Assessing Immunity by Quantitative Measurement of SARS-CoV-2 IgG Antibodies in Fingerstick Samples.

COVID-19 has affected billions of people around the world directly or indirectly. The response to the pandemic has focused on preventing the spread of the disease and improving treatment options. Diagnostic technologies have played a key role in this response since the beginning of the pandemic. As vaccines and other treatments have been developed and deployed, interest in understanding and measuring the individual level of immune protection has increased. Historically, use of antibody titers to measure systemic immunity has been constrained by an incomplete understanding of the relationship between antibodies and immunity, the lack of international standards for antibody concentration to enable cross-study comparisons, and insufficient clinical data to allow for the development of robust antibody-immunity models. However, these constraints have recently shifted. With a deeper understanding of antibodies, the promulgation of WHO antibody standards, and the development of immunity models using datasets from multiple COVID-19 vaccine trials, certain types of quantitative antibody tests may now provide a way to monitor individual or community immunity against COVID-19. Specifically, tests that quantitate the concentration of anti-RBD IgG -antibodies that target the receptor binding domain of the S1 spike protein component of the SARS-CoV-2 virus - show promise as a useful and scalable measure of the COVID-19 immunity of both individuals and communities. However, to fulfill this promise, a rapid and easy-to-administer test is needed. To address this important clinical need, Brevitest deployed its point-of-care-capable technology platform that can run a rapid (<15 minute), quantitative antibody test with a sample of 10 µl of whole blood from a fingerstick. The test we validated on this platform measures the concentration of anti-RBD IgG in Binding Antibody Units per milliliter (BAU/mL) per WHO Reference Standard NIBSC 20/136. In this paper, we present studies used to characterize the Brevitest anti-RBD IgG assay and evaluate its clinical performance, lower limits of measurement, precision, linearity, interference, and cross-reactivity. The results demonstrate the ability of this assay to measure a patient's anti-RBD IgG concentration. This information, together with models developed from recent COVID-19 vaccine clinical trials, can provide a means of assessing the current level of immune protection of an individual or community against COVID-19 infection.

Salivary anti-SARS-CoV-2 IgA as an accessible biomarker of mucosal immunity against COVID-19.

Background: Mucosal immunity, including secretory IgA (sIgA), plays an important role in early defenses against respiratory pathogens. Salivary testing, the most convenient way to measure sIgA, has been used to characterize mucosal immune responses to many viral infections including SARS, MERS, influenza, HIV, and RSV. However, its role has not yet been characterized in the COVID-19 pandemic. Here, we report development and validation of a rapid immunoassay for measuring salivary IgA against the SARS-CoV-2 virus, and report quantitative results in both pre-COVID-19 and muco-converted subjects. Methods: We developed and refined a specific test for salivary IgA against SARS-CoV-2 on the Brevitest platform, a rapid immunoassay system designed for point-of-care use. A qualitative test was validated as per FDA guidelines with saliva obtained from subjects prior to the emergence of COVID-19, and from PCR-confirmed COVID-19 patients. We also generated a quantitative measure of anti-SARS-CoV-2 salivary IgA. Time taken for saliva self-collection was measured and its ease-of-use assessed. Results: We successfully validated a qualitative salivary assay for SARS-CoV-2 IgA antibodies, with positive and negative predictive values of 92% and 97%, respectively, and no observable cross-reactivity with any of seven potential confounders. Pre-COVID-19 saliva samples showed an 8-fold range of IgA concentrations, suggesting a broad continuum of natural antibody resistance against the novel virus, though at levels lower than that observed in COVID-19 PCR-confirmed subjects. Samples from muco-positive subjects also shown a ~9-fold variation in salivary IgA levels, with elevated salivary IgA observed beyond three months after onset of symptoms. We observed a correlation (r=0.4405) between salivary IgA levels and COVID-19 disease severity. In anecdotal observations, we observed individuals who exhibited antibodies early in the course of their disease, contemporaneously with a positive PCR test, as well as individuals who muco-converted despite no known direct exposure to a COVID-19 patient, no symptoms, and negative molecular and/or serum antibody tests. Salivary collection took 5-10 minutes, and was reported as being easy (mean of 1.1 on a scale of 1 to 10). Implications: Mucosal immunity, including secretory IgA, plays an important role in host defense against respiratory pathogens, and our early data suggest it may do so in COVID-19. Salivary IgA, an accessible marker of mucosal immunity, may be a useful indicator of several key parameters including individual and community immune response, disease severity, clinical risk, and herd immunity. The non-invasive nature and ease of saliva collection facilitates its potential use as a biomarker for ongoing patient assessment and management, as well as a community surveillance tool. By measuring mucosal immune responses directly and systemic immune responses indirectly, salivary IgA could be useful in developing and deploying a vaccine(s) against COVID-19. Quantitative IgA assessment could also potentially serve as a tool to segment the population into different risk categories and inform individual and collective decisions relating to appropriate activities and vaccine prioritization/delivery. These data reinforce the importance of further investigation into the role of mucosal immunity and IgA in host responses against COVID-19.

Small molecule targeting of the STAT5/6 Src homology 2 (SH2) domains to inhibit allergic airway disease.

Asthma is a chronic inflammatory disease of the lungs and airways and one of the most burdensome of all chronic maladies. Previous studies have established that expression of experimental and human asthma requires the IL-4/IL-13/IL-4 receptor α (IL-4Rα) signaling pathway, which activates the transcription factor STAT6. However, no small molecules targeting this important pathway are currently in clinical development. To this end, using a preclinical asthma model, we sought to develop and test a small-molecule inhibitor of the Src homology 2 domains in mouse and human STAT6. We previously developed multiple peptidomimetic compounds on the basis of blocking the docking site of STAT6 to IL-4Rα and phosphorylation of Tyr641 in STAT6. Here, we expanded the scope of our initial in vitro structure-activity relationship studies to include central and C-terminal analogs of these peptides to develop a lead compound, PM-43I. Conducting initial dose range, toxicity, and pharmacokinetic experiments with PM-43I, we found that it potently inhibits both STAT5- and STAT6-dependent allergic airway disease in mice. Moreover, PM-43I reversed preexisting allergic airway disease in mice with a minimum ED50 of 0.25 µg/kg. Of note, PM-43I was efficiently cleared through the kidneys with no long-term toxicity. We conclude that PM-43I represents the first of a class of small molecules that may be suitable for further clinical development against asthma.

Safety, tolerability, and biomarkers of the treatment of mice with aerosolized Toll-like receptor ligands.

We have previously discovered a synergistically therapeutic combination of two Toll-like receptor ligands, an oligodeoxynucleotide (ODN) and Pam2CSK4. Aerosolization of these ligands stimulates innate immunity within the lungs to prevent pneumonia from bacterial and viral pathogens. Here we examined the safety and tolerability of this treatment in mice, and characterized the expression of biomarkers of innate immune activation. We found that neutrophils appeared in lung lavage fluid 4 h after treatment, reached a peak at 48 h, and resolved by 7 days. The peak of neutrophil influx was accompanied by a small increase in lung permeability. Despite the abundance of neutrophils in lung lavage fluid, only rare neutrophils were visible histopathologically in the interstitium surrounding bronchi and veins and none were visible in alveolar airspaces. The cytokines interleukin 6 (IL-6), tumour necrosis factor, and Chemokine (C-X-C motif) ligand 2 rose several hundred-fold in lung lavage fluid 4 h after treatment in a dose-dependent and synergistic manner, providing useful biomarkers of lung activation. IL-6 rose fivefold in serum with delayed kinetics compared to its rise in lavage fluid, and might serve as a systemic biomarker of immune activation of the lungs. The dose-response relationship of lavage fluid cytokines was preserved in mice that underwent myeloablative treatment with cytosine arabinoside to model the treatment of hematologic malignancy. There were no overt signs of distress in mice treated with ODN/Pam2CSK4 in doses up to eightfold the therapeutic dose, and no changes in temperature, respiratory rate, or behavioral signs of sickness including sugar water preference, food disappearance, cage exploration or social interaction, though there was a small degree of transient weight loss. We conclude that treatment with aerosolized ODN/Pam2CSK4 is well tolerated in mice, and that innate immune activation of the lungs can be monitored by the measurement of inflammatory cytokines in lung lavage fluid and serum.

Randomized, double-blind, placebo-controlled phase II study of single-agent oral talactoferrin in patients with locally advanced or metastatic non-small-cell lung cancer that progressed after chemotherapy.

PURPOSE: To investigate the activity and safety of oral talactoferrin (TLF) in patients with stages IIIB to IV non-small-cell lung cancer (NSCLC) for whom one or two prior lines of systemic anticancer therapy had failed. PATIENTS AND METHODS: Patients (n = 100) were randomly assigned to receive either oral TLF (1.5 g in 15 mL phosphate-based buffer) or placebo (15 mL phosphate-based buffer) twice per day in addition to supportive care. Oral TLF or placebo was administered for a maximum of three 14-week cycles with dosing for 12 consecutive weeks followed by 2 weeks off. The primary objective was overall survival (OS) in the intent-to-treat (ITT) patient population. Secondary objectives included progression-free survival (PFS), disease control rate (DCR), and safety. RESULTS: TLF was associated with improvement in OS in the ITT patient population, meeting the protocol-specified level of significance of a one-tailed P = .05. Compared with the placebo group, median OS increased by 65% in the TLF group (3.7 to 6.1 months; hazard ratio, 0.68; 90% CI, 0.47 to 0.98; P = .04 with one-tailed log-rank test). Supportive trends were also observed for PFS and DCR. TLF was well tolerated and, generally, there were fewer adverse events (AEs) and grade ≥ 3 AEs reported in the TLF arm. AEs were consistent with those expected in late-stage NSCLC. CONCLUSION: TLF demonstrated an apparent improvement in OS in patients with stages IIIB to IV NSCLC for whom one or two prior lines of systemic anticancer therapy had failed and was well tolerated. These results should be confirmed in a global phase III trial.

A randomized, double-blind, placebo-controlled, phase II study of oral talactoferrin in combination with carboplatin and paclitaxel in previously untreated locally advanced or metastatic non-small cell lung cancer.

INTRODUCTION: The aim of the study is to investigate the activity and safety of oral talactoferrin (TLF) plus carboplatin and paclitaxel (C/P) in patients with previously untreated stage IIIB/IV non-small cell lung cancer. METHODS: Patients (n = 110) were randomly assigned to receive C/P plus either TLF (C/P/T) or placebo (C/P/P). The primary objective of this exploratory study was assessment of confirmed response rate (RR) in the prospectively defined evaluable population with a one-tailed p = 0.05. Secondary objectives included assessment of progression-free survival (PFS), duration of response, overall survival (OS), and safety. RESULTS: The trial met the primary end point of improvement in confirmed RR in the prospectively defined evaluable population. Compared with the C/P/P group, RR increased in the C/P/T group by 18% (29-47%; p = 0.05) and 15% (27-42%; p = 0.08) in the evaluable and intent-to-treat populations, respectively. Compared with the C/P/P group, the C/P/T group had a longer median PFS (4.2 versus 7.0 months), OS (8.5 versus 10.4 months), and duration of response (5.5 versus 7.6 months), although the differences were not statistically significant. Adverse events (AEs) were consistent with C/P therapy. There were fewer total AEs (472 versus 569; two-tailed p = 0.003) and grade 3/4 AEs (78 versus 105; p = 0.05) in the C/P/T group compared with the C/P/P group. CONCLUSION: TLF, in combination with C/P, demonstrated an apparent improvement in RR, PFS, and OS in patients with previously untreated stage IIIB/IV non-small cell lung cancer and appears to enhance activity without significant additional toxicity. These results need to be confirmed in a phase III trial.

Phase IB trial of oral talactoferrin in the treatment of patients with metastatic solid tumors.

PURPOSE: We evaluated safety and activity of talactoferrin, a novel immunomodulatory protein in a phase IB trial of patients with refractory solid tumors. METHODS: Thirty-six patients with metastatic cancer who had progressed on, or were ineligible for, standard chemotherapy received single-agent oral talactoferrin. Following dose-escalation, with no DLTs , patients were randomized to 4.5 or 9 g/day talactoferrin. RESULTS: Talactoferrin was well tolerated with apparent anti-cancer activity, particularly in NSCLC and RCC. One patient had a PR (RECIST) and 17 patients (47%) had stable disease (50% disease control rate). Median PFS in the twelve NSCLC and seven RCC patients was 4.2 and 7.3 months, respectively. There was no apparent difference in anti-tumor activity or adverse events between talactoferrin doses. CONCLUSIONS: Oral talactoferrin was well tolerated. Although evaluated in a small number of patients, talactoferrin appeared to have anti-cancer activity, particularly in NSCLC and RCC and should be evaluated further.

Talactoferrin stimulates wound healing with modulation of inflammation.

BACKGROUND: Inflammation is an acute, early process during normal wound healing. Talactoferrin, a recombinant human lactoferrin, can induce the secretion of inflammatory mediators. MATERIALS AND METHODS: We measured wound healing activity of topical talactoferrin in full-thickness wounds of normal mice and diabetic (db(-)/db(-)) mice, systemic bioavailability, and the potential to modulate inflammation through in vitro and in vivo binding assays and inflammatory mediator measurements. RESULTS: Talactoferrin significantly increased the closure rate during 12 to 19 d (maximally on d 3 to 6), the 75% closure incidence, and the time to 50% closure versus vehicle or becaplermin (recombinant human platelet-derived growth factor). Systemic bioavailability was less than 0.5% following administration to open wounds. Talactoferrin bound local dermal cells in vivo and human dermal fibroblasts in vitro, and it induced the migration of dermal fibroblasts, THP-1 macrophages, Jurkat T cells, and mouse granulocytes in vitro. Competition binding assays suggested the involvement of IL-8RB and CCR2 chemokine receptors in binding and/or cell migration. Consistently, the induction of migration was partially inhibited in interleukin (IL)-8RB deficient granulocytes. Talactoferrin also enhanced the production of key repair inflammatory mediators IL-8, IL-6, macrophage inflammatory protein-1 alpha, and tumor necrosis factor alpha in d 3 wounds, and IL-8, IL-6, and monocyte chemotactic protein-1 in cultured dermal fibroblasts. CONCLUSION: Talactoferrin promotes wound repair in vivo, correlating with a modulated enhancement of the early inflammatory phase of wound healing. Based on this data, talactoferrin was subsequently tested clinically in a Phase II trial in patients with diabetic ulcers and was found to be effective and safe. Talactoferrin should be further evaluated in patients with diabetic and other types of ulcers.

Phase 2 trial of talactoferrin in previously treated patients with metastatic renal cell carcinoma.

BACKGROUND: Talactoferrin (TLF), a recombinant form of human lactoferrin (hLF), is an immunomodulatory iron-binding glycoprotein first identified in breast milk. Its immunomodulatory functions include activation of natural killer (NK) and lymphokine-activated killer cells and enhancement of polymorphonuclear cells and macrophage cytotoxicity. Studies in animal models have shown promising anticancer activity, and clinical antitumor activity has been observed in nonsmall cell lung cancer and other tumor types. The purpose of the current study was to evaluate the activity and safety of TLF in patients with refractory metastatic renal cell carcinoma (RCC). METHODS: Forty-four adult patients with progressive advanced or metastatic RCC who had failed prior systemic therapy received oral talactoferrin at a dose of 1.5 g twice daily on a 12-week-on 2-week-off schedule. Patients were evaluated for progression-free survival at 14 weeks, overall response rate, and progression-free and overall survival. RESULTS: TLF was well tolerated. No significant hematologic, hepatic, or renal toxicities were reported. The study met its predefined target with a 14-week progression-free survival rate of 59%. The response rate was 4.5%. The mMedian progression-free survival was 6.4 months and the median overall survival was 21.1 months. CONCLUSIONS: TLF is a well-tolerated new agent that has demonstrated preliminary signs of clinical activity. Given the lack of toxicity, the lack of rapid disease progression in this cohort, and the preclinical data on immune activation, a randomized study assessing its effects on disease progression in patients with metastatic RCC is rational.

Lactoferrin acts as an alarmin to promote the recruitment and activation of APCs and antigen-specific immune responses.

Lactoferrin is an 80-kDa iron-binding protein present at high concentrations in milk and in the granules of neutrophils. It possesses multiple activities, including antibacterial, antiviral, antifungal, and even antitumor effects. Most of its antimicrobial effects are due to direct interaction with pathogens, but a few reports show that it has direct interactions with cells of the immune system. In this study, we show the ability of recombinant human lactoferrin (talactoferrin alfa (TLF)) to chemoattract monocytes. What is more, addition of TLF to human peripheral blood or monocyte-derived dendritic cell cultures resulted in cell maturation, as evidenced by up-regulated expression of CD80, CD83, and CD86, production of proinflammatory cytokines, and increased capacity to stimulate the proliferation of allogeneic lymphocytes. When injected into the mouse peritoneal cavity, lactoferrin also caused a marked recruitment of neutrophils and macrophages. Immunization of mice with OVA in the presence of TLF promoted Th1-polarized Ag-specific immune responses. These results suggest that lactoferrin contributes to the activation of both the innate and adaptive immune responses by promoting the recruitment of leukocytes and activation of dendritic cells.

Lactoferrin, a major defense protein of innate immunity, is a novel maturation factor for human dendritic cells.

Lactoferrin (LF) is an important protein component of the innate immune system that is broadly distributed within the body fluids. LF is endowed with multiple biological activities. Talactoferrin (TLF), a recombinant human LF, is in clinical development as an anticancer agent and is entering Phase III clinical trials. Here, we show that TLF induces the maturation of human dendritic cells (DCs) derived from monocytes. TLF, at physiologically relevant concentrations (100 microg/ml) up-regulates the expression of human leukocyte antigen (HLA) class II, CD83, CD80, and CD86 costimulatory molecule and CXCR4 and CCR7 chemokine receptors, acting primarily through the p38 MAPK signaling pathway. DCs matured by TLF displayed an enhanced release of IL-8 and CXCL10, as well as a significantly reduced production of IL-6, IL-10, and CCL20. They also display a reduced ability to take up antigen and increased capacity to trigger proliferation and release IFN-gamma in the presence of allogeneic human T cells. TLF-matured DCs are able to prime naive T cells to respond to KLH antigen and display a significantly increased capacity to present Flu-MA(58-66) peptide to HLA-A2-matched T cells. These data suggest that a key immunomodulatory function that may be mediated by TLF is to link the innate with adaptive immunity through DC maturation.

Requirement for IFN-gamma, CD8+ T lymphocytes, and NKT cells in talactoferrin-induced inhibition of neu+ tumors.

We have previously shown that talactoferrin-alfa (TLF), a recombinant human lactoferrin, is an immunomodulatory protein that is active against implanted tumors, both as a single agent and in combination with chemotherapy. In this study, we show that talactoferrin is active against autochthonous tumors in a transgenic mouse line, which is more analogous to human cancers, and identify key mechanistic steps involved in the anticancer activity of oral TLF. BALB/c mice transgenic for the rat neu (ErbB2) oncogene (BALB-neuT) treated with oral TLF showed a significant delay in carcinogenesis, with 60% tumor protection relative to vehicle-treated mice at week 21. Oral TLF also showed tumor growth inhibition in wild-type BALB/c mice implanted with neu(+) mammary adenocarcinoma, with one third displaying a long-lasting or complete response. Oral TLF induces an increase in intestinal mucosal IFN-gamma production and an increase in Peyer's patch cellularity, including expansion of CD8(+) T lymphocytes and NKT cells, and the enhancement of CD8(+) T-cell cytotoxicity. In IFN-gamma knockout mice, there is an absence of the TLF-induced Peyer's patch cellularity, no expansion of CD8(+) T lymphocytes and NKT cells, and loss of TLF anticancer activity. TLF antitumor activity is also lost in mice depleted of CD8(+) T cells and in CD1 knockout mice, which lack NKT activity. Thus, the inhibition of distant tumors by oral TLF seems to be mediated by an IFN-gamma-dependent enhancement of CD8(+) T- and NKT cell activity initiated within the intestinal mucosa.

Oral lactoferrin results in T cell-dependent tumor inhibition of head and neck squamous cell carcinoma in vivo.

PURPOSE: Human lactoferrin is a naturally occurring glycoprotein that inhibits cancer growth. Our purpose was to evaluate recombinant human lactoferrin as a chemotherapeutic agent against head and neck squamous cell carcinoma. EXPERIMENTAL DESIGN: Controlled experiments both in vitro and in the murine model evaluating both the effect and mechanism of lactoferrin on cancer growth. RESULTS: In both human and murine cell lines, lactoferrin induced dose-dependent growth inhibition. Using flow cytometric analysis, lactoferrin was shown to induce G(1)-G(0) growth arrest. This arrest seemed to be modulated by down-regulation of cyclin D1. In the in vitro model, luminex data revealed that lactoferrin inhibited cellular release of proinflammatory and prometastatic cytokines, including interleukin-8, interleukin-6, granulocyte macrophage colony-stimulating factor, and tumor necrosis factor-alpha. Lactoferrin up-regulated the cellular activation of nuclear factor-kappaB within 4 h of cellular exposure. In C3h/HeJ mice implanted with SCCVII tumors, orally delivered lactoferrin inhibited tumor growth by 75% compared with control mice. Immunohistochemical analysis of harvested tumors revealed up to 20-fold increases of lymphocytes within treated animals. When mice were depleted of CD3(+) cells, all lactoferrin-induced tumor inhibition was abrogated. CONCLUSION: We conclude that human recombinant lactoferrin can inhibit the growth of head and neck squamous cell carcinoma via direct cellular inhibition as well as systemically via immunomodulation. Our data support the study of human lactoferrin as an immunomodulatory compound with therapeutic potential.

Phase I trial of oral talactoferrin alfa in refractory solid tumors.

BACKGROUND: Lactoferrin is an iron-binding glycoprotein first identified in breast milk as a protein product of mammary epithelial cells. Its immunomodulatory functions include activation of NK and lymphokine-activated killer cells and enhancement of PMN and macrophage cytotoxicity. Studies in animal models have shown promising anti-cancer activity. The purpose of the present study was to evaluate the safety and tolerability of talactoferrin alfa (talactoferrin; TLF) in humans, as well as pharmacokinetics and pharmacodynamics. METHODS: Ten adult patients with progressive advanced solid tumors who had failed conventional chemotherapy were administered oral TLF at doses from 1.5 to 9 g/day, using a 2 weeks on, 2 weeks off schedule. Patients were evaluated for drug toxicity, tumor growth rate, talactoferrin pharmacokinetics and cytokine markers. RESULTS: Talactoferrin was very well tolerated. No hematological, hepatic, or renal toxicities were reported. A single patient had Grade 2 diarrhea, and there were no Grade 3 or 4 toxicities. Following oral administration, significant levels of talactoferrin were undetectable in circulation, but a statistically significant increase in circulating IL-18, a pharmacodynamic indicator of talactoferrin activity, was observed. Of the eight patients who were radiologically evaluable, five (63%) had stable disease by RECIST criteria two months after start of therapy, including one patient with a minor response. Seven patients (88%) had a decrease in their tumor growth rate. The three patients with non-small cell lung cancer (NSCLC) all survived for at least one year following the start of talactoferrin monotherapy. CONCLUSIONS: Talactoferrin is a promising, well-tolerated new agent that should be evaluated further in patients with refractory metastatic cancer.

Oral lactoferrin inhibits growth of established tumors and potentiates conventional chemotherapy.

In this work, we investigated the anticancer activity of orally administered recombinant human lactoferrin (rhLF) alone and in combination with chemotherapy in tumor-bearing mice. rhLF inhibited the growth of squamous cell carcinoma (O12) tumors in T cell-immunocompromised nu/nu mice by 80% when administered at 1,000 mg/kg (2.9 g/m2) by oral gavage twice daily for 8 days (p < 0.001). Similar activity was observed in syngeneic, immunocompetent BALB/c mice, where orally administered rhLF (1,000 mg/kg, 2.9 g/m2 once daily) halted the growth of mammary adenocarcinoma TUBO. Oral rhLF (200 mg/kg, 0.57 g/m2) was also used alone and in combination with cis-platinum (5 mg/kg) to treat head-and-neck squamous cell carcinoma in a syngeneic murine model. Monotherapy with oral rhLF or cis-platinum caused 61% or 66% tumor growth inhibition over placebo, respectively. Mice receiving both therapies showed 79% growth inhibition, a statistically significant improvement over each drug alone. We then demonstrated that administration of oral rhLF (300 mg/kg, 0.86 g/m2) to tumor-bearing or naive mice resulted in (i) significantly increased production of IL-18 in the intestinal tract, (ii) systemic NK cell activation and (iii) circulating CD8+ T-cell expansion. These data suggest that oral rhLF is an immunomodulatory agent active against cancer as a single agent and in combination chemotherapy, exerting its systemic effect through stimulation of IL-18 and other cytokines in the gut enterocytes. rhLF has been administered orally to 211 people without a single serious drug-related adverse event. Thus, rhLF shows promise as a safe and well-tolerated novel immunomodulatory anticancer agent.