2021, Temple et al. JITC 2023) that we are moving to clinical translation. However, we observed reduced efficacy of H24 nanoCAR versus tumor with low CD72 antigen density. We hypothesized that affinity matured CD72 nanoCAR-Ts could target lower antigen thresholds in lymphoma models. Affinity maturation and humanization of CAR-Ts was described in Temple et al. 2023. Humanized affinity matured NbD4.13-H24 CAR was generated by altering the framework region of clone NbD4.13 (KD = 0.8 nM) to that in clone H24 (KD = 33 nM). We previously described engineered JeKo1-CD72 low cell line (Izgutdina et al. ASH 2023). Flow cytometry confirmed ubiquitous CD72 expression on 20 primary B-NHL samples. Two B-ALL patient samples relapsed after CD19 CAR therapy showed downregulation of both surface CD19 and CD22, but surface CD72 was retained, raising the hypothesis that CD72 is a preferential second-line antigen after CD19 relapse. To evaluate efficacy versus CD72 antigen low model, we tested affinity matured NbD4.13 CAR and humanized affinity matured NbD4.13-H24, in comparison to H24 (clinical candidate), against an engineered JeKo1-CD72 low cell line. NbD4.13 and NbD4.13-H24 demonstrated enhanced cytokine secretion compared to H24. Upon repetitive restimulation with JeKo1-CD72 low, NbD4.13 CAR showed more rapid elimination of tumor than H24. However, NbD4.13 and H24 demonstrated equal efficacy in vivo versus mantle cell lymphoma PDX. In an in vivo experiment with JeKo1-low, NbD4.13 showed improved early tumor control than H24, but no survival benefit, while NbD4.13-H24 showed a modest survival benefit (p=0.0442 by Log-rank test). As an alternate strategy to address low antigen density, we showed that the protein kinase C inhibitor bryostatin can lead to increased surface CD72 when applied to the CD19 resistant model Nalm6-R. To understand the mechanism of action, we performed bulk RNAseq of CD72 antigen-low lymphoma cell line SC1 treated with bryostatin. GSEA showed that bryostatin broadly increases B cell lineage specific expression programs. CD72 is a potential second line antigen post-CD19 relapse in B-cell malignancies. We found that affinity maturation did not demonstrably improve CD72 nanoCAR efficacy. Our results support findings demonstrated for scFv-based CARs, that moderate affinity for surface antigen may lead to optimal CAR performance in vivo, even when nanobodies are used as the recognition element. Adila Izgutdina, Tasfia Rashid, Bonell Patino-Escobar, Sujata Walunj, Huimin Geng, Amrik Kang, Szu-Ying Chen, Haley Johnson, William C. Temple, Matthew A. Nix, Akul Naik, Emilio Ramos, Hiroyuki Takamatsu, Daniel Gil-Alós, Joaquin Martinez-Lopez, Antonio Valeri, Constance Yuan, Hao-Wei Wang, Sarah Aminov, Srabani Sahu, Christopher Carpenter, Fernando Salangsang, Paul Phojanakong, Juan Antonio Camara Serrano, Isa Tariq, Veronica Steri, Samir Parekh, Amit Verma, Nirali N. Shah, Arun P. Wiita. Anti-CD72 nanobody-based CAR-T affinity maturation leads to improved in vitro efficacy versus low antigen density lymphoma models, but limited benefit in vivo [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 4820.

ABSTRACT:Despite the success of B-cell maturation antigen (BCMA)-targeting chimeric antigen receptor (CAR) T cells (CAR-Ts) in multiple myeloma, patients with high-risk cytogenetic features continue to relapse most quickly and are in urgent need of additional therapeutic options. Here, we identify CD70, widely recognized as a favorable immunotherapy target in other cancers, as a specifically upregulated cell surface antigen in high-risk myeloma tumors. We use a structure-guided design to define a CD27-based anti-CD70 CAR-T design that outperforms all tested single-chain variable fragment-based CARs, leading to >80-fold improved CAR-T expansion in vivo. Epigenetic analysis via machine learning predicts key transcription factors and transcriptional networks driving CD70 upregulation in high-risk myeloma. Dual-targeting CAR-Ts against either CD70 or BCMA demonstrate a potential strategy to avoid antigen escape-mediated resistance. Together, these findings support the promise of targeting CD70 with optimized CAR-Ts in myeloma as well as future clinical translation of this approach.

The surface antigen landscape of acute myeloid leukemia (AML) displays significant heterogeneity and overlap with healthy hematopoietic cells. This imparts a substantial hurdle to the development of AML-targeting chimeric antigen receptor (CAR) T-cells that can avoid on-target, off-tumor toxicity. Here, we develop a dual-antigen targeting CAR-T against CD70 and the active conformation of integrin β2 (aITGB2), each previously reported as promising AML targets due to minimal off-tumor expression. We show an OR-gated approach for these antigens significantly increases the proportion of AML blasts that can be targeted, in part using a novel ex vivo co-culture method to restore surface protein homeostasis following a freeze-thaw cycle. We test dual-targeting CAR-T constructs with different combinations of costimulatory domains, identifying constructs with superior anti-tumor cytotoxicity in vitro against AML cell line and patient derived xenograft models. We further show significantly improved in vivo tumor clearance and survival for a dual targeting CAR in murine models of AML tumor heterogeneity. Finally, we show that this dual-targeting CAR does not increase off-tumor toxicity, especially against hematopoietic stem and progenitor cells. Together, these findings demonstrate a promising clinically-translatable approach for the treatment of AML without the notable toxicity liabilities associated with other leading CAR-T targets for this disease.

Despite the success of BCMA CAR-Ts, many multiple myeloma patients relapse and require additional therapeutic options. Our group previously identified the chemokine receptor CCR10 as a potential alternate target to address this need. Here, we validated CCR10 expression on primary myeloma tumors and sought to develop CAR T-cells against CCR10, utilizing its natural ligand CCL27 as a CAR binding element. However, CARs based on the native CCL27 sequence were ineffective. We thus utilized computational modeling and structure-guided engineering to inform rational mutations along the CCL27-CCR10 interface, exploiting a hydrophobic pocket on CCR10. This effort identified CCL27 mutants with an additional N-terminal aromatic amino acid that dramatically improved the efficacy of CCL27-based CAR-Ts to near that of current anti-BCMA CAR-Ts. We validated key amino acid contacts at the CCL27-CCR10 interface, which contribute to increased CAR binding avidity, predicted to be influenced by increased Van der Waals interactions. Lastly, we found that the CCL27 mutants have no toxicity in the hematopoietic compartment. This work illustrates the potential of engineering natural ligand CAR-Ts beyond their wild-type sequences and underscores the translational potential of engineered CCL27 mutant CAR-Ts.

Despite the success of BCMA-targeting CAR-Ts in multiple myeloma, patients with high-risk cytogenetic features still relapse most quickly and are in urgent need of additional therapeutic options. Here, we identify CD70, widely recognized as a favorable immunotherapy target in other cancers, as a specifically upregulated cell surface antigen in high risk myeloma tumors. We use a structure-guided design to define a CD27-based anti-CD70 CAR-T design that outperforms all tested scFv-based CARs, leading to >80-fold improved CAR-T expansion in vivo. Epigenetic analysis via machine learning predicts key transcription factors and transcriptional networks driving CD70 upregulation in high risk myeloma. Dual-targeting CAR-Ts against either CD70 or BCMA demonstrate a potential strategy to avoid antigen escape-mediated resistance. Together, these findings support the promise of targeting CD70 with optimized CAR-Ts in myeloma as well as future clinical translation of this approach.

Introduction: Acute myeloid leukemia (AML) remains a devastating disease with a great need for new therapies. While CAR-T therapy has achieved remarkable success in B-cell leukemias, lymphomas, and multiple myeloma, successful cellular therapy for AML remains elusive. A major contributing factor is thought to be antigen expression heterogeneity in AML, preventing complete tumor clearance with single antigen-targeting. Additionally, most leading AML targets (e.g. CD33, CD123) are also expressed on normal hematopoietic stem and progenitor cells (HSPCs), resulting in a limited therapeutic window and clinically deleterious hematologic toxicity. Our group previously identified the active conformation of integrin β2 (aITGB2) as an AML-specific target and generated a CAR-T therapy that eliminated AML tumors while sparing normal HSPCs (Mandal et al., Nat Cancer 2023). More recently, our group developed a potent natural ligand-based CAR-T therapy targeting CD70 (Kasap et al., ASH 2023), another antigen expressed on many AML cells but absent on normal HSPCs. Both aITGB2 and CD70 have heterogeneous expression on AML blasts, however, preventing complete tumor clearance. Here, we combine the potent efficacy and low toxicity of these two single-antigen CAR-Ts into a dual-antigen targeting approach capable of eliminating a larger proportion of AML cells, thereby driving deeper remissions while achieving an acceptable therapeutic index. Methods: Primary AML patient samples were co-cultured with the HS-5 stromal cell line in RPMI media with 20% FBS and 25 ng/ml IL-3, SCF, and FLT3L. Primary AML cells were assessed for expression of CD70 and aITGB2 by flow cytometry. CAR constructs were lentivirally transduced into CD3-selected T-cells. For In vitro cytotoxicity assays, luciferase-expressing target cells were co-cultured with CAR-Ts for 24 hours, with leukemia cell killing measured by endpoint luminescence. Results: To first verify the AML-specificity of CD70, we analyzed the TARGET AML RNA-seq dataset, observing significantly higher expression in AML compared to normal CD34+ and bone marrow cells (p<0.0001). We then profiled 10 primary AML samples for co-expression of CD70 and aITGB2 using flow cytometry. To better simulate the tumor microenvironment, we co-cultured these AML cells with a bone marrow stromal cell line and exogenous cytokines for 7 days (see Methods). This approach resulted in a notable increase in the percentage of AML blasts displaying CD70 and aITGB2, possibly by reestablishing cell surface homeostasis after shedding of surface proteins with a freeze-thaw cycle. We showed that a dual-targeting, OR-gated approach could eliminate a mean 87±11% of blasts, compared to 72±28% targeting aITGB2 alone or 44±26% for CD70 alone (p= 0.015, 1-way ANOVA). Utilizing our previously validated aITGB2 and CD70 binders, we designed 8 dual-targeting CAR-T constructs, differing in their costimulatory domains and relative CAR positions on the plasmid. Despite similar overall designs, we found surprisingly large differences in transduction and anti-tumor efficacy. We showed that an anti-aITGB2 CAR with a 4-1BB costimulatory domain, followed by an anti-CD70 CAR with CD28 costimulatory domain, demonstrated the best expression in T-cells. To prevent fratricide in CAR-Ts, which express both aITGB2 and CD70 when activated, we optimized a CRISPR-Cas9 protocol using a library of guide RNAs to knock out both targets in T-cells, with no defect in CAR transduction or killing ability observed. Our best dual-targeting construct showed dose-dependent tumor killing (p<0.0001, 2-way ANOVA) in vitro against the AML cell lines NOMO-1 and THP-1, outperforming the other dual-targeting CAR designs and maintaining similar efficacy to single-targeting CARs. We then generated CD70 and ITGB2 knockouts for each cell line and observed that our dual-targeting CARs preserved tumor killing while single antigen-targeting CARs could no longer target their respective knockout cell lines (P<0.0001, 2-way ANOVA). Conclusion: Overall, using an ex vivo co-culture system for target validation and strategies for optimizing a bicistronic CAR design, we demonstrate that a dual-antigen targeted CAR-T therapy directed against both aITGB2 and CD70 is a highly promising approach for AML. We are continuing preclinical development of this strategy, including in vivo mouse model studies, to further assess efficacy and safety.

Background: Even in the BCMA CAR-T era, many multiple myeloma (MM) patients often relapse and need additional treatment options. Our group identified CCR10 as a therapeutic target for MM and showed it is correlated with significantly worse outcomes and relapse. Additionally, we developed anti-CCR10 CAR-Ts that utilized the natural ligand CCL27 as the binder (Ferguson et al., Nat Comm 2022). However, these proof-of-concept CAR-Ts only had moderate efficacy in in vitro experiments. Here, we further validated CCR10 as a therapeutic target for MM and utilized structure-guided and computational modeling approaches to improve the performance of these CCL27 CAR-Ts to the level of current BCMA CAR-Ts. Methods: CAR-Ts were generated by lentiviral transduction of CD3+ human T cells. In vitro and in vivo performance of CAR-Ts was assessed against cell lines engineered to express luciferase. Statistical analysis on cytotoxicity was performed by two-way ANOVA. We used the Seurat and Harmony packages in R for data analysis and integration of single-cell RNAseq data. AlphaFold3 was utilized for generating predicted structural models, visualized by ChimeraX. Murine studies were performed with 1e6 MM.1S intravenously implanted in NSG mice, with 3e6 CAR-Ts administered 8 days after tumor inoculation (n=3 mice/arm). Results: Flow cytometry of 16 primary MM samples, 10 previously published and 6 new cases, showed that all samples were positive for CCR10. We also confirmed the expression of CCR10 on 7 MM cell lines by flow cytometry. Analyzing single-cell RNAseq data from 9 MM patients and 7 healthy donors, we found CCR10 was highly expressed in myeloma patient plasma cells, with lower expression in healthy donor plasma cells. To improve on our proof-of-concept CAR-Ts, we used homologous chemokine-chemokine receptor structures, the limited literature on CCL27, and AlphaFold models to identify the importance of the N-terminus of CCL27 in its interaction with CCR10. This structure-guided approach suggested that there was scope to improve the binding of CCL27 by modifying N-terminal residues and that our N-terminal MYC tag may have prevented CCR10 binding. From this, we created a small mutational library of 10 unique CCL27 CAR-T constructs with modified N-terminal residues, while moving the MYC tag to the C-terminus. Screening this library for in vitro cytotoxicity against the MM cell lines MM.1S and AMO1, we identified two mutants that performed similarly to BCMA CAR-Ts in one or both cell lines. These were both N-terminal additions of a single aromatic amino acid, tryptophan (W-CCL27) or phenylalanine (F-CCL27). Further validation in two new T-cell donors indicated that these mutants performed significantly better than the wild-type natural ligand (WT-CCL27) in MM.1S, AMO1, and LP-1 (p<0.05). Efficacy was correlated with CCR10 expression and equivalent to BCMA CAR in cell lines with high levels of CCR10. In these experiments, WT-CCL27 had minimal efficacy. Knocking out CCR10 in the AMO1 cell line, we observed no cytotoxicity, showing the specificity of these mutants. In an ongoing murine study with an orthotopic MM.1S model, we have observed no signs of toxicity and an increased efficacy of CCL27 mutants compared to WT-CCL27, similar to BCMA CAR. We further investigated these mutant CCL27s using AlphaFold3 to model their interaction with CCR10. The confident models indicated that the additional aromatic N-terminal amino acid on the mutants is interacting with a region of CCR10 not accessed by the WT-CCL27. We identified four amino acids in this binding pocket of CCR10 (V47, S48, Y120, and L300) that are predicted to interact with the mutant CCL27s. Finally, we evaluated on-target off-tumor toxicity of these CAR-Ts. Profiling GM-CSF-mobilized peripheral blood, we found no CCR10 on CD34+ HSPCs (n=3). Despite no signal in single-cell and bulk RNAseq data, we surprisingly observed expression of CCR10 by flow cytometry on monocytes in both healthy donors and MM primary samples. However, we saw minimal cytotoxicity of the mutant CCL27 CAR-Ts against the THP-1 cell line, a model of monocytes that expresses high levels of CCR10. We are now working to validate the potential MM cell-type specificity of these CCL27-based CAR-Ts. Conclusion: These anti-CCR10 mutant CCL27 CAR-Ts represent a promising therapeutic candidate for MM that indicates the potential of using a structure-guided design to optimize natural ligand-based binders.

The processes that govern human haematopoietic stem cell (HSC) self-renewal and engraftment are poorly understood and challenging to recapitulate in culture to reliably expand functional HSCs1-3. Here we identify MYC target 1 (MYCT1; also known as MTLC) as a crucial human HSC regulator that moderates endocytosis and environmental sensing in HSCs. MYCT1 is selectively expressed in undifferentiated human haematopoietic stem and progenitor cells (HSPCs) and endothelial cells but becomes markedly downregulated during HSC culture. Lentivirus-mediated knockdown of MYCT1 prevented human fetal liver and cord blood (CB) HSPC expansion and engraftment. By contrast, restoring MYCT1 expression improved the expansion and engraftment of cultured CB HSPCs. Single-cell RNA sequencing of human CB HSPCs in which MYCT1 was knocked down or overexpressed revealed that MYCT1 governs important regulatory programmes and cellular properties essential for HSC stemness, such as ETS factor expression and low mitochondrial activity. MYCT1 is localized in the endosomal membrane in HSPCs and interacts with vesicle trafficking regulators and signalling machinery. MYCT1 loss in HSPCs led to excessive endocytosis and hyperactive signalling responses, whereas restoring MYCT1 expression balanced culture-induced endocytosis and dysregulated signalling. Moreover, sorting cultured CB HSPCs on the basis of lowest endocytosis rate identified HSPCs with preserved MYCT1 expression and MYCT1-regulated HSC stemness programmes. Our work identifies MYCT1-moderated endocytosis and environmental sensing as essential regulatory mechanisms required to preserve human HSC stemness. Our data also pinpoint silencing of MYCT1 as a cell-culture-induced vulnerability that compromises human HSC expansion.

Hematopoietic stem cells (HSC) integrate diverse environmental cues to balance between quiescence, self-renewal and differentiation. However, the molecular programs governing HSC stemness become dysregulated during culture, compromising HSC self-renewal and engraftment ability. Despite recent advances, our ability to expand functional human HSCs in culture for therapeutic use is still limited. We uncovered MYCT1 as a novel regulator of human HSCs that becomes drastically downregulated during culture, concomitantly with loss of engraftment potential. We discovered that MYCT1 is essential for human HSPC ex vivo expansion and engraftment upon transplantation. Immunoprecipitation-mass spectrometry revealed that MYCT1 is a membrane-associated endosomal protein that interacts with signaling receptors with critical functions in HSC biology. MYCT1 knockdown (KD) in human ECs and HSPCs led to hyperactivation of endocytosis, a crucial regulatory step determining the responsiveness to extracellular cues and governing cell fate decisions. Phospho-proteomics, single-cell RNAseq, and functional assays revealed that MYCT1 KD causes widespread dysregulation of signaling pathways, including hypersensitivity to the cytokines in the media, loss of stemness programs, and defective proliferation. Importantly, overexpression of MYCT1 in human HSPCs rescued endocytosis, signaling, and transcriptomic programs that were dysregulated in cultured human HSPCs and further disrupted by MYCT1 KD. These data suggest that MYCT1 governs human HSC stemness by moderating environmental sensing through the control of endocytosis. As MYCT1 expression is downregulated in cultured HSPCs, the inability to properly sense microenvironmental signals may be a key mechanism contributing to culture-associated HSC dysfunction that will need to be overcome to restore transplantability of ex vivo expanded human HSCs.