Adoptive transfer of T cells engineered with tumor-specific T cell receptors (TCRs) has shown limited efficacy in solid tumors, hindered by insufficient persistence, tumor trafficking, and dependence on tumor-associated co-stimulatory ligands. In a phase I trial (NCT 04639245) for patients with metastatic MAGE-A1-expressing tumors and adequate organ function; one participant received treatment, which was well-tolerated. In this case and NSG murine models, infusion of CD4/CD8 T cells co-expressing a class-I MAGE-A1-specific TCR and CD8αβ, failed to control tumor progression. To enhance function downstream of TCR signaling, here we investigate the adaptability of TCR components to synthetic modification. Leveraging the obligate co-expression of CD8αβ required for class-I TCR function in CD4 T cells, we identify CD8β as a tractable site for engineering without loss of function. In vitro screening demonstrates incorporation of the CD28 intracellular tail, yielding a CD8/CD28 chimeric co-receptor, most effectively enhances cytokine production, T cell persistence, and tumor control in immunodeficient murine models while preserving stem-like transcriptional features compared to native CD8β. Further rational modification of the CD28 binding motifs improves tumor control in vivo with increased intratumoral accumulation and reduced exhaustion. This benefit also extends to PRAME and WT1-specific TCRs in vitro supporting generalizability. TCR-engineered T cells have shown limited efficacy in part due to the absence of co-stimulation leading to limited accumulation in solid tumors. The authors here show engineering the CD8β coreceptor with an intracellular CD28 domain enhances cytokine production, persistence, and tumor control in vivo independent of tumor-associated co-stimulatory ligand encounter.

Adoptive therapies using T cells genetically modified with T cell receptors (TCR)s have shown limited efficacy in the solid tumor setting. Although functional CD4 + and CD8 + T cells transduced with a TCR specific for HLA-A2-restricted melanoma-associated antigen A1 (MAGE-A1, T TCR−MA1−CD8αβ ) could be detected post-transfer and were safe in one patient who subsequently progressed, T TCR−MA1−CD8αβ were insufficient to sustain antitumor activity in “stress” mouse tumor models. Leveraging the obligate co-expression of CD8αβ required for engagement of CD4 + T cells expressing the TCR, we screened positive co-stimulatory signals tethered to the intracellular tail of CD8β and identified that CD28 reduced exhaustion, enhanced tumor infiltration and improved murine tumor control. Further modifications of the CD28 intracellular domain produced a mutant CD8β-CD28 construct that conferred superior therapeutic control across tumor models. Thus, integrating co-stimulatory signals downstream of the TCR signaling complex can enhance TCR-engineered T cell function, independent of tumor-associated co-stimulatory ligand expression.

CD47 expression on canine cancer samples.

Recombinant production and evaluation of canine SIRPalpha protein.

Supplementary Table S1. Similarity between CD47 variants across species. Supplementary Table S2. Similarity between SIRPα variants across species. Supplementary Table S3. Summary of tumor necrosis scores following treatment with the indicated therapies. Supplementary Figure Legends. Supplementary Materials and Methods.

Combination therapy with CD47-blockade and anti-CD20 antibodies produces in vivo efficacy with no overt toxicity.

The success of T cell immunotherapies in the solid tumor is impeded by challenges in part created by the secretion of metabolic byproducts from the tumor, which generate a hostile and immunosuppressive tumor microenvironment (TME) (Tan et al., 2021). Dysregulated tumor metabolism results in the accumulation of metabolites that are exported into the interstitial space in the tumor, where they act as signaling molecules promoting the immunosuppressive landscape, ultimately reducing anti-tumor responses to T cell therapies (Jiang et al., 2020; Joyce & Fearon, 2015). Tumor cells exhibit several advantages through their high metabolic plasticity, but metabolic aggression deprives surrounding immune cells of nutrients, leading to exhausted, non-functional, and suppressive phenotypes (Lim et al., 2020). Regulatory T cells (Tregs) are a central immunosuppressive component of the TME that can rewire their metabolism and obstruct anti-tumor immunity by suppressing proliferation and activation of CD8+ T cells and CD4+ helper T cells in the TME (Betts et al., 2012; McNally et al., 2011). High Treg accumulation is associated with reduced anti-tumor responses, reduced efficacy of TCR therapies and poor clinical outcomes (Onda et al., 2019; Preston et al., 2013; Tang et al., 2014). Therefore, there is an imminent clinical need for the development of more synergistic therapies that specifically target Tregs in the TME to successfully overcome the barriers of infiltration and function of antigen-specific T cells. Accumulation of succinate, a key mitochondrial metabolite, occurs in conditions of low oxygen and increased energy demand, such the TME of solid tumors, and mutations in the gene that encoding succinate dehydrogenase ( Sdh) have been identified in a wide variety of solid tumors (Killian et al., 2013; Roh et al., 2019),resulting further an elevation of succinate levels. Accumulated succinate is secreted into the interstitial space (Garrigue et al., 2017) where it can stimulate the membrane bound succinate receptor (SUCNR1) on neighboring cells, further polarizing their immunosuppressive phenotype, and skewing macrophages to an M2 anti-inflammatory phenotype (Trauelsen et al., 2021; Wu et al., 2020). However, the effect of succinate on Tregs has not been explored. We hypothesized that succinate drives immunosuppression in the solid tumor by promoting Treg proliferation and function, leading to reduced anti-tumor responses to antigen-specific T cells. Consistent with this hypothesis, we confirmed that CD4+ T cells express the SUCNR1 and demonstrated that succinate promotes Treg numbers in CD4+ T cells isolated from healthy donor PBMCs ( Figure 1A). This effect was blocked upon administration of the SUCNR1 antagonist, NF-56-EJ40 ( Figure 1A). Deletion of the Sdhb gene, which encodes a subunit of the succinate processing enzyme, leads to intracellular accumulation of succinate. We deleted Sdhb in the non-small cell lung carcinoma cell line (H1299) and a melanoma cells line (A375) and confirmed that Sdhb -/- tumors secreted higher levels of succinate than WT. We translated these findings to a pre-clinical immunodeficient mouse model and determined that high succinate producing H1299 and A375 tumors had higher numbers of infiltrated Tregs in the TME compared with WT tumors ( Figure 1B), indicating a central role for succinate in the immunosuppressive landscape of the TME and identifying the suppression of succinate signaling as a potential therapeutic strategy to decrease immunosuppressive Tregs. Targeting Tregs in the TME is an attractive therapeutic strategy and regulating the induction and function of Tregs has the potential to increase the efficacy of effector T cells, thus improving anti-tumor responses by removing a critical suppressive barrier. Based on these findings, we propose a novel mechanism of Treg-mediated immunosuppression driven by succinate, whereby succinate promotes a Treg-rich environment in the TME and pushes Tregs towards a Th1-suppressing phenotype. These studies identify a previously unknown molecular regulation of Tregs by extracellular succinate and provide a novel therapeutic target that has the potential to enhance the effectiveness of TCR therapy in solid tumors thereby laying a framework for translational therapeutic development and innovation, that will limit suppression of and enhance tumor-specific T cell cytotoxicity.

Human CD34 + hematopoietic stem and progenitor cells (HSPCs) are a standard source of cells for clinical HSC transplantations as well as experimental xenotransplantation to generate “humanized mice”. To further extend the range of applications of these humanized mice, we developed a protocol to efficiently edit the genomes of human CD34 + HSPCs before transplantation. In the past, manipulating HSPCs has been complicated by the fact that they are inherently difficult to transduce with lentivectors, and rapidly lose their stemness and engraftment potential during in vitro culture. However, with optimized nucleofection of sgRNA:Cas9 ribonucleoprotein complexes, we are now able to edit a candidate gene in CD34 + HSPCs with almost 100% efficiency, and transplant these modified cells in immunodeficient mice with high engraftment levels and multilineage hematopoietic differentiation. The result is a humanized mouse from which we knocked out a gene of interest from their human immune system.

1E4 antibody binds canine CD20 extracellular domain 2.

CV1 does not inhibit proliferation of CLBL-1 cells in vitro.

Historically, the role of radiation in gynecological metastatic disease involved palliation for pain or bleeding. Stereotactic Body Radiation Therapy (SBRT) has shown survival benefits in oligometastatic disease from varying primary histologies in recent randomized trials. However, gynecologic primary oligometastases have been underrepresented in these trials. Recent studies across gynecological malignancy types have similarly shown favorable outcomes and acceptable toxicities from treating recurrent or oligometastatic gynecologic cancer (ROMGC) patients with definitive radiation therapy. The largest body of literature reported on the use of SBRT in ovarian cancer, which was found to be an effective option, especially in the setting of chemo-resistant disease. Despite the encouraging outcomes using SBRT in oligometastatic gynecologic malignancies, SBRT remains underutilized given the lack of randomized studies studying ROMGC with long term follow-up. While waiting for future prospective trials to establish the role of SBRT as the standard of care in ROMGC patients, this review focuses on reporting the advantages and drawbacks of this technique and examines the current literature to help guide patient centered treatment decisions.

Canine macrophages respond to CD47-blocking therapies in vitro.

Microscopic visualization demonstrates macrophage engulfment of CLBL-1 cells.

Despite recent therapeutic progress, advanced melanoma remains lethal for many patients. The composition of the immune tumor microenvironment (TME) has decisive impacts on therapy response and disease outcome, and high-dimensional analyses of patient samples reveal the heterogeneity of the immune TME. Macrophages infiltrate TMEs and generally associate with tumor progression, but the underlying mechanisms are incompletely understood. Because experimental systems are needed to elucidate the functional properties of these cells, we developed a humanized mouse model reconstituted with human immune cells and human melanoma. We used two strains of recipient mice, supporting or not supporting the development of human myeloid cells. We found that human myeloid cells favored metastatic spread of the primary tumor, thereby recapitulating the cancer-supportive role of macrophages. We next analyzed the transcriptome of human immune cells infiltrating tumors versus other tissues. This analysis identified a cluster of myeloid cells present in the TME, but not in other tissues, which do not correspond to canonical M2 cells. The transcriptome of these cells is characterized by high expression of glycolytic enzymes and multiple chemokines and by low expression of gene sets associated with inflammation and adaptive immunity. Compared with humanized mouse results, we found transcriptionally similar myeloid cells in patient-derived samples of melanoma and other cancer types. The humanized mouse model described here thus complements patient sample analyses, enabling further elucidation of fundamental principles in melanoma biology beyond M1/M2 macrophage polarization. The model can also support the development and evaluation of candidate antitumor therapies.

Since the late 1980s, mice have been repopulated with human hematopoietic cells to study the fundamental biology of human hematopoiesis and immunity, as well as a broad range of human diseases in vivo. Multiple mouse recipient strains have been developed and protocols optimized to efficiently generate these “humanized” mice. Here, we review three guiding principles that have been applied to the development of the currently available models: (1) establishing tolerance of the mouse host for the human graft; (2) opening hematopoietic niches so that they can be occupied by human cells; and (3) providing necessary support for human hematopoiesis. We then discuss four remaining challenges: (1) human hematopoietic lineages that poorly develop in mice; (2) limited antigen-specific adaptive immunity; (3) absent tolerance of the human immune system for its mouse host; and (4) sub-functional interactions between human immune effectors and target mouse tissues. While major advances are still needed, the current models can already be used to answer specific, clinically-relevant questions and hopefully inform the development of new, life-saving therapies.

e17035 Background: CD47 is an antiphagocytic signal and macrophage checkpoint that bladder and other cancer cells over-express to evade innate immunity. Magrolimab (Hu5F9-G4) a CD47 blocking antibody, promotes phagocytosis of cancer cells by macrophages and is being tested in several clinical trials (NCT02953509, NCT03248479, NCT02953782, NCT03558139). Chemotherapies synergize with magrolimab by increasing “eat me” signals on cancer cells, and thus enhancing phagocytosis. This synergy has been shown in MDS and AML, whereby magrolimab+azacitidine has shown encouraging efficacy in pre-clinical and clinical studies. This study aimed to investigate the effect of magrolimab as monotherapy and in combination with gemcitabine-cisplatin chemotherapy in bladder cancer. Methods: Phagocytosis of urothelial bladder cancer cells (639V) was evaluated in vitro with magrolimab alone and in combination with chemotherapy (gemcitabine + cisplatin). Treatment in vivo was evaluated in a xenograft mouse model. 639V cells were transplanted into NSG mice and upon confirmation of engraftment mice were randomized into 4 treatment cohorts: control (PBS), magrolimab, chemotherapy (cisplatin + gemcitabine), and magrolimab in combination with chemotherapy. In the first experimental setup treatment was started early in small tumors and in the second experimental setup treatment was started late after tumors have grown to large size. Tumor growth was monitored by in vivo bioluminescent imaging. Metastases were evaluated postmortem. Results: Chemotherapy increased calreticulin on bladder cancer cells. Magrolimab enhanced phagocytosis of bladder cancer cells in vitro and combination of magrolimab with chemotherapy further increased phagocytosis compared to either therapy alone. Magrolimab and chemotherapy, each alone decreased tumor growth in vivo but only combination of magrolimab with chemotherapy showed a strong inhibition of tumor growth, resulting in a significantly prolonged survival compared to all other treatment cohorts. This was shown for both, small tumors and large tumors. Metastases formation in liver and lungs was completely inhibited by treatment with magrolimab, whereas mice treated with chemotherapy alone or PBS control showed metastases in these organs. Conclusions: Magrolimab treatment in combination with chemotherapy was efficacious in preclinical in vitro and in vivo studies in bladder cancer and provides a novel treatment opportunity for patients with bladder cancer and other solid tumors.

CD47 is an anti-phagocytic signal and macrophage checkpoint that acute myeloid leukemia (AML) and other cancer cells utilize to evade innate immunity and establish disease. 5F9 is a humanized IgG4 monoclonal antibody (mAb) that binds to human CD47 and blocks its interaction with its macrophage receptor SIRPα, thereby promoting phagocytosis of cancer cells. We have found in numerous preclinical studies that anti-CD47 Abs synergize with targeted Abs (such as rituximab and cetuximab) by promoting phagocytosis, and also enable antigen cross-presentation and activation of cytotoxic T cells. These preclinical findings are being translated into clinical results as we have established in several clinical trials promising preliminary evidence of 5F9's therapeutic potential.

CD47 is an anti-phagocytic (i.e. “don't eat me”) signal and macrophage checkpoint that cancer cells utilize to evade innate immunity and establish disease. 5F9 is a humanized IgG4 monoclonal antibody (mAb) that binds to human CD47 and blocks its interaction with SIRPα, its cognate inhibitory receptor.5F9 is undergoing investigation in several clinical trials and preliminary analyses have revealed encouraging therapeutic potential.

Peritoneal adhesions are fibrous tissues that tether organs to one another or to the peritoneal wall and are a major cause of postsurgical and infectious morbidity. The primary molecular chain of events leading to the initiation of adhesions has been elusive, chiefly due to the lack of an identifiable cell of origin. Using clonal analysis and lineage tracing, we have identified injured surface mesothelium expressing podoplanin (PDPN) and mesothelin (MSLN) as a primary instigator of peritoneal adhesions after surgery in mice. We demonstrate that an anti-MSLN antibody diminished adhesion formation in a mouse model where adhesions were induced by surgical ligation to form ischemic buttons and subsequent surgical abrasion of the peritoneum. RNA sequencing and bioinformatics analyses of mouse mesothelial cells from injured mesothelium revealed aspects of the pathological mechanism of adhesion development and yielded several potential regulators of this process. Specifically, we show that PDPN+MSLN+ mesothelium responded to hypoxia by early up-regulation of hypoxia-inducible factor 1 alpha (HIF1α) that preceded adhesion development. Inhibition of HIF1α with small molecules ameliorated the injury program in damaged mesothelium and was sufficient to diminish adhesion severity in a mouse model. Analyses of human adhesion tissue suggested that similar surface markers and signaling pathways may contribute to surgical adhesions in human patients.