InVivoMAb anti-mouse/human TYRP1/TRP1 (gp75)

• Cancer Research
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Clustered macrophages cooperate to eliminate tumors via coordinated intrudopodia

Preprint on BioRxiv : the Preprint Server for Biology on 20 September 2024 by Dooling, L. J., Anlaş, A. A., et al.

Macrophages often pervade solid tumors, but their nearest neighbor organization is understudied and potentially enables key functions such as phagocytosis. Here, we observe dynamic macrophage clusters in tumors under conditions that maximize cancer cell phagocytosis and use reductionist approaches to uncover pathways to cluster formation and roles for tumor-intrusive pseudopodia, which we term ‘intrudopodia’. Macrophage clusters form over hours on low- adhesion substrates after M1 polarization with interferons, including T cell-derived cytokines, and yet clusters prove fluid on timescales of minutes. Clusters also sort from M2 macrophages that disperse on the same substrates. M1 macrophages upregulate specific cell-cell adhesion receptors but suppress actomyosin contractility, and while both pathways contribute to cluster formation, decreased cortical tension was predicted to unleash pseudopodia. Macrophage neighbors in tumor spheroids indeed extend intrudopodia between adjacent cancer cell junctions – at least when phagocytosis conditions are maximized, and coordinated intrudopodia help detach and individualize cancer cells for rapid engulfment. Macrophage clusters thereby provide a cooperative advantage for phagocytosis to overcome solid tumor cohesion.

• Mus musculus (House mouse)
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• Cancer Research
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• Immunology and Microbiology

Noncovalently particle-anchored cytokines with prolonged tumor retention safely elicit potent antitumor immunity.

In Science Advances on 19 April 2024 by Niu, L., Jang, E., et al.

Preclinical studies have shown that immunostimulatory cytokines elicit antitumor immune responses but their clinical use is limited by severe immune-related adverse events upon systemic administration. Here, we report a facile and versatile strategy for noncovalently anchoring potent Fc-fused cytokine molecules to the surface of size-discrete particles decorated with Fc-binding peptide for local administration. Following intratumoral injection, particle-anchored Fc cytokines exhibit size-dependent intratumoral retention. The 1-micrometer particle prolongs intratumoral retention of Fc cytokine for over a week and has minimal systemic exposure, thereby eliciting antitumor immunity while eliminating systemic toxicity caused by circulating cytokines. In addition, the combination of these particle-anchored cytokines with immune checkpoint blockade antibodies safely promotes tumor regression in various syngeneic tumor models and genetically engineered murine tumor models and elicits systemic antitumor immunity against tumor rechallenge. Our formulation strategy renders a safe and tumor-agnostic approach that uncouples cytokines' immunostimulatory properties from their systemic toxicities for potential clinical application.

• Cancer Research

Cooperative phagocytosis of solid tumours by macrophages triggers durable anti-tumour responses.

In Nature Biomedical Engineering on 1 September 2023 by Dooling, L. J., Andrechak, J. C., et al.

In solid tumours, the abundance of macrophages is typically associated with a poor prognosis. However, macrophage clusters in tumour-cell nests have been associated with survival in some tumour types. Here, by using tumour organoids comprising macrophages and cancer cells opsonized via a monoclonal antibody, we show that highly ordered clusters of macrophages cooperatively phagocytose cancer cells to suppress tumour growth. In mice with poorly immunogenic tumours, the systemic delivery of macrophages with signal-regulatory protein alpha (SIRPα) genetically knocked out or else with blockade of the CD47-SIRPα macrophage checkpoint was combined with the monoclonal antibody and subsequently triggered the production of endogenous tumour-opsonizing immunoglobulin G, substantially increased the survival of the animals and helped confer durable protection from tumour re-challenge and metastasis. Maximizing phagocytic potency by increasing macrophage numbers, by tumour-cell opsonization and by disrupting the phagocytic checkpoint CD47-SIRPα may lead to durable anti-tumour responses in solid cancers. © 2023. The Author(s), under exclusive licence to Springer Nature Limited.

• Cancer Research
,
• Mus musculus (House mouse)

In PNAS Nexus on 1 August 2023 by Hayes, B., Zhu, H., et al.

PubMed

Phagocytic elimination of solid tumors by innate immune cells seems attractive for immunotherapy, particularly because of the possibilities for acquired immunity. However, the approach remains challenging, with blockade of the macrophage checkpoint CD47 working in immunodeficient mice and against highly immunogenic tumors but not in the clinic where tumors are poorly immunogenic. Even when mouse tumors of poorly immunogenic B16F10 melanoma are opsonized to drive engulfment with a suitable monoclonal antibody (mAb), anti-CD47 blockade remains insufficient. Using both in vitro immuno-tumoroids and in vivo mouse models, we show with CRISPR interference (CRISPRi) that a relatively uniform minimum repression of CD47 by 80% is needed for phagocytosis to dominate net growth when combined with an otherwise ineffective mAb (anti-Tyrp1). Heterogeneity enriches for CD47-high cells, but mice that eliminate tumors generate prophagocytic IgGs that increase in titer with CD47 repression and with tumor accumulation of macrophages, although deeper repression does not improve survival. Given well-known limitations of antibody permeation into solid tumors, our studies clarify benchmarks for CD47 disruption that should be more clinically feasible and safer but just as effective as complete ablation. Additionally, safe but ineffective opsonization in human melanoma trials suggests that combinations with deep repression of CD47 could prove effective and initiate durable immunity. © The Author(s) 2023. Published by Oxford University Press on behalf of National Academy of Sciences.

• Cancer Research
,
• Genetics

Preprint on BioRxiv : the Preprint Server for Biology on 9 May 2023 by Glassbrook, J. E., Hackett, J. B., et al.

PubMed

Background Antitumor antibody, or targeted immunotherapy, has revolutionized cancer treatment and markedly improved patient outcomes. A prime example is the monoclonal antibody (mAb) trastuzumab, which targets human epidermal growth factor receptor 2 (HER2). However, like many targeted immunotherapies, only a subset of patients benefit from trastuzumab long-term. In addition to tumor-intrinsic factors, we hypothesize that host genetics may influence subsequent immune activation. Methods To model the human population, we produced F1 crosses of genetically heterogeneous Diversity Outbred (DO) mice with BALB/c mice (DOCF1). Distinct DOCF1 mice were orthotopically implanted with the BALB/c-syngeneic TUBO mammary tumor line, which expresses the HER2 ortholog rat neu. Treatment with anti-neu mAb clone 7.16.4 began once tumors reached ∼200 mm 3 . Genetic linkage and quantitative trait locus (QTL) effects analyses in R/qtl2 identified loci associated with tumor growth rates. Locus validation was performed with BALB/c F1 crosses with recombinant-inbred Collaborative Cross (CC) strains selected for therapy-associated driver genetics (CCxCF1). The respective roles of natural killer (NK) cells and macrophages were investigated by selective depletion in vivo. Ex vivo macrophage antibody-dependent phagocytosis (ADCP) assays were evaluated by confocal microscopy using 7.16.4-opsonized E2Crimson-expressing TUBO tumor cells. Results We observed a divergent response to anti-tumor antibody therapy in DOCF1 mice. Genetic linkage analysis detected a locus on chromosome 10 that correlates to a robust response to therapy, which was validated in CCxCF1 models. Single-cell RNA sequencing of tumors from responder and non-responder models identified key differences in tumor immune infiltrate composition, particularly within macrophage (Mφ) subsets. This is further supported by ex vivo analysis showing Mφ ADCP capacity correlates to in vivo treatment outcomes in both DOCF1 and CCxCF1 models. Conclusions Host genetics play a key regulatory role in targeted immunotherapy outcomes, and putative causal genes are identified in murine chromosome 10 which may govern Mφ function during ADCP.

• Cancer Research
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• Genetics
,
• Immunology and Microbiology

Chromosomal instability can favor macrophage-mediated immune response and induce a broad, vaccination-like anti-tumor IgG response

Preprint on BioRxiv : the Preprint Server for Biology on 4 April 2023 by Hayes, B. H., Wang, M., et al.

Chromosomal instability (CIN), a state in which cells undergo mitotic aberrations that generate chromosome copy number variations, generates aneuploidy and is thought to drive cancer evolution. Although associated with poor prognosis and reduced immune response, CIN generates aneuploidy-induced stresses that could be exploited for immunotherapies. In such contexts, macrophages and the CD47-SIRPα checkpoint are understudied. Here, CIN is induced pharmacologically induced in poorly immunogenic B16F10 mouse melanoma cells, generating persistent micronuclei and diverse aneuploidy while skewing macrophages towards an anti-cancer M1-like phenotype, based on RNA-sequencing profiling, surface marker expression and short-term antitumor studies. These results further translate to in vivo efficacy: Mice bearing CIN-afflicted tumors with wild-type CD47 levels survive only slightly longer relative to chromosomally stable controls, but long-term survival is maximized when combining macrophage-stimulating anti-tumor IgG opsonization and some form of disruption of the CD47-SIRPα checkpoint. Survivors make multi-epitope, de novo anti-cancer IgG that promote macrophage-mediated phagocytosis of CD47 knockout B16F10 cells and suppress tumoroids in vitro and growth of tumors in vivo . CIN does not greatly affect the level of the IgG response compared to previous studies but does significantly increase survival. These results highlight an unexpected therapeutic benefit from CIN when paired with maximal macrophage anti-cancer activity: an anti-cancer vaccination-like antibody response that can lead to more durable cures and further potentiate cell-mediated acquired immunity.

• In Vivo
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• Mus musculus (House mouse)
,
• Cancer Research

In Cancer Cell on 13 February 2023 by Linde, I. L., Prestwood, T. R., et al.

PubMed

Despite their cytotoxic capacity, neutrophils are often co-opted by cancers to promote immunosuppression, tumor growth, and metastasis. Consequently, these cells have received little attention as potential cancer immunotherapeutic agents. Here, we demonstrate in mouse models that neutrophils can be harnessed to induce eradication of tumors and reduce metastatic seeding through the combined actions of tumor necrosis factor, CD40 agonist, and tumor-binding antibody. The same combination activates human neutrophils in vitro, enabling their lysis of human tumor cells. Mechanistically, this therapy induces rapid mobilization and tumor infiltration of neutrophils along with complement activation in tumors. Complement component C5a activates neutrophils to produce leukotriene B4, which stimulates reactive oxygen species production via xanthine oxidase, resulting in oxidative damage and T cell-independent clearance of multiple tumor types. These data establish neutrophils as potent anti-tumor immune mediators and define an inflammatory pathway that can be harnessed to drive neutrophil-mediated eradication of cancer. Copyright © 2023 Elsevier Inc. All rights reserved.

• Cancer Research

Titrating CD47 by mismatch CRISPRi reveals incomplete repression can eliminate IgG-opsonized tumors but CD47 heterogeneity limits induction of anti-tumor IgG

Preprint on BioRxiv : the Preprint Server for Biology on 28 September 2022 by Hayes, B., Zhu, H., et al.

Phagocytic elimination of solid tumors is an attractive mechanism for immunotherapy – particularly because of the possible induction of anti-cancer immunity. The phagocytic potential of macrophages is limited, however, by the CD47-SIRPα checkpoint, and how much CD47 disruption is needed for efficacy remains unclear, even when tumors are opsonized by a pro-phagocytic antibody. Here, CRISPR-interference (CRISPRi) is applied with a large set of sgRNAs to produce a broad range of CD47 knockdowns in B16F10 melanoma, which is generally found to be resistant to the heavily studied PD-1 blockade. Guided by 3D immuno-tumoroid results, we identify a critical CD47 density below which macrophage-mediated phagocytosis dominates proliferation in the presence of an otherwise ineffective pro-phagocytic antibody (anti-Tyrp1). Growing tumors and immuno-tumoroids generally show selection for CD47-positive cells, but some mice reject tumors having >97% mean repression of CD47 or even having 80% repression – unless mixed with 50% repressed cells. Interestingly, long-term survivors have de novo pro-phagocytic IgG antibodies that increase in titer with depth of repression and also with early accumulation of tumor macrophages. Given well-known limitations of antibody permeation into solid tumors, our studies set a benchmark for anti-CD47 blockade and suggest deep disruption favors acquired immunity.

• Cancer Research
,
• Immunology and Microbiology

Preprint on BioRxiv : the Preprint Server for Biology on 2 January 2022 by Andrechak, J. C., Dooling, L. J., et al.

PubMed

Macrophages are abundant in solid tumours and typically associate with poor prognosis, but macrophage clusters in tumour nests have also been reported as beneficial even though dispersed macrophages would have more contacts with cancer cells. Here, by maximizing both phagocytic activity and macrophage numbers, we discover cooperative phagocytosis by low entropy clusters in rapidly growing engineered immuno-tumouroids. The results fit the calculus of proliferation-versus-engulfment, and rheological measurements and molecular perturbations provide a basis for understanding phagocytic disruption of a tumour’s cohesive forces in soft cellular phases. The perturbations underscore the utility of suppressing a macrophage checkpoint in combination with an otherwise ineffective tumour-opsonizing monoclonal antibody, and the approach translates in vivo to tumour elimination that durably protects mice from re-challenge and metastasis. Adoptive transfer of engineered macrophages increases the fraction of mice that eliminate tumours and potentially overcomes checkpoint blockade challenges in solid tumours like insufficient permeation of blocking antibodies and on-target, off-tumour binding. Finally, anti-cancer IgG induced in vivo are tumour-specific but multi-epitope and contribute to a phagocytic feedback that drives macrophage clustering in vitro . Given that solid tumours remain challenging for immunotherapies, durable anti-tumour responses here illustrate unexpected advantages in maximizing net phagocytic activity.

In IScience on 24 September 2021 by Shen, Z., Xiang, Y., et al.

PubMed

Therapeutic and diagnostic efficacies of small biomolecules and chemical compounds are hampered by suboptimal pharmacokinetics. Here, we developed a repertoire of robust and high-affinity antihuman serum albumin nanobodies (NbHSA) that can be readily fused to small biologics for half-life extension. We characterized the thermostability, binding kinetics, and cross-species reactivity of NbHSAs, mapped their epitopes, and structurally resolved a tetrameric HSA-Nb complex. We parallelly determined the half-lives of a cohort of selected NbHSAs in an HSA mouse model by quantitative proteomics. Compared to short-lived control nanobodies, the half-lives of NbHSAs were drastically prolonged by 771-fold. NbHSAs have distinct and diverse pharmacokinetics, positively correlating with their albumin binding affinities at the endosomal pH. We then generated stable and highly bioactive NbHSA-cytokine fusion constructs "Duraleukin" and demonstrated Duraleukin's high preclinical efficacy for cancer treatment in a melanoma model. This high-quality and versatile Nb toolkit will help tailor drug half-life to specific medical needs. © 2021 The Authors.

• IHC
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• Mus musculus (House mouse)
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• Cancer Research
,
• Immunology and Microbiology

In The Journal of Clinical Investigation on 16 August 2021 by Veatch, J. R., Singhi, N., et al.

PubMed

Therapeutic vaccines that augment T cell responses to tumor antigens have been limited by poor potency in clinical trials. In contrast, the transfer of T cells modified with foreign transgenes frequently induces potent endogenous T cell responses to epitopes in the transgene product, and these responses are undesirable, because they lead to rejection of the transferred T cells. We sought to harness gene-modified T cells as a vaccine platform and developed cancer vaccines composed of autologous T cells modified with tumor antigens and additional adjuvant signals (Tvax). T cells expressing model antigens and a broad range of tumor neoantigens induced robust and durable T cell responses through cross-presentation of antigens by host DCs. Providing Tvax with signals such as CD80, CD137L, IFN-β, IL-12, GM-CSF, and FLT3L enhanced T cell priming. Coexpression of IL-12 and GM-CSF induced the strongest CD4+ and CD8+ T cell responses through complimentary effects on the recruitment and activation of DCs, mediated by autocrine IL-12 receptor signaling in the Tvax. Therapeutic vaccination with Tvax and adjuvants showed antitumor activity in subcutaneous and metastatic preclinical mouse models. Human T cells modified with neoantigens readily activated specific T cells derived from patients, providing a path for clinical translation of this therapeutic platform in cancer.

• Immunology and Microbiology

In Proceedings of the National Academy of Sciences of the United States of America on 29 June 2021 by Ibarlucea-Benitez, I., Weitzenfeld, P., et al.

PubMed

Given the role of myeloid cells in T cell activation and in the antitumor response, targeting checkpoint molecules expressed on this population represents a promising strategy to augment antitumor immunity. However, myeloid checkpoints that can be effectively used as immunotherapy targets are still lacking. Here, we demonstrate the therapeutic potential of targeting the myeloid receptors Siglec-7 and Siglec-9 in vivo. By using a humanized immunocompetent murine model, we demonstrate that human Siglec-7 and Siglec-9, in addition to the murine homolog Siglec-E, inhibit the endogenous antitumor immune response, as well as the response to tumor-targeting and immune checkpoint inhibiting antibodies in vivo. The impact of these Siglecs on tumor progression is highly dependent on the anatomical distribution of the tumor and, as a consequence, the local tumor microenvironment, as tumors with a more immune-suppressive tumor microenvironment are less sensitive to Siglec perturbation. Finally, to assess the potential of these two receptors as targets for immunotherapy, we developed Fc engineered blocking antibodies to Siglec-7 and Siglec-9 and demonstrate that Siglec-7 and Siglec-9 blockade can significantly reduce tumor burden in vivo, demonstrating the therapeutic potential of targeting these two receptors.

• Cell Biology

In Journal of Cell Science on 6 March 2020 by Hayes, B., Tsai, R. K., et al.

PubMed

The macrophage checkpoint receptor SIRPα signals against phagocytosis by binding CD47 expressed on all cells - including macrophages. Here, we found that inhibiting cis interactions between SIRPα and CD47 on the same macrophage increased engulfment ('eating') by approximately the same level as inhibiting trans interactions. Antibody blockade of CD47, as pursued in clinical trials against cancer, was applied separately to human-derived macrophages and to red blood cell (RBC) targets for phagocytosis, and both scenarios produced surprisingly similar increases in RBC engulfment. Blockade of both macrophages and targets resulted in hyper-phagocytosis, and knockdown of macrophage-CD47 likewise increased engulfment of 'foreign' cells and particles, decreased the baseline inhibitory signaling of SIRPα, and linearly increased binding of soluble CD47 in trans, consistent with cis-trans competition. Many cell types express both SIRPα and CD47, including mouse melanoma B16 cells, and CRISPR-mediated deletions modulate B16 phagocytosis, consistent with cis-trans competition. Additionally, soluble SIRPα binding to human CD47 displayed on Chinese hamster ovary (CHO) cells was suppressed by SIRPα co-display, and atomistic computations confirm SIRPα bends and binds CD47 in cis Safety and efficacy profiles for CD47-SIRPα blockade might therefore reflect a disruption of both cis and trans interactions. © 2020. Published by The Company of Biologists Ltd.

• Cell Biology

In Journal of Cell Science on 1 January 2020 by Hayes, B. H., Tsai, R. K., et al.

PubMed

The macrophage checkpoint receptor SIRPα signals against phagocytosis by binding CD47 expressed on all cells - including macrophages. Here, inhibiting cis interactions between SIRPα and CD47 on the same macrophage increases eating approximately the same as inhibiting trans interactions. Antibody blockade of CD47, as pursued in clinical trials against cancer, is applied separately to human-derived macrophages and to red blood cell (RBC) targets for phagocytosis, and both scenarios produce surprisingly similar increases in RBC engulfment. Blockade of both macrophages and targets results in hyper-phagocytosis, and knockdown of macrophage-CD47 likewise increases eating of 'foreign' cells and particles, decreases SIRPα's baseline inhibitory signaling, and linearly increases binding of soluble-CD47 in trans, consistent with cis-trans competition. Many cell types express both SIRPα and CD47, including mouse melanoma B16 cells, and CRISPR-mediated deletions modulate B16 phagocytosis consistent with cis-trans competition. Additionally, soluble SIRPα binding to human-CD47 displayed on Chinese hamster ovary (CHO) cells is suppressed by SIRPα co-display, and atomistic computations confirm SIRPα bends and binds CD47 in cis. Safety and efficacy profiles for CD47-SIRPα blockade might therefore reflect a disruption of both cis and trans interactions. © 2020. Published by The Company of Biologists Ltd.

• Cancer Research

In Scientific Reports on 27 December 2019 by Jayaraman Rukmini, S., Bi, H., et al.

PubMed

Tumors can escape from the immune system by overexpressing CD47 and other checkpoint blockades. CD47 is expressed ubiquitously by all cells in the body, posing an obstacle for CD47 blocking treatments due to their systemic toxicity. We performed a study to determine how the tumor microenvironment changes after vaccination with genome edited CD47-/- syngeneic tumor cells. We discovered that inactivated CD47-depleted mouse melanoma cells can protect mice from melanoma. Our animal study indicated that 33% of vaccinated mice remained tumor-free, and 100% of mice had 5-fold reduced growth rates. The characterization of immunomodulatory effects of the vaccine revealed a highly anti-tumorigenic and homogenous microenvironment after vaccination. We observed consistently that in the tumors that failed to respond to vaccines, there were reduced natural killer cells, elevated regulatory T cells, M2-type macrophages, and high PD-L1 expression in these cells. These observations suggested that the tumor microenvironments became more suppressive to tumor growth after vaccination, suggesting a potential new immunotherapy for solid tumors.

• Cancer Research
,
• Immunology and Microbiology

In Nature Communications on 6 November 2019 by Medina-Echeverz, J., Hinterberger, M., et al.

PubMed

Virus-based vaccines and appropriate costimulation potently enhance antigen-specific T cell immunity against cancer. Here we report the use of recombinant modified vaccinia virus Ankara (rMVA) encoding costimulatory CD40L against solid tumors. Therapeutic treatment with rMVA-CD40L-expressing tumor-associated antigens results in the control of established tumors. The expansion of tumor-specific cytotoxic CD8+ T cells is essential for the therapeutic antitumor effects. Strikingly, rMVA-CD40L also induces strong natural killer (NK) cell activation and expansion. Moreover, the combination of rMVA-CD40L and tumor-targeting antibodies results in increased therapeutic antitumor efficacy relying on the presence of Fc receptor and NK cells. We describe a translationally relevant therapeutic synergy between systemic viral vaccination and CD40L costimulation. We show strengthened antitumor immune responses when both rMVA-CD40L-induced innate and adaptive immune mechanisms are exploited by combination with tumor-targeting antibodies. This immunotherapeutic approach could translate into clinical cancer therapies where tumor-targeting antibodies are employed.

In The Journal of Clinical Investigation on 1 October 2019 by Rasoulouniriana, D., Santana-Magal, N., et al.

PubMed

While a high frequency of Th1 cells in tumors is associated with improved cancer prognosis, this benefit has been attributed mainly to support of cytotoxic activity of CD8+ T cells. By attempting to potentiate antibody-driven immunity, we found a remarkable synergy between CD4+ T cells and tumor-binding antibodies. This surprising synergy was mediated by a small subset of tumor-infiltrating CD4+ T cells that express the high-affinity Fcγ receptor for IgG (FcγRI) in both mouse and human patients. These cells efficiently lyse tumor cells coated with antibodies through concomitant crosslinking of their T cell receptor (TCR) and FcγRI. By expressing FcγRI and its signaling chain in conventional CD4+ T cells, we successfully employed this mechanism to treat established solid cancers. Overall, this discovery sheds new light on the biology of this T cell subset, their function during tumor immunity, and the means to utilize their unique killing signals in immunotherapy.

• Cancer Research

In Cell Reports on 26 June 2018 by Matlung, H. L., Babes, L., et al.

PubMed

Destruction of cancer cells by therapeutic antibodies occurs, at least in part, through antibody-dependent cellular cytotoxicity (ADCC), and this can be mediated by various Fc-receptor-expressing immune cells, including neutrophils. However, the mechanism(s) by which neutrophils kill antibody-opsonized cancer cells has not been established. Here, we demonstrate that neutrophils can exert a mode of destruction of cancer cells, which involves antibody-mediated trogocytosis by neutrophils. Intimately associated with this is an active mechanical disruption of the cancer cell plasma membrane, leading to a lytic (i.e., necrotic) type of cancer cell death. Furthermore, this mode of destruction of antibody-opsonized cancer cells by neutrophils is potentiated by CD47-SIRPα checkpoint blockade. Collectively, these findings show that neutrophil ADCC toward cancer cells occurs by a mechanism of cytotoxicity called trogoptosis, which can be further improved by targeting CD47-SIRPα interactions. Crown Copyright © 2018. Published by Elsevier Inc. All rights reserved.