Overexpression of the proto-oncogene Src is common to a wide variety of cancers. In this work, we found that Src is noncanonically translocated and inverted onto the cell surface in cancer, both in vitro and in vivo. We identified autophagolysosomal exocytosis (ALE) as a secretory mechanism prominent in cancer cell lines. Src represents the prototypical example of a family of membrane-anchored proteins that are transported by this process. Furthermore, this extracellular membrane-associated Src (eSrc) was found in primary tumors, and anti-Src antibody-based therapies mediated tumor cell killing in cell culture systems and in mouse xenograft models. Thus, intracellular N-myristoylated proteins, prototypically Src, can be topologically inverted onto the cell surface in cancer and targeted with antibody therapeutics.

Multiple myeloma (MM) is a malignancy of plasma cells characterized by bone destruction, anemia, and renal failure. Although therapeutic advances have improved outcomes, patients frequently relapse, and a definitive cure remains elusive. A major barrier to curative drug development is the lack of widely-available patient-derived xenograft (PDX) models that accurately replicate MM biology and clinical presentation. Existing models, particularly those using cell lines, often fail to reflect the slower growth kinetics and therapeutic resistance seen in primary disease. While humanized cytokine mouse models have shown promise, their complexity and limited accessibility have hindered widespread adoption. Here, we demonstrate a simplified and commercially-available human IL-6–expressing NOG mouse model enables consistent engraftment of primary MM cells and recapitulates hallmark clinical disease features. Six super-immunodeficient NOG mice expressing human IL-6 (NOD.Cg-Prkdc^scid Il2rg^tm1Sug Tg(CMV-IL6)1-1Jic/JicTac, Taconic Biosciences®) were used. The hIL-6 transgene, driven by the CMV promoter, was introduced via microinjection into fertilized NOD oocytes, followed by backcrossing with NOG mice. Serum IL-6 was quantified using an immunoassay (IDEXX BioAnalytics), based on Luminex xMAP technology. Mice were conditioned with 30 mg/kg busulfan (IP) 24 hours prior to tail vein injection of 1×10⁶ CD138⁺ frozen MACS-enriched primary MM cells harboring a t(4;14) translocation. T cells were inactivated using OKT3 (1 μg/million cells) on ice for 30 minutes prior to injection. Tumor engraftment was monitored biweekly from week 12 using serum protein electrophoresis (SPEP; Helena® QuickGel). At endpoint, bone marrow (BM) was collected for flow cytometry, imaging, and complete blood counts. ELISA confirmed stable hiL-6 expression in plasma, with a mean of 58.03 pg/mL (range: 49.16–68.75 pg/mL). Four of six mice developed a monoclonal gamma spike (M-spike) by SPEP, with a median detection time of day 141 (range: 117–159), and progressive increase over time. Flow cytometry on BM revealed ~45% CD138⁺ plasma cells with immunophenotype CD45-/CD19-/CD138+/CD38+/CD56+, consistent with MM (mean 45.2%, range 32.2%-60.1%). Therapeutic targets including BCMA and CD70 were expressed at levels typically observed in primary samples. Minimal amount of plasma cells was detected in spleen. Complete blood counts confirmed anemia in engrafted mice compared to NSG controls: Hemoglobin: 10.7 vs. 14.3 g/dL (p < 0.05), Hematocrit: 29.2 vs. 38.8% (p < 0.001), Red blood cell count: 6.0 vs. 8.24 × 10⁶/μL (p < 0.0001). BM processing revealed brittle femurs, suggestive of underlying bone disease. This was further supported by micro-CT imaging, which demonstrated medullary bone loss in the vertebrae. This human IL-6–expressing NOG PDX model enables robust engraftment of primary MM cells and recapitulates key clinical features, including anemia, bone marrow plasma cell infiltration, and bone disease. The model represents a valuable and accessible tool for studying MM biology and testing emerging therapies. Ongoing work includes renal function assessment, cytospin-based morphological validation, and genomic profiling (e.g., exome sequencing) pre- and post-engraftment. Future studies will evaluate CAR-T cell therapies, including strategies targeting CD70 in high-risk MM.

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.

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.

Background: Natural killer (NK) cells play a key role in immune surveillance of multiple myeloma (MM), but their function becomes progressively impaired as the disease advances. CD48, one of the most abundant NK-cell ligands on MM cells, engages the receptor CD244 (2B4), transmitting either activating or inhibitory signals depending on downstream adaptors such as SAP or inhibitory phosphatases like SHIP-1. However, the net effect of CD48–CD244 interaction (whether it promotes NK-cell activation or dysfunction) remains unclear. Methods: We combined bulk and single-cell RNA-seq (scRNA-seq), ATAC-seq, machine learning, genome-wide CRISPRa/KO screens, in vitro functional assays, and in vivo murine modeling to investigate CD48's regulation and role in NK-cell immunity. Results: In CoMMpass RNA-seq data (n=774), CD48 expression was highest in high-risk MM subsets (t(4;14), gain(1q21), p=<0.0001) and associated with inferior survival (p=0.0044). Unsupervised clustering of NK-ligand expression in tumor cells (including HLA-A/B/C/E, PCNA, MICA/B, PVR, ULBP1–3, NECTIN2, in addition to CD48) stratified patients into groups with distinct cytogenetics and outcomes, with broader NK-ligand patterns, such as Cluster B characterized by High expression of HLA-A/B/C/E and MICA/B, linked to improved survival. To uncover CD48 regulation, we analyzed ATAC-seq and ENCODE ChIP-seq data and trained an XGBoost model to predict CD48 expression from transcription factor activity. IRF4 and TFAP2A emerged as top positive regulators, while MEF2B acted as a negative regulator (R² = 0.40). Genome-wide CRISPRa/KO screens in MM cell lines identified 52 candidate regulators of surface CD48 expression, which clustered into functional categories including steroid receptor signaling, ER trafficking, and GPI-anchor biosynthesis. CRISPR-mediated validation confirmed that ESRRA represses and TFAP2A promotes CD48 surface expression. To test functional consequences, we engineered Vk*MYC MM cells to overexpress or knock out CD48 and co-cultured them with murine KIL C.2 NK cells. CD48 overexpression enhanced NK cytotoxicity and cytokine secretion in vitro, while CD48KO impaired activation. SHIP1 deletion in NK cells failed to restore cytotoxicity, suggesting deeper exhaustion mechanisms. Human scRNA-seq analysis (GSE223060, n=54 MM, 8 HD) revealed CD48–CD244 signaling from plasma cells and monocytes to NK cells, inferred using CellChat, was dominant in MM but not HD. MM-NK cells exhibited transcriptional exhaustion: upregulated SHIP1, SAP, and HLA-inhibitory receptors (e.g., NKG2A), alongside downregulation of AP-1 family members (FOS, JUN, JUNB). An CMV “adaptive-like” CD56-dim NK subset in MM co-expressed FGFBP2, KLRC2/3, and downregulated KLRB1/KLRF1, consistent with chronic stimulation. An independent scRNA-seq cohort (n>200) confirmed AP-1 family members suppression and elevated CD48 expression in MM over MGUS, SMM, or controls. In vivo, in the immunocompetent Vk*MYC model, CD48-overexpressing tumors showed delayed growth and extended survival vs. WT, while NK-cell depletion accelerated disease. NK cells isolated ex vivo from tumor-bearing mice showed functional exhaustion with reduced cytokine secretion and degranulation, even after PMA/ionomycin stimulation. Finally, murine scRNA-seq of Vk*MYC bone marrow samples across disease stages (ND, MM, AMM) revealed stepwise NK dysfunction: decreasing cytotoxicity, rising exhaustion and inflammatory module scores, and upregulation of HLA-inhibitory receptors, paralleling human MM data. Conclusion: CD48 exerts dual effects in MM, potentiating acute NK activation while driving chronic exhaustion via sustained CD244 engagement. Our multi-platform analysis uncovers transcriptional and post-translational CD48 regulators, validates immunocompetent Vk*MYC as a preclinical model of NK dysfunction, and identifies CD48–CD244 signaling as a key immunosuppressive axis. These insights suggest that modulating CD48 or its downstream effectors could restore NK surveillance and inform new immunotherapies for MM.

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.

Introduction: The balance between activating and inhibitory receptors on NK cells regulates their ability to recognize and eliminate target cells. This balance's impact on myeloma (MM) progression and immune surveillance remains unclear. The 2B4 (CD244) receptor modulates NK cell cytotoxicity and cytokine production, containing four Immunoreceptor Tyrosine-based Switch Motifs (ITSMs) that interact with various adaptor molecules to relay both inhibitory and activating signals. CD244's ligand, CD48, is a surface glycoprotein overexpressed in MM cells (Ferguson et al., Nat Comm 2022). While high CD48 expression in solid tumors often links to poor prognosis, its role in hematological malignancies is less clear. CD48 is believed to activate NK cells, and its absence may impair NK cell function, though this is not well elucidated. Methods: We assessed the role of CD48 in MM using RNA-seq data from the coMMpass (IA19) study, analyzed with DESeq2, survminer, and survival packages in R. Public scRNA-seq data (GSE223060) was also analyzed with Seurat v4.3.0, comparing 53 MM patients (MMPt) to 7 healthy donor (HD) bone marrow samples. Harmony package was used for data integration. To identify transcription factors (TFs) binding to the CD48 locus, MMPt ATAC-seq and ENCODE ChIP-seq data were analyzed, informing an eXtreme Gradient Boosting (XGBoost) model. NK cells' role in MM progression was examined using an in vivo mouse model with the Vk*MYC cell line, where NK cell depletion was achieved with anti-NK1.1 antibody, and MM progression was monitored via bioluminescence. Results: RNA-seq analysis of the CoMMpass dataset showed elevated CD48 expression in high-risk MM subtypes, such as t(4;14) and gain 1q21 (p-value < 0.0001). Higher CD48 expression correlated with poorer overall survival (top 20% vs. bottom 20% expression, p-value = 0.0044). This suggests that increased CD48 expression might impair NK cell immunosurveillance of MM, contrary to cell line-based CRISPR screens indicating CD48 can activate NK cells (Dufva et al., Immunity (2023); Liu et al., Nat Commun (2024)). Analysis of scRNA-seq data from 150,256 cells revealed a trend toward higher CD244 expression in MMPt NK cells compared to HD, with elevated levels of inhibitory phosphatases binding to ITSMs and triggering inhibitory NK cell signaling through CD244, such as SHIP1 (INPP5D), SHP1 (PTPN6), and EAT-2 (SH2D1B). SAP (SH2D1A), an activating phosphatase, was also higher in MMPt NK cells. Distinct NK cell subclusters were observed in MM patients, showing a phenotype similar to the adaptive NK CD56-dim phenotype with high expression of cytotoxic markers (GNLY, PRF1, NKG7, FGFBP2) and altered receptor expression, including downregulation of KLRB1 and KLRF1, and upregulation of inhibitory receptors KLRC2 and KLRC3. Notably, KLRC2 was predominantly upregulated in NK cell subclusters from MM patients absent in healthy donors. To explore potential drivers of CD48 expression in MM, ATAC-seq and ENCODE ChIP-seq data identified 89 TFs potentially binding the CD48 locus in primary MM samples. An XGBoost model indicated TFs associated with MM progression, such as IRF4, predicted CD48 expression (R^2 = 0.40). Using the syngeneic Vk*MYC mouse model, in vitro co-culture of CD48 knockout or CD48 overexpression Vk*MYC cells suggested that increased CD48 modestly enhanced KIL.C2 murine NK cell killing of tumor cells. In the immunocompetent Vk*MYC mouse model, NK cell depletion led to accelerated and more aggressive MM progression, highlighting the critical role of NK cells in tumor control. Conclusions: NK cells play a crucial role in controlling MM progression. Although previous studies have focused on CD48's role in NK cell activation using cells from HD, our findings suggest that CD48's effects on NK cells are complex and might be a significant prognostic factor in the MM microenvironment. Further research is needed to investigate NK cell surface markers related to dysfunction, such as CD244 and KLRC2, and to understand how increased CD48 influences MM evasion of NK cell-mediated surveillance, particularly in relation to adaptive NK cells and inhibitory phosphatases like SHIP1 and EAT-2.

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 Approximately 50% of patients who receive CD19 chimeric antigen receptor (CAR)-T cells relapse at one year. We previously found that CD72 is a novel immunotherapy target expressed on B-cell acute lymphoblastic leukemia (B-ALL) and B-cell non-Hodgkin lymphoma (B-NHL), and that anti-CD72 nanobody CAR-T cells (CD72 nanoCARs) were highly potent. Further work revealed that humanized CD72 nanoCARs (H24 nanoCARs) unexpectedly had enhanced anti-tumor potency due to increased binding affinity for CD72. Here we further characterize H24 nanoCARs, our leading preclinical candidate, to translate this promising novel cellular therapy from the bench to the bedside. Objectives Present the first data regarding preclinical efficacy, toxicity, CAR expansion, and pharmacologic modulation of CD72 antigen density. Methods CD72 nanoCARs were produced by transducing T cells with a lentivirus containing an anti-CD72 transgene. CD19 CAR-T cells were generated with a tisagenlecleucel backbone. Empty CAR-T cells do not contain an antigen binding domain and were a negative control. Results We identified B-ALL blasts from patients that relapsed after CD19 CAR-Ts and found CD72 expression is retained on blasts with CD19 antigen escape (Fig. 1A). Furthermore, H24 nanoCARs potently eliminate these tumors (Fig. 1B). To understand transcriptional differences between CD19 CAR-Ts and H24 nanoCARs, RNA-sequencing was performed and revealed significant transcriptional changes. H24 nanoCARs have similar proliferation kinetics in vitro (Fig. 1C) and in vivo compared to CD19 CAR-T cells. To explore the safety profile of H24 nanoCARs, we found there is no predicted off target binding with our anti-CD72 nanobody binder, and there is no apparent in vivo organ toxicity on autopsy in immunocompromised murine models with SEM tumors that received H24 nanoCARs. We also found that bryostatin1, a protein kinase C inhibitor, increases CD72 antigen density on the surface of a mantle cell lymphoma cell line (JeKo-1) (Fig 1D). Conclusion This is the first demonstration that H24 nanoCARs have a reassuring preclinical toxicity profile with robust CAR expansion and preserved anti-tumor efficacy against B-ALL that relapsed after CD19 CAR-Ts. This work supports clinical translation of this novel cellular therapy.

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.

Foreign epitopes for immune recognition provide the basis of anticancer immunity. Due to the high concentration of extracellular adenosine triphosphate in the tumor microenvironment, we hypothesized that extracellular kinases (ectokinases) could have dysregulated activity and introduce aberrant phosphorylation sites on cell surface proteins. We engineered a cell-tethered version of the extracellular kinase CK2α, demonstrated it was active on cells under tumor-relevant conditions, and profiled its substrate scope using a chemoproteomic workflow. We then demonstrated that mice developed polyreactive antisera in response to syngeneic tumor cells that had been subjected to surface hyperphosphorylation with CK2α. Interestingly, these mice developed B cell and CD4+ T cell responses in response to these antigens but failed to develop a CD8+ T cell response. This work provides a workflow for probing the extracellular phosphoproteome and demonstrates that extracellular phosphoproteins are immunogenic even in a syngeneic system.

Acquired resistance to targeted FLT3 tyrosine kinase inhibitors (TKIs) remains a major barrier to durable clinical responses for patients with FLT3-mutant AML. Multiple resistance mechanisms to next generation FLT3 TKIs converge upon RAS/MAPK pathway activation, including protective cytokine stimuli of the bone marrow microenvironment or through secondary oncogenic RAS mutations. RAS mutations also decrease efficacy of the BCL2 inhibitor venetoclax now widely used in AML, suggesting an anti-apoptotic influence of MAPK signaling. These findings highlight a significant unmet need for novel therapeutic combinations to address MAPK-mediated resistance in AML. We report results from a genome-wide CRISPR interference (CRISPRi) apoptosis screen using FLT3-ITD mutant MOLM-14 cells treated with the FLT3 TKI gilteritinib while cultured in HS5 conditioned media (CM) to mimic cell-extrinsic MAPK-driven TKI resistance. We identified RNA pol II and Mediator kinase/CDK8 ascritical nodes for AML cell survival in the setting of FLT3 inhibition. We further demonstrated that CDK8 inhibition with the small-molecule SEL120 re-sensitized multiple FLT3-mutant AML cell lines and primary samples to gilteritinib. We also performed a second CRISPRi apoptosis screen to identify hits that sensitize to gilteritinib in a model of cell-intrinsic MAPK-mediated resistance using MOLM-14 cells that harbor secondary NRAS-G12C mutation.We used Enrichr to perform an integrated analysis and validated the Core Mediator Complex and CDK8 knockdown genes as cellular components significantly enriched among sensitizing hits in both screens. The Mediator subunit MED12 was identified as a top gene level hit that strongly sensitized cells to gilteritinib in both screens, further emphasizing that Mediator kinase and its transcriptional regulation may be a central pathway engaged in MAPK-dependent survival in FLT3-mutant AML. To better understand potential mechanisms of increased combinatorial CDK8i/FLT3i activity, we performed RNA-seq in MOLM-14 cells treated with gilteritinib, SEL120, or the combination in HS5 CM. Using GSEA we found that gilteritinib stimulated adaptive interferon/inflammatory gene signatures at 16h drug treatment, but this response was restrained by addition of SEL120. Additionally, we identified a striking upregulation of the lineage-controlling transcription factor IRF8 and its gene expression signature with concomitant silencing of SPI1 transcriptional output in cells treated with gilteritinib/SEL120. CDK8i treatment alone in AML has previously been shown to increase expression of super-enhancer (SE) associated genes, including IRF8. Interestingly, SPI1was also a significant gene-level hit in both CRISPRi screens. Using CRISPRi we repressed IRF8 expression in MOLM-14, MOLM-14 NRAS-G12C, and MV411 (another FLT3-mutant line) cells and found this significantly blunted the combinatorial pro-apoptotic effect of SEL120, implying that induction of IRF8 expression plays a significant role in driving response to combined gilteritinib/SEL120. We finally assessed in vivo activity of gilteritinib combined with SEL120. We first performed pilot toxicity studies in NSG mice and determined that gilteritinib 30 mg/kg, SEL120 30 mg/kg, or the combination dosed 5 days/week by oral gavage (OG) were regimens well tolerated by mice. We then engrafted luciferase-tagged MOLM-14 and MOLM-14 NRAS-G12C cells into NSG mice and treated for 4-weeks with gilteritinib, SEL120, or the combination. We found gilteritinib/SEL120 extended survival in both MOLM-14 and MOLM-14 NRAS-G12C models. Similar efficacy studies are ongoing in FLT3-mutant patient-derived xenograft (PDX) models with NSGS mice and will be reported. Our results indicate that FLT3-mutant AML cells employ an adaptive inflammatory response to evade FLT3i-induced apoptosis, and CDK8i represses this adaptation while concomitantly increasing the ratio of IRF8 to SPI1-driven gene expression programs. We speculate this latter effect may in part alter an AML cell's differentiation state to sensitize cells to apoptosis, though further study to explore this hypothesis is needed. Our in vitro and in vivo efficacy data further validated combined FLT3i/CDK8i as a promising investigational strategy to pre-empt or overcome MAPK-mediated FLT3 TKI resistance.

Background: Even in the CD19 CAR-T era, many patients with relapsed/refractory B cell non-Hodgkin lymphoma (B-NHL) need additional therapeutic options. Our group has identified CD72 as a therapeutic target for high-risk B-cell malignancies and validated anti-CD72 CAR-Ts (“nanoCAR”s) for these indications in relevant preclinical models (Nix et al., Cancer Discovery 2021, Temple et al., ASH 2022). However, patient B-NHL tumors may express a wide distribution of surface CD72, and our current CAR-T designs may be less efficacious for tumors with low antigen density. We previously described the development of affinity-matured binders extending from our initial published anti-CD72 nanobody clone (NbD4), with ~20-60x higher target affinity (Temple et al.). Here, we tested the hypothesis that these higher affinity binders would better eliminate low antigen density B-NHL models, both in vitro and in vivo, and impact other properties including avidity, and antigen binding epitopes. Methods: Anti-CD72 affinity-matured binders were developed by random mutagenesis of CDR regions of the nanobody clone NbD4 (described in Temple et al.). CAR-T cells were generated by lentiviral transduction. CAR-T cell efficacy was tested by in vitro cytotoxicity assays and Incucyte live-cell imaging against lymphoma cell lines. For in vivo studies, JeKo-1 tumor cells were implanted at 1e6 per mouse and then mice were treated with 3.5e6 CAR+ T-cells at day 7. Tumors from mice bearing JeKo-1 that relapsed on CAR-T treatment were isolated and used in Incucyte assays. Results: We analyzed publicly available datasets of patient cohorts and found that CD72 is widely expressed, though with a broad distribution, across diffuse large B cell lymphoma (DLBCL), follicular lymphoma, and Burkitt lymphoma patient samples. In both DLBCL and chronic lymphocytic leukemia (CLL), high expression of CD72 correlated with significantly shorter overall survival (DLBCL: GSE10846, n=233, p = 0.0382; CLL: GSE22762, n=107, p = 0.00124, log-rank test). We confirmed expression of CD72 on 8 lymphoma cell lines by flow cytometry. In vitro against a subset of these lines, affinity-matured clone NbD4.13 showed improved cytotoxicity compared to parental NbD4 and similar cytotoxicity to a humanized variant (H24 clone). We developed a JeKo-1 model of low CD72 antigen density (4.7x MFI reduction) by CRISPR/Cas9 knockout of endogenous CD72 and re-expression of a recombinant construct. In this “CD72 low” model, affinity matured clone NbD4.13 led to improved cytotoxicity vs. H24 ( p = 5.1e-4 by t-test). We previously showed that tumor isolated from JeKo-1 mice treated with H24 CAR appear to have reduced antigen expression at relapse (Temple et al.). We cocultured these post-relapse JeKo-1 tumors with various CD72 nanoCARs and found that NbD4.13 eliminates tumor significantly faster at 24 hours than H24 by live-cell imaging (p = 0.0054 by t-test). In vivo in a JeKo-1 model, NbD4.13 also out-performed the H24 clone with respect to survival benefit (p = 0.011 by log-rank for survival) ( Fig. 1). To assess if additional differences beyond affinity may determine the efficacy of NbD4.13, we performed structural modeling using AlphaFold and HADDOCK. However, this analysis predicts NbD4, NbD4.13 and H24 bind a similar non-linear epitope on the CD72 monomer. Furthermore, acoustic force microscopy did not reveal a significant difference in binding avidity for any of the compared CD72 nanoCAR constructs. Finally, we evaluated whether it could be possible to pharmacologically increase CD72, as co-treatment strategy to enhance efficacy of CD72 nanoCARs. We found that the Protein Kinase C inhibitor Bryostatin-1, previously demonstrated to increase CD22 antigen density (Ramakrishna et al., Clin Cancer Res 2019), could also significantly increase surface CD72 in vitro in both B-ALL and B-NHL models ( Fig. 2). Conclusion: NbD4.13-based anti-CD72 CAR is a promising candidate for further preclinical development for treatment of relapsed/refractory B-cell non-Hodgkin lymphoma. Given anticipated heterogeneity in CD72 expression in various lymphomas, pharmacologic co-treatment strategies may be considered to modulate tumor antigen density.

Background Approximately 50% of patients who receive anti-CD19 CAR-T cells relapse, and new immunotherapeutic targets are urgently needed. We recently described CD72 as a promising target in B-cell malignancies and developed nanobody-based CAR-T cells (nanoCARs) against it. This cellular therapy design is understudied compared with scFv-based CAR-T cells, but has recently become of significant interest given the first regulatory approval of a nanoCAR in multiple myeloma.Methods We humanized our previous nanobody framework regions, derived from llama, to generate a series of humanized anti-CD72 nanobodies. These nanobody binders were inserted into second-generation CD72 CAR-T cells and were evaluated against preclinical models of B cell acute lymphoblastic leukemia and B cell non-Hodgkin’s lymphoma in vitro and in vivo. Humanized CD72 nanoCARs were compared with parental (“NbD4”) CD72 nanoCARs and the clinically approved CD19-directed CAR-T construct tisangenlecleucel. RNA-sequencing, flow cytometry, and cytokine secretion profiling were used to determine differences between the different CAR constructs. We then used affinity maturation on the parental NbD4 construct to generate high affinity binders against CD72 to test if higher affinity to CD72 improved antitumor potency.Results Toward clinical translation, here we humanize our previous nanobody framework regions, derived from llama, and surprisingly discover a clone (“H24”) with enhanced potency against B-cell tumors, including patient-derived samples after CD19 CAR-T relapse. Potentially underpinning improved potency, H24 has moderately higher binding affinity to CD72 compared with a fully llama framework. However, further affinity maturation (KD<1 nM) did not lead to improvement in cytotoxicity. After treatment with H24 nanoCARs, in vivo relapse was accompanied by CD72 antigen downregulation which was partially reversible. The H24 nanobody clone was found to have no off-target binding and is therefore designated as a true clinical candidate.Conclusion This work supports translation of H24 CD72 nanoCARs for refractory B-cell malignancies, reveals potential mechanisms of resistance, and unexpectedly demonstrates that nanoCAR potency can be improved by framework alterations alone. These findings may have implications for future engineering of nanobody-based cellular therapies.

Safely expanding indications for cellular therapies has been challenging given a lack of highly cancer-specific surface markers. Here we explore the hypothesis that tumor cells express cancer-specific surface protein conformations that are invisible to standard target discovery pipelines evaluating gene or protein expression, and these conformations can be identified and immunotherapeutically targeted. We term this strategy integrating cross-linking mass spectrometry with glycoprotein surface capture ‘structural surfaceomics’. As a proof of principle, we apply this technology to acute myeloid leukemia (AML), a hematologic malignancy with dismal outcomes and no known optimal immunotherapy target. We identify the activated conformation of integrin β 2 as a structurally defined, widely expressed AML-specific target. We develop and characterize recombinant antibodies to this protein conformation and show that chimeric antigen receptor T cells eliminate AML cells and patient-derived xenografts without notable toxicity toward normal hematopoietic cells. Our findings validate an AML conformation-specific target antigen and demonstrate a tool kit for applying these strategies more broadly.

Background: Even in the BCMA CAR-T era, multiple myeloma (MM) patients with high-risk genotypes have the poorest outcomes. We thus leveraged MMRF CoMMpass data to identify potential novel surface immunotherapeutic targets in high-risk MM. As described below, we focused on CD70, an immunotherapeutic target known to be upregulated on many hematologic and solid tumors but minimally expressed on normal tissue. While CD70 was previously explored in MM (McEarchern et al, Clin Cancer Res (2008)), it was not further investigated due to heterogeneous expression in newly diagnosed samples. Here we revisit this target to suggest cellular therapies against CD70 could be highly beneficial in high-risk MM patients, currently the greatest unmet need in the field. Methods: Primary patient samples were obtained under an IRB-approved protocol. CAR-T lentiviral transduction was performed on paired CD4/CD8 human T cells from at least 3 healthy donors. Effector function was assessed by cytotoxicity against MM cell lines and CAR-T profiling for memory and exhaustion markers. In vivo efficiency was assessed using bioluminescence. ATAC-seq peaks were called using Promo and HOMER (data from Jin et al. Blood (2018)). Machine learning was performed using XGBoost approach applied to CoMMpass RNA-seq data with 80% of samples for training and 20% as test set. Results: Analyzing CoMMpass mRNA data (release IA19) we first evaluated surface protein encoding-genes upregulated in tumors at relapse compared to diagnosis. Among >100 significantly upregulated genes, we focused on CD70 given known promise as an immunotherapy target. Further analysis across genomic subtypes revealed a specific, marked upregulation of CD70 in t(4;14) and gain 1q ( Fig. 1), as well as R-ISS 3 at diagnosis. Flow cytometry profiling of 8 cell line models, including t(4;14) models KHM11, KMS26, and LP1, revealed consistent CD70 protein expression with antigen density similar to BCMA. Profiling of relapsed primary MM samples confirmed that 19 of 28 samples had CD138+ plasma cells positive for CD70 expression. Of 5 patients in whom both BCMA and CD70 were measured, antigen densities were also similar. To target CD70, we then designed 10 unique anti-CD70 CAR-T constructs. 9 of these were scFv-based binders derived from therapeutic antibody fragments currently in preclinical or clinical development. By a structure-guided design based on the CD70:CD27 co-crystal structure (PDB: 7KX0), we also derived a unique truncated fragment of CD27 as an alternative CD70 binding element. All CD70 CARs were efficacious in in vitro cytotoxicity assays targeting MM cell lines AMO1, MM1.S, LP1, and RPMI8226. We then selected the top 3 performing scFv based-CARs as well as our natural ligand (CD27) CAR-T for in vivo studies. All 4 CD70 CAR-Ts prolonged survival of MM.1S-bearing mice compared to empty CAR ( n=5 mice/arm, p<0.005 by log-rank), similar to BCMA CAR-T controls. Remarkably, the CD27-based CAR showed superior expansion (50-85x, p< 0.0001 by ANOVA) and persistence compared to scFv-based ( Fig. 2). These results underscore that targeting CD70 using a natural ligand-based CAR-T represents a promising preclinical candidate for treatment of relapsed and high risk MM. We further investigated why CD70 may be upregulated in high risk MM. Analysis of ChIP-seq in ENCODE and primary MM tumor ATAC-seq was used to nominate a set of transcription factors that bind the CD70 locus. Extending from CoMMpass data, we built a predictive machine learning model for CD70 expression as a function of transcription factor gene expression, nominating key transcription factors TFAP2A, LEF1, MYB, and PAX5 as CD70 regulators; validation is now underway. Additionally, we showed that knockout of NSD2 in t(4;14) models KMS34 and KMS11 led to significantly decreased CD70 surface expression, implicating this central epigenetic driver in CD70 overexpression in this high-risk MM subtype. Finally, we also demonstrated that treatment with the hypomethylating agent azacytidine can drive increased CD70 expression, similar to that shown in AML models. Conclusion: Taken together, targeting CD70 appears to be a highly promising cellular therapeutic strategy for MM, with particularly high utility in high-risk patients after BCMA CAR-T relapse. We are continuing preclinical and future clinical development of this CAR-T strategy, using our natural ligand CD27 design with extended in vivo persistence.

Targeted degradation of cell surface and extracellular proteins via lysosomal delivery is an important means to modulate extracellular biology. However, these approaches have limitations due to lack of modularity, ease of development, restricted tissue targeting and applicability to both cell surface and extracellular proteins. We describe a lysosomal degradation strategy, termed cytokine receptor-targeting chimeras (KineTACs), that addresses these limitations. KineTACs are fully genetically encoded bispecific antibodies consisting of a cytokine arm, which binds its cognate cytokine receptor, and a target-binding arm for the protein of interest. We show that KineTACs containing the cytokine CXCL12 can use the decoy recycling receptor, CXCR7, to target a variety of target proteins to the lysosome for degradation. Additional KineTACs were designed to harness other CXCR7-targeting cytokines, CXCL11 and vMIPII, and the interleukin-2 (IL-2) receptor-targeting cytokine IL-2. Thus, KineTACs represent a general, modular, selective and simple genetically encoded strategy for inducing lysosomal delivery of extracellular and cell surface targets with broad or tissue-specific distribution.