DLL3-guided therapies in small-cell lung cancer: from antibody-drug conjugate to precision immunotherapy and radioimmunotherapy.

DLL3 acts as an inhibitory ligand that downregulates Notch signaling and is upregulated by ASCL1, a transcription factor prevalent in the small-cell lung cancer (SCLC) subtype SCLC-A. Currently, the therapeutic strategies targeting DLL3 are varied, including antibody-drug conjugates (ADCs), bispecific T-cell engagers (BiTEs), and chimeric antigen receptor (CAR) T-cell therapies. Although rovalpituzumab tesirine (Rova-T) showed promise in a phase II study, it failed to produce favorable results in subsequent phase III trials, leading to the cessation of its development. Conversely, DLL3-targeted BiTEs have garnered significant clinical interest. Tarlatamab, for instance, demonstrated enhanced response rates and progression-free survival compared to the standard of care in a phase II trial; its biologics license application (BLA) is currently under US Food and Drug Administration (FDA) review. Numerous ongoing phase III studies aim to further evaluate tarlatamab's clinical efficacy, alongside the development of novel DLL3-targeted T-cell engagers, both bispecific and trispecific. CAR-T cell therapies targeting DLL3 have recently emerged and are undergoing various preclinical and early-phase clinical studies. Additionally, preclinical studies have shown promising efficacy for DLL3-targeted radiotherapy, which employs ß-particle-emitting therapeutic radioisotopes conjugated to DLL3-targeting antibodies. DLL3-targeted therapies hold substantial potential for SCLC management. Future clinical trials will be crucial for comparing treatment outcomes among various approaches and exploring combination therapies to improve patient survival outcomes.

mRNA vaccines against SARS-CoV-2 induce divergent antigen-specific T-cell responses in patients with lung cancer.

BACKGROUND: The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) Omicron variant is highly transmissible and evades pre-established immunity. Messenger RNA (mRNA) vaccination against ancestral strain spike protein can induce intact T-cell immunity against the Omicron variant, but efficacy of booster vaccination in patients with late-stage lung cancer on immune-modulating agents including anti-programmed cell death protein 1(PD-1)/programmed death-ligand 1 (PD-L1) has not yet been elucidated. METHODS: We assessed T-cell responses using a modified activation-induced marker assay, coupled with high-dimension flow cytometry analyses. Peripheral blood mononuclear cells (PBMCs) were stimulated with various viral peptides and antigen-specific T-cell responses were evaluated using flow cytometry. RESULTS: Booster vaccines induced CD8+ T-cell response against the ancestral SARS-CoV-2 strain and Omicron variant in both non-cancer subjects and patients with lung cancer, but only a marginal induction was detected for CD4+ T cells. Importantly, antigen-specific T cells from patients with lung cancer showed distinct subpopulation dynamics with varying degrees of differentiation compared with non-cancer subjects, with evidence of dysfunction. Notably, female-biased T-cell responses were observed. CONCLUSION: We conclude that patients with lung cancer on immunotherapy show a substantial qualitative deviation from non-cancer subjects in their T-cell response to mRNA vaccines, highlighting the need for heightened protective measures for patients with cancer to minimize the risk of breakthrough infection with the Omicron and other future variants.

Mean platelet volume, thrombocytosis, and survival in non-small cell lung cancer patients treated with first-line pembrolizumab alone or with chemotherapy.

INTRODUCTION: Patients treated with immune checkpoint inhibitors (ICIs) may not response to treatment and are at risk for immune-related adverse events (irAEs). Platelet function has been linked to both oncogenesis and immune evasion. We studied the association between the change in mean platelet volume (MPV), platelet count, survival, and the risk of developing irAEs in patients with metastatic non-small cell lung cancer (NSCLC) who have received first-line ICI. METHODS: In this retrospective study, delta (∆) MPV was defined as the difference between cycle 2 and baseline MPV. Patient data were collected via chart review, and Cox proportional hazard and Kaplan-Meier method were used to assess the risk and estimate median overall survival. RESULTS: We identified 188 patients treated with first-line pembrolizumab, with or without concurrent chemotherapy. There were 80 (42.6%) patients received pembrolizumab monotherapy, and 108 (57.4%) received pembrolizumab in combination with platinum-based chemotherapy. Patients whose MPV (∆MPV ≤ 0) decreased had hazard ratio (HR) = 0.64 (95% CI 0.43-0.94) for death with p = 0.023. Patients with ∆MPV ≤ - 0.2 fL (median), there was a 58% increase in the risk of developing irAE (HR = 1.58, 95% CI 1.04-2.40, p = 0.031). Thrombocytosis at baseline and cycle 2 was associated with shorter OS with p = 0.014 and 0.039, respectively. CONCLUSION: Change in MPV after 1 cycle of pembrolizumab-based treatment was significantly associated with overall survival as well as the occurrence of irAEs in patients with metastatic NSCLC in the first-line setting. In addition, thrombocytosis was associated with poor survival.

Selective suppression of de novo SARS-CoV-2 vaccine antibody responses in patients with cancer on B cell-targeted therapy.

We assessed vaccine-induced antibody responses to the SARS-CoV-2 ancestral virus and Omicron variant before and after booster immunization in 57 patients with B cell malignancies. Over one-third of vaccinated patients at the pre-booster time point were seronegative, and these patients were predominantly on active cancer therapies such as anti-CD20 monoclonal antibody. While booster immunization was able to induce detectable antibodies in a small fraction of seronegative patients, the overall booster benefit was disproportionately evident in patients already seropositive and not receiving active therapy. While ancestral virus- and Omicron variant-reactive antibody levels among individual patients were largely concordant, neutralizing antibodies against Omicron tended to be reduced. Interestingly, in all patients, including those unable to generate detectable antibodies against SARS-CoV-2 spike, we observed comparable levels of EBV- and influenza-reactive antibodies, demonstrating that B cell-targeting therapies primarily impair de novo but not preexisting antibody levels. These findings support rationale for vaccination before cancer treatment.

Selective targeting of GARP-LTGFß axis in the tumor microenvironment augments PD-1 blockade via enhancing CD8+ T cell antitumor immunity.

BACKGROUND: Immune checkpoint blockade (ICB) has revolutionized cancer immunotherapy. However, most patients with cancer fail to respond clinically. One potential reason is the accumulation of immunosuppressive transforming growth factor ß (TGFß) in the tumor microenvironment (TME). TGFß drives cancer immune evasion in part by inducing regulatory T cells (Tregs) and limiting CD8+ T cell function. Glycoprotein-A repetitions predominant (GARP) is a cell surface docking receptor for activating latent TGFß1, TGFß2 and TGFß3, with its expression restricted predominantly to effector Tregs, cancer cells, and platelets. METHODS: We investigated the role of GARP in human patients with cancer by analyzing existing large databases. In addition, we generated and humanized an anti-GARP monoclonal antibody and evaluated its antitumor efficacy and underlying mechanisms of action in murine models of cancer. RESULTS: We demonstrate that GARP overexpression in human cancers correlates with a tolerogenic TME and poor clinical response to ICB, suggesting GARP blockade may improve cancer immunotherapy. We report on a unique anti-human GARP antibody (named PIIO-1) that specifically binds the ligand-interacting domain of all latent TGFß isoforms. PIIO-1 lacks recognition of GARP-TGFß complex on platelets. Using human LRRC32 (encoding GARP) knock-in mice, we find that PIIO-1 does not cause thrombocytopenia; is preferentially distributed in the TME; and exhibits therapeutic efficacy against GARP+ and GARP- cancers, alone or in combination with anti-PD-1 antibody. Mechanistically, PIIO-1 treatment reduces canonical TGFß signaling in tumor-infiltrating immune cells, prevents T cell exhaustion, and enhances CD8+ T cell migration into the TME in a C-X-C motif chemokine receptor 3 (CXCR3)-dependent manner. CONCLUSION: GARP contributes to multiple aspects of immune resistance in cancer. Anti-human GARP antibody PIIO-1 is an efficacious and safe strategy to block GARP-mediated LTGFß activation, enhance CD8+ T cell trafficking and functionality in the tumor, and overcome primary resistance to anti-PD-1 ICB. PIIO-1 therefore warrants clinical development as a novel cancer immunotherapeutic.

Treatment with soluble CD24 attenuates COVID-19-associated systemic immunopathology.

BACKGROUND: Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) causes coronavirus disease 2019 (COVID-19) through direct lysis of infected lung epithelial cells, which releases damage-associated molecular patterns and induces a pro-inflammatory cytokine milieu causing systemic inflammation. Anti-viral and anti-inflammatory agents have shown limited therapeutic efficacy. Soluble CD24 (CD24Fc) blunts the broad inflammatory response induced by damage-associated molecular patterns via binding to extracellular high mobility group box 1 and heat shock proteins, as well as regulating the downstream Siglec10-Src homology 2 domain-containing phosphatase 1 pathway. A recent randomized phase III trial evaluating CD24Fc for patients with severe COVID-19 (SAC-COVID; NCT04317040) demonstrated encouraging clinical efficacy. METHODS: Using a systems analytical approach, we studied peripheral blood samples obtained from patients enrolled at a single institution in the SAC-COVID trial to discern the impact of CD24Fc treatment on immune homeostasis. We performed high dimensional spectral flow cytometry and measured the levels of a broad array of cytokines and chemokines to discern the impact of CD24Fc treatment on immune homeostasis in patients with COVID-19. RESULTS: Twenty-two patients were enrolled, and the clinical characteristics from the CD24Fc vs. placebo groups were matched. Using high-content spectral flow cytometry and network-level analysis, we found that patients with severe COVID-19 had systemic hyper-activation of multiple cellular compartments, including CD8+ T cells, CD4+ T cells, and CD56+ natural killer cells. Treatment with CD24Fc blunted this systemic inflammation, inducing a return to homeostasis in NK and T cells without compromising the anti-Spike protein antibody response. CD24Fc significantly attenuated the systemic cytokine response and diminished the cytokine coexpression and network connectivity linked with COVID-19 severity and pathogenesis. CONCLUSIONS: Our data demonstrate that CD24Fc rapidly down-modulates systemic inflammation and restores immune homeostasis in SARS-CoV-2-infected individuals, supporting further development of CD24Fc as a novel therapeutic against severe COVID-19.

Impaired neutralizing antibody response to COVID-19 mRNA vaccines in cancer patients.

There is currently a critical need to determine the efficacy of SARS-CoV-2 vaccination for immunocompromised patients. In this study, we determined the neutralizing antibody response in 160 cancer patients diagnosed with chronic lymphocytic leukemia (CLL), lung cancer, breast cancer, and various non-Hodgkin's lymphomas (NHL), after they received two doses of mRNA vaccines. Serum from 46 mRNA vaccinated health care workers (HCWs) served as healthy controls. We discovered that (1) cancer patients exhibited reduced neutralizing antibody titer (NT50) compared to HCWs; (2) CLL and NHL patients exhibited the lowest NT50 levels, with 50-60% of them below the detection limit; (3) mean NT50 levels in patients with CLL and NHL was ~2.6 fold lower than those with solid tumors; and (4) cancer patients who received anti-B cell therapy exhibited significantly reduced NT50 levels. Our results demonstrate an urgent need for novel immunization strategies for cancer patients against SARS-CoV-2, particularly those with hematological cancers and those on anti-B cell therapies.

Impaired Neutralizing Antibody Response to COVID-19 mRNA Vaccines in Cancer Patients.

There is currently a critical need to determine the efficacy of SARS-CoV-2 vaccination for immunocompromised patients. In this study, we determined the neutralizing antibody response in 160 cancer patients diagnosed with chronic lymphocytic leukemia (CLL), lung cancer, breast cancer, and various non-Hodgkin's lymphomas (NHL), after they received two doses of mRNA vaccines. Serum from 46 mRNA vaccinated health care workers (HCWs) served as healthy controls. We discovered that (1) cancer patients exhibited reduced neutralizing antibody titer (NT 50 ) compared to HCWs; (2) CLL and NHL patients exhibited the lowest NT 50 levels, with 50-60% of them below the detection limit; (3) mean NT 50 levels in patients with CLL and NHL was ∼2.6 fold lower than those with solid tumors; and (4) cancer patients who received anti-B cell therapy exhibited significantly reduced NT 50 levels. Our results demonstrate an urgent need for novel immunization strategies for cancer patients against SARS-CoV-2, particularly those with hematological cancers and those on anti-B cell therapies.

TREATMENT WITH SOLUBLE CD24 ATTENUATES COVID-19-ASSOCIATED SYSTEMIC IMMUNOPATHOLOGY.

BACKGROUND: SARS-CoV-2 causes COVID-19 through direct lysis of infected lung epithelial cells, which releases damage-associated molecular patterns (DAMPs) and induces a pro-inflammatory cytokine milieu causing systemic inflammation. Anti-viral and anti-inflammatory agents have shown limited therapeutic efficacy. Soluble CD24 (CD24Fc) is able to blunt the broad inflammatory response induced by DAMPs in multiple models. A recent randomized phase III trial evaluating the impact of CD24Fc in patients with severe COVID-19 demonstrated encouraging clinical efficacy. METHODS: We studied peripheral blood samples obtained from patients enrolled at a single institution in the SAC-COVID trial (NCT04317040) collected before and after treatment with CD24Fc or placebo. We performed high dimensional spectral flow cytometry analysis of peripheral blood mononuclear cells and measured the levels of a broad array of cytokines and chemokines. A systems analytical approach was used to discern the impact of CD24Fc treatment on immune homeostasis in patients with COVID-19. FINDINGS: Twenty-two patients were enrolled, and the clinical characteristics from the CD24Fc vs. placebo groups were matched. Using high-content spectral flow cytometry and network-level analysis, we found systemic hyper-activation of multiple cellular compartments in the placebo group, including CD8+ T cells, CD4+ T cells, and CD56+ NK cells. By contrast, CD24Fc-treated patients demonstrated blunted systemic inflammation, with a return to homeostasis in both NK and T cells within days without compromising the ability of patients to mount an effective anti-Spike protein antibody response. A single dose of CD24Fc significantly attenuated induction of the systemic cytokine response, including expression of IL-10 and IL-15, and diminished the coexpression and network connectivity among extensive circulating inflammatory cytokines, the parameters associated with COVID-19 disease severity. INTERPRETATION: Our data demonstrates that CD24Fc treatment rapidly down-modulates systemic inflammation and restores immune homeostasis in SARS-CoV-2-infected individuals, supporting further development of CD24Fc as a novel therapeutic against severe COVID-19. FUNDING: NIH.

In Vitro Aerosol Exposure to Nanomaterials: From Laboratory to Environmental Field Toxicity Testing.

Exposure to nanomaterials (NMs) is inevitable, requiring robust toxicological assessment to understand potential environmental and human health effects. NMs are favored in many applications because of their small size; however, this allows them to easily aerosolize and, subsequently, expose humans via inhalation. Toxicological assessment of NMs by conventional methods in submerged cell culture is not a relevant way to assess inhalation toxicity of NMs because of particle interference with bioassays and changes in particokinetics when dispersed in medium. Therefore, an in vitro aerosol exposure chamber (AEC) was custom designed and used for direct deposition of NMs from aerosols in the environment to the air-liquid interface of lung cells. Human epithelial lung cell line, A549, was used to assess the toxicity of copper, nickel, and zinc oxide nanopowders aerosolized by acoustic agitation in laboratory study. Post optimization, the AEC was used in the field to expose the A549 cells to NM aerosols generated from firing a hand gun and rifle. Toxicity was assessed using nondestructive assays for cell viability and inflammatory response, comparing the biologic effect to the delivered mass dose measured by inductively coupled plasma-mass spectrometry. The nanopowder exposure to submerged and ALI cells resulted in dose-dependent toxicity. In the field, weapon exhaust from the M4 reduced cell viability greater than the M9, while the M9 stimulated inflammatory cytokine release of IL-8. This study highlights the use of a portable chamber with the capability to assess toxicity of NM aerosols exposed to air-liquid interface in vitro lung cell culture.