Dose IL-2 Research Articles

Anti-IL12p40 autoantibodies in a teenage girl with multiple recurrent abscesses

More frequent among adults, phenocopies may be caused by somatic mutations or anti-cytokine autoantibodies, mimicking the phenotypes of primary immunodeficiencies. A fourteen-year-old girl was referred for a two-year history of weight loss and multiple recurrent abscesses, complicated recurrent pneumonia, pyelonephritis, osteomyelitis, and septic shock, without fever. She had started with nausea, hyporexia, and weight loss, then with abscesses in her hands, knee, ankle, and spleen. She also developed a rib fracture and left thoracic herpes zoster. The patient was cachectic, with normal vital signs, bilateral crackles on chest auscultation, tumefaction of the knee joint, and poorly healed wounds in hands and chest, oozing a yellowish fluid. Chest computed tomography revealed multiple bilateral bronchiectases. Laboratory workup reported chronic anemia, leukocytosis, neutrophilia, mild lymphopenia, thrombocytosis, pan-hypergammaglobulinemia, and elevated acute serum reactants. Lymphocyte subsets were low but present. Mycobacterium tuberculosis was detected via polymerase chain reaction in a bone biopsy specimen from ankle osteomyelitis. Whole-exome sequencing failed to identify a monogenic defect. Interleukin-12 was found markedly elevated in the serum of the patient. Phosphorylation of STAT4, induced by increasing doses of IL-12, was neutralized by patient serum, confirming the presence of anti-IL12 autoantibodies. IL-12 and IL-23 are crucial cytokines in the defense against intracellular microorganisms, the induction of interferon-gamma production by lymphocytes, and other inflammatory functions. Patients who develop neutralizing serum autoantibodies against IL12 manifest late in life with weight loss, multiple recurrent abscesses, poor wound healing, and fistulae. Treatment with anti-CD20 monoclonal antibodies was effective.

De novo design of mini-binder proteins against IL-2 receptor β chain

IL-2 regulates the immune response by interacting with different IL-2 receptor (IL-2R) subunits. High dose of IL-2 binds to IL-2Rβγc heterodimer, which induce various side effects while activating immune function. Disrupting IL-2 and IL-2R interactions can block IL-2 mediated immune response. Here, we used a computational approach to de novo design mini-binder proteins against IL-2R β chain (IL-2Rβ) to block IL-2 signaling. The hydrophobic region where IL-2 binds to IL-2Rβ was selected and the promising binding mode was broadly explored. Three mini-binders with amino acid numbers ranging from 55 to 65 were obtained and binder 1 showed the best effects in inhibiting CTLL-2 cells proliferation and STAT5 phosphorylation. Molecular dynamics simulation showed that the binding of binder 1 to IL-2Rβ was stable; the free energy of binder1/IL-2Rβ complex was lower, indicating that the affinity of binder 1 to IL-2Rβ was higher than that of IL-2. Free energy decomposition suggested that the ARG35 and ARG131 of IL-2Rβ might be the key to improve the affinity of binder. Our efforts provided new insights in developing of IL-2R blocker, offering a potential strategy for ameliorating the side effects of IL-2 treatment.

A phase 1b study of tbio-4101 (autologous selected and expanded tumor-infiltrating lymphocytes [TILs]) with pembrolizumab in patients with anti-PD-1–resistant, advanced head and neck squamous cell carcinoma (aHNSCC).

TPS6115 Background: Despite the excitement and approval of anti-PD1 therapy in first line setting of patients with aHNSCC, prognosis remains poor, with the majority experiencing disease progression after therapy. Consequently, novel strategies to improve treatment activity represent a critical and unmet need for this patient population. Cell therapies offer the advantage of a more personalized therapy for patients over standard immune checkpoint inhibitors. Historically, cell therapies have relied on bulk products that may not effectively select a tumor-reactive T cell population. TBio-4101 is produced by an industrialized manufacturing approach leveraging single-cell sorting of patient-specific neoepitope-reactive TIL with the potential to address immunologically ‘cold’ tumors. The TBio-4101 process specifically enriches polyclonal, polyfunctional, tumor-reactive T cells with an endogenous neoantigen-reactive T cell receptor repertoire that can target multiple tumor antigens. Non-tumor-reactive bystander T cells, which may negatively impact treatment efficacy via product dilution, and typically account for ≥90% of bulk TIL, are substantially reduced by this process. Inspired by the early academic clinically efficacious NCI selection and enrichment strategies (Tran et al 2014, 2016; Zacharakis et al, 2022), TBio-4101 is refined to better identify and potentially enrich to >70% tumor-reactive CD8+ and CD4+ T cells (median < 3% in bulk TIL). Methods: This is a Phase 1b, open-label, single-center study of TBio-4101 and pembrolizumab. Eligible patients are 18 to 75 years of age with aHNSCC. Prior to receiving pembrolizumab with or without chemotherapy as first line standard-of-care (SOC), these patients undergo tumor harvest for TILs followed by apheresis to collect monocytes. TILs are isolated from the tumor and expanded in vitro. Patient-specific neoepitopes are defined by whole exome and RNA sequencing of tumor versus germline to manufacture a peptide pool representing potential tumor-specific mutations. Monocytes isolated from apheresis are differentiated into dendritic cells, pulsed with the neoepitope peptides, and co-cultured with the TILs. Tumor-reactive T cells are specifically activated and selected based on upregulation of activation markers prior to expansion in culture. This process results in a patient-specific T cell product enriched for tumor-reactive T cells. Enrolled patients who progress on first line SOC therapy are preconditioned with 5 days of nonmyeloablative lymphodepletion followed by TBio-4101 infusion. Thereafter, patients receive ≤6 doses of IL-2 (600,000 IU/kg) every 8h, and pembrolizumab (200 mg) every 3 weeks until disease progression. The trial is currently open, with a target enrollment of 15 patients (NCT06236425). Clinical trial information: NCT06236425 .

Efficacy and safety of lifileucel, an autologous tumor-infiltrating lymphocyte cell therapy, and pembrolizumab in patients with immune checkpoint inhibitor-naive unresectable or metastatic melanoma: Updated results from IOV-COM-202 cohort 1A.

9505 Background: Immune checkpoint inhibitors (ICI) are front-line standard-of-care treatment (tx) for advanced (unresectable or metastatic) melanoma. Despite recent advances in front-line tx, a majority of patients (pts) do not achieve long-term benefit. Lifileucel, a one-time autologous tumor-infiltrating lymphocyte (TIL) cell therapy, potentially induces durable responses in pts with advanced melanoma previously treated with ICI. This study evaluates lifileucel combined with anti-PD-1 therapy in front-line advanced melanoma. Methods: IOV-COM-202 (NCT03645928) Cohort 1A assesses the efficacy and safety of lifileucel and pembrolizumab (pembro) in pts with ICI-naive unresectable or metastatic melanoma. Pts may have received BRAF/MEK inhibitor tx if BRAF mutation-positive. Eligible pts must have ≥1 resectable lesion (≥1.5 cm diameter) for 22-day cryopreserved lifileucel manufacturing, and ≥1 measurable lesion for response assessment per RECIST v1.1. The tx regimen consists of pembro, nonmyeloablative lymphodepletion (cyclophosphamide and fludarabine), a single lifileucel infusion (1 × 109 – 150 × 109 cells), ≤6 doses of IL-2 (600,000 IU/kg IV), and continued pembro until disease progression, or unacceptable toxicity for ≤24 months. The endpoints are investigator-assessed objective response rate (ORR) and incidence of grade ≥3 treatment-emergent adverse events (TEAE). Results: As of 22-Dec-2023, 22 pts with a median (range) age of 48.5 (18–68) years received lifileucel and pembro. At baseline, the median (range) target lesion sum of diameters was 54.5 (14–355) mm; 7 (31.8%) pts had liver lesions. Metastatic staging at study entry was as follows: 4 (18.2%) had M1a, 2 (9.1%) had M1b, 10 (45.5%) had M1c, and 2 (9.1%) had M1d. Eight (36.4%) pts had V600 BRAF mutations; 3 (13.6%) pts had prior BRAF/MEK inhibitor tx. Confirmed ORR was 63.6% (14/22), including 22.7% (5/22) CR and 40.9% (9/22) PR; 6 pts (27.3%) had SD. Median time to initial response was 2.5 months. All response evaluable pts demonstrated regression of target lesions. At a median follow-up of 17.2 months, median duration of response was not reached. Responses deepened over time; 10/14 (71.4%) pts had ongoing response and 8/14 (36.4%) pts had response ≥12 months. TEAEs were consistent with the underlying disease and known safety profiles of pembro, nonmyeloablative lymphodepletion, and IL-2. Most common grade ≥3 TEAEs were thrombocytopenia (68.2%), neutropenia (50.0%), and anemia (45.5%). Conclusions: These results demonstrate encouraging efficacy and durability for the combination of lifileucel and pembro and support its further evaluation in pts with untreated advanced melanoma in the phase 3 study TILVANCE-301 (NCT05727904). Clinical trial information: NCT03645928 .

A pilot trial of autologous tumor infiltrating lymphocytes (lifileucel, LN-144) for patients with asymptomatic melanoma brain metastases.

TPS9603 Background: Melanoma brain metastases (MBM) are a leading cause of morbidity and mortality for patients with advanced melanoma. Modern systemic therapies are insufficient at controlling MBM, resulting in median overall survival (OS) of <1 year. Adoptive T cell therapy, using tumor infiltrating lymphocytes (TIL), has demonstrated efficacy in treating advanced melanoma. Lifileucel (LN-144), an autologous TIL product, was recently shown to be safe and effective for patients with PD-1 refractory melanoma. However, no trials have yet explored lifileucel in patients with active MBM. Methods: This single-center pilot trial (NCT05640193) aims to enroll 10 pts with asymptomatic MBM from non-uveal melanoma to receive lifileucel. Patients must have ≥1 intracranial lesion measuring 5-30mm visible on MRI to be used as a target lesion for modified (m)RECIST measurement, progression on prior anti-PD-1 therapy (with or without anti-CTLA-4), progression on targeted therapy (if BRAF V600E/K-mutated), ECOG PS ≤1, ≥1 resectable lesion(s) (≥1.5 cm), recovered from prior surgery/anticancer treatment-related AEs (grade ≤1). Patients are ineligible if they have symptomatic MBM and/or require corticosteroids of ≥10 mg/day of prednisone or equivalent. Lifileucel is generated from resected tumor in a centralized GMP process. The regimen includes nonmyeloablative lymphodepletion with cyclophosphamide 750 mg/m2 on days 1-3 and fludarabine 30 mg/kg on days 1-4 (reduced dose compared with standard lifileucel protocol), lifileucel infusion, and up to 6 doses of high-dose IL-2. The primary endpoint is feasibility, defined as ≥7/10 patients who undergo tumor resection successfully receiving lifileucel infusion. Secondary endpoints are safety, feasibility of manufacturing lifileucel in patients with MBM, and brain metastasis response rate (BMRR) per mRECIST 1.1. Exploratory endpoints include overall objective response rate by mRECIST 1.1, best extracranial response rate, intracranial progression-free survival (PFS), overall PFS, OS. Extensive correlative analyses of peripheral blood mononuclear cells, plasma, tumors, and cerebrospinal fluid are planned to better understand MBM growth, treatment resistance, and response of the central nervous system to lifileucel. Clinical trial information: NCT05640193 .

A first-in-human study of CRISPR/Cas9-engineered tumor infiltrating lymphocytes (TILs) product GT316 as monotherapy in advanced solid tumors.

2549 Background: A next-generation TIL product GT316 was engineered with CRISPR/Cas9 to genetically disrupt two key immunoregulatory targets identified from a genome-wide CRISPR screening platform and demonstrated significantly enhanced anti-tumor efficacy and persistence in pre-clinical animal studies with ameliorated TIL exhaustion profile. A first-in-human trial was initiated to evaluate the preliminary safety and efficacy of GT316 in advanced solid tumors. Methods: The first-in-class study is to enroll patients (pts) with advanced treatment-refractory solid tumors, especially gynecological tumors. Once the optimal biological dose (OBD) is identified, a monotherapy expansion phase will be initiated for pts with various solid tumors. Enrolled pts were preconditioned with a nonmyeloablative (NMA) lymphodepletion regimen and treated by infusion of the GMP-grade G316 product followed by IL-2 administration. Results: As of January 22, 2024, the dose escalation study enrolled 4 pts, all of whom were female with a median age of 58 years with 1 to 9 prior lines of systemic treatment. The median number of infused TIL cells was 1.0E+10. All pts experienced TRAEs with the majority being grade 1 or 2. Grade 3/4 TRAEs included lymphocyte count decreased, white blood cell count decreased, monocyte count decreased and neutrophil count decreased, which were related to the NMA regimen. No dose-limiting toxicities (DLTs) or TIL-related grade≥3 TRAEs were observed. Median time on study was 33.4 wks (8–35 wks). One pt with treatment-refractory cervical cancer experienced a confirmed complete response (CR) after 4 wks and the duration of response is 32 wks by now (RECIST 1.1). One ovarian cancer pt with 9 lines of previous systemic therapies experienced a confirmed partial response (PR) after 4 wks and the duration of response is 22 wks by now (RECIST 1.1). Response data will be continuously collected. The other two ovarian cancer pts experienced stable disease (SD) and progressive disease (PD), respectively. Despite varying doses of TILs (3.2E+09 to 1.90E+10) and IL-2 (up to 3.0E+05 IU/kg), all pts receiving GT316 showed robust TIL expansion post-infusion, which is positively correlated with administrated IL-2 doses. Enhanced IFN-γ secretion could be detected in the serum of the CR and PR pts compared to the SD and PD pts, indicating potential tumor antigen-specific response. Conclusions: The first-in-human study of GT316, a CRISPR/Cas9-dual KO anti-exhaustion TIL product, demonstrates good tolerability with encouraging preliminary anti-tumor efficacy as a monotherapy in pts with advanced solid cancers. The infusion of GT316 was associated with robust TIL expansion and IFN-γ secretion. Updated data with additional follow-up will be continuously collected, and OBD will be determined based on the overall data of safety and preliminary efficiency. Clinical trial information: NCT06145802 .

Abstract PO2-03-10: Association of pathologic complete response with the 27-gene IO score and week 3 IOpath response following neoadjuvant pembrolizumab +/- intralymphatic cytokines in the neoIRX trial

Abstract Introduction: Novel biomarker strategies are needed to identify immunotherapy (IO)-sensitive early-stage triple-negative breast cancers (TNBC). We recently reported a phase II trial (neoIRX, NCT04373031) evaluating induction IO (pembrolizumab +/- intralymphatic cytokines, IRX-2, comprised of physiologic doses of IL-2, IFNγ, and other cytokines derived from activated donor lymphocytes) preceding de-escalated neoadjuvant chemotherapy (NAC). Here, we report associations of response with the 27-gene IO score (DetermaIO), a commercially available RT-qPCR assay shown to predict IO response in the neoPACT, I-SPY2, and neoTRIP trials. We also propose a novel surrogate endpoint, entitled “IOpath” response, that characterizes radiographic and pathologic response following single-cycle IO and may be useful for guiding NAC de-escalation or omission. Methods: Subjects with stage II-III TNBC were randomized 1:1 (n=12) to receive induction pembrolizumab (200mg IV) +/- peri-areolar IRX-2 (IRX-2 arm: 1 ml SQ x2 daily for 10 days + cyclophosphamide 300 mg/m2 IV x 1) preceding initiation of pembrolizumab + NAC. The primary endpoint was pathologic complete response (pCR) rate. Post-induction IO response (IOpath) was assessed by week 3 ultrasound (US) and biopsy. IOpath response was designated as IOpath-NR (non-response: radiographic progression and/or biopsy non-response [neither treatment effect nor TILs expansion]), IOpath-PR (partial response: viable tumor with either treatment effect or TILs expansion), IOpath-CR (complete response: treatment effect with no viable tumor), or IOpath-NE (not evaluable). Results: The IRX-2 arm achieved 83% pCR (n=5/6, CI 36-100%) compared to 33% pCR with pembrolizumab alone (n=2/6, CI 4-78%) and was well tolerated (67% grade I skin bruise). Baseline 27-gene IO score predicted pCR (AUC 0.77, IO+: 5/6 pCR; IO-: 2/6 pCR) and week 3 IOpath response (AUC 0.85, IO+: 3/5 IOpath-CR/PR; IO-: 1/4 IOpath-PR), and outperformed TILs (AUC for pCR: 0.66; AUC for IOpath: 0.68) and CPS score (AUC for pCR: 0.63, AUC for IOpath: 0.58). Week 3 IOpath response was associated with 100% pCR (IOpath+: 4/4 pCR; IOpath-: 2/5 pCR). Conclusion: A subset of TNBCs experience brisk IO response following single-cycle IO (IOpath response), which is associated with 100% pCR in this preliminary dataset, highlighting IOpath as a potential surrogate biomarker to guide NAC de-escalation or omission in early-stage TNBC. Baseline 27-gene IO score predicts pCR and week 3 IOpath response. A future study is planned (neoINBRX) to evaluate the feasibility of combining baseline IO score with week 3 IOpath response to identify and treat IO-sensitive tumors with chemotherapy-sparing IO combination therapy. Acknowledgements: The study was funded in part by Merck, Sharpe, & Dohme as part of the Merck Investigator Studies Program, and by BrooklynImmunotherapeutics. Citation Format: David Page, Alan Su, Nicole Moxon, Staci Mellinger, Tracy Kelly, Nicole Fredrich, Amanda Seino, Zheng Topp, Philippa Newell, Kristen Massimino, Shaghayegh Aliabadi, Yaping Wu, Maritza Martel, William Redmond, Yoshinobu Kogushi, Grace Mcgee, Alejandra Mendez-Torres, Zhaoyu Sun, James Imatani, Alison Conlin, Kelly Perlewitz, Tyler Nielsen, Sasha Stanton. Association of pathologic complete response with the 27-gene IO score and week 3 IOpath response following neoadjuvant pembrolizumab +/- intralymphatic cytokines in the neoIRX trial [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO2-03-10.

Highly efficient IL-2-dependent gene activation by a single wave of IL-2R signaling in human regulatory T cells

Abstract Low-dose IL-2 is being advanced as a therapy for autoimmunity based on selectivity toward Tregs. Regarding human CD4+ Treg and effector memory (TEM) T cells, the IL-2 sensitivity of Tregs is 10- and 100-fold greater for proximal signaling and down-stream gene activation, respectively. To further investigate the efficiency of IL-2R signaling in Tregs, we monitored the persistence of a single wave of pSTAT5 and assessed if this supported IL-2-dependent transcription. When Treg and TEM cells were pulsed with a high dose of IL-2 to normalize initial STAT5 activation, pSTAT5 persisted for >2 hr only in Tregs. Lower amounts of pulsed-IL-2 also led to persistent pSTAT5 in Tregs. The short persistence of pSTAT5 in TEM cells is not cell intrinsic because pulsing TEM cells with IL-7 also led to lasting pSTAT5. Thus, the high expression of CD25 and CD127 in Treg and TEM cells likely contributed to persistent pSTAT5. This conclusion implies that a component of proximal STAT5-dependent signaling is limiting in comparison to the numbers of bound receptors. RNA Prime Flow demonstrated that transient pulse of IL-2 or IL-7 in Treg and TEM cells caused upregulation of CISH mRNA. These results indicate that a limited exposure of T cells expressing high amounts of the IL-2R and IL-7R are linked to a mechanism to sustain proximal signaling to support gene transcription. This mechanism may fine tune gene expression enabling cells to maximize utilization of limiting cytokines in vivo.

Low-dose IL-2 treatment confers anti-inflammatory effect against subarachnoid hemorrhage in mice

ObjectiveSubarachnoid hemorrhage (SAH) was a stroke with high occurrence and mortality. At the early stage, SAH patients have severe cerebral injury which is contributed by inflammation. In this study, we aimed to explore the anti-inflammation effect of low-dose IL-2 in SAH mice. MethodsThe 12-week-old C57BL/6J male mice were conducted with SAH surgery (Internal carotid artery puncture method). Different dose of IL-2 was injected intraperitoneally for 1 h, 1 day, and 2 days after SAH. Single-cell suspension and flow cytometry were used for the test of regulatory T (Treg) cells. Immunofluorescence staining was used to investigate the phenotypic polarization of microglia and inflammation response around neurons. Enzyme-Linked Immuno-sorbent Assay (ELISA) was applied to detect the level of pro-inflammatory factors. ResultsLow-dose IL-2 could enrich the Treg cells and drive the microglia polarizing to M2. The level of pro-inflammatory factors, IL-1α, IL-6, and TNF-α decreased in the low-dose IL-2 group. The inflammation response around neurons was attenuated. Low-dose IL-2 could increase the number of Treg cells, which could exert a neuroprotective effect against inflammation after SAH. ConclusionLow-dose IL-2 had the potential to be an effective clinical method to inhibit inflammation after SAH.

Abstract CT286: TILVANCE-301, a phase 3 study of lifileucel tumor-infiltrating lymphocyte (TIL) cell therapy in combination with pembrolizumab vs pembrolizumab alone in treatment-naive unresectable or metastatic melanoma

Abstract Introduction: Novel early-line therapies for advanced (unresectable or metastatic) melanoma are needed to improve the rate of deep and durable responses and increase the proportion of patients with long-term benefit. TILVANCE-301 will evaluate the efficacy and safety of lifileucel autologous TIL cell therapy plus pembrolizumab in patients with untreated advanced melanoma. Methods: TILVANCE-301 (NCT05727904) is a phase 3, multicenter, randomized, open-label, parallel group study that will randomize ~670 patients (1:1; Day 0) to Arm A: lifileucel plus pembrolizumab (Day 3: tumor tissue resection for TIL manufacturing; Day 5: pembrolizumab 200 mg; Day 26: pembrolizumab 400 mg; Day 28-29: cyclophosphamide 60 mg/kg; Day 28-32: fludarabine 25 mg/m2; Day 33: lifileucel; Day 34-37: ≤6 doses of high-dose IL-2; Week 10: pembrolizumab 400 mg Q6W) or Arm B: pembrolizumab alone (same pembrolizumab dosing as Arm A). Patients in Arm B with confirmed progressive disease verified by blinded independent review committee (BIRC) have the option to receive lifileucel monotherapy as immediate next treatment and may continue pembrolizumab until start of nonmyeloablative lymphodepletion. Eligible adults have histologically confirmed advanced melanoma, Eastern Cooperative Oncology Group performance status of 0-1, estimated life expectancy >6 months, ≥1 resectable lesion to generate lifileucel, and ≥1 remaining measurable lesion. Prior neoadjuvant or adjuvant treatment including immune checkpoint inhibitors may be allowed. Prior therapy for metastatic disease, symptomatic untreated brain metastases, organ allograft or prior cell therapy, uveal/ocular melanoma, and chronic systemic steroid therapy are not permitted. The dual primary efficacy endpoints are BIRC-assessed (RECIST v1.1) objective response rate (ORR) and progression-free survival (PFS). Key secondary efficacy endpoint is overall survival. Additional secondary efficacy endpoints include BIRC-assessed complete response (CR) rate, duration of response (DOR), and event-free survival (EFS); investigator-assessed ORR, PFS, CR rate, DOR, EFS, and PFS2; and safety as characterized by severity and seriousness of treatment-emergent adverse events and relationship to study drug. Exploratory endpoints include in vivo T-cell persistence (unique CDR3 sequences in peripheral blood mononuclear cell [PBMC] over time) and correlative biomarkers (eg, lifileucel phenotypic and functional characteristics; lifileucel, tumor, and PBMC gene expression profiles; tumor mutational landscape). The study will enroll globally, with initial sites in Europe, North America, and Australia. Trial registration: NCT05727904 Citation Format: Sajeve Thomas, Young Ki Hong, Yazan Samhouri, James Larkin, David J. Olson, Gino K. In, Victoria Atkinson, Philip Lammers, Andrew J. Furness, Juan Martin-Liberal, Patrick Terheyden, Andrew S. Poklepovic, Ryan H. Nguyen, Idit Peretz, Marcus Butler, Adnan Khattak, Lavinia Spain, E.M. Gaughan, Melissa Wilson, John W. Dubay, Anja Williams, Stephanie Goff, Gary C. Doolittle, Jason Chesney, Friedrich Graf Finckenstein, Jeffrey Chou, Xiao Wu, Giri Sulur, Wen Shi, John Haanen. TILVANCE-301, a phase 3 study of lifileucel tumor-infiltrating lymphocyte (TIL) cell therapy in combination with pembrolizumab vs pembrolizumab alone in treatment-naive unresectable or metastatic melanoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr CT286.

Mathematical model for IL-2-based cancer immunotherapy

A basic mathematical model for IL-2-based cancer immunotherapy is proposed and studied. Our analysis shows that the outcome of therapy is mainly determined by three parameters, the relative death rate of CD4+ T cells, the relative death rate of CD8+ T cells, and the dose of IL-2 treatment. Minimal equilibrium tumor size can be reached with a large dose of IL-2 in the case that CD4+ T cells die out. However, in cases where CD4+ and CD8+ T cells persist, the final tumor size is independent of the IL-2 dose and is given by the relative death rate of CD4+ T cells. Two groups of in silico clinical trials show some short-term behaviors of IL-2 treatment. IL-2 administration can slow the proliferation of CD4+ T cells, while high doses for a short period of time over several days transiently increase the population of CD8+ T cells during treatment before it recedes to its equilibrium. IL-2 administration for a short period of time over many days suppresses the tumor population for a longer time before approaching its steady-state levels. This implies that intermittent administration of IL-2 may be a good strategy for controlling tumor size.

Abstract 3605: Phase II study of pembrolizumab in conjunction with lymphodepletion, TIL ACT, and high- or low-dose IL-2 in patients with metastatic melanoma

Abstract Background: In metastatic melanoma (MM) patients (pts), adoptive cell transfer (ACT) using tumor-infiltrating lymphocytes (TIL) can yield lasting responses. However, TIL ACT with high-dose IL-2 has notable toxicities. Anti-PD1 (pembrolizumab) is FDA approved for MM treatment. Our study aimed to merge TIL infusion with anti-PD1 in two arms, comparing high (HD, arm 1) and low (LD, arm 2) IL-2 doses. Methods: After tumor harvest and TIL expansion, pts were lymphodepleted with cyclophosphamide and fludarabine, followed by infusion with their ex-vivo expanded TIL combined with IL-2 (arm 1: 720,000 IU/kg IV q 8 hrs up to 15 doses; arm 2: 2 million IU SC for 14 days). Pts were given 200 mg of IV anti-PD1 starting 21 days post-TIL infusion, followed by doses every 3 weeks for up to 2 years. Blood was taken before lymphodepletion and before the first two anti-PD1 doses for flow cytometry and cytokine analysis The primary endpoint of the study was determination of overall response rate by RECISTS 1.1 criteria. Enrollment aimed for 18 pts per arm, monitoring the primary ORR endpoint using a Bayesian rule. Enrollment would halt in any arm if the ORR dropped below 40%. Results: The median age was 50 years (n=14: 6 F and 8 M). Of these 14 pts, 8, 2, 2, and 2 had cutaneous, uveal, mucosal, or unknown primary melanoma. Pts had a median of 3 lines of prior therapies (range 1-6), including anti-PD-1 (n=13). In arm 1, one pt had a partial response (PR) for 10 months, 2 had stable disease (SD), 3 had progressive disease (PD), and 1 was not evaluable (NE). In arm 2, one pt had an ongoing PR for over 76 months, 1 had SD, and 5 had PD. Due to ORR <40% in each arm, enrollment was stopped after treatment of 14 pts. There was no significant different in progression-free survival or overall survival between the two arms (p=0.99 and p=0.71, respectively). In general, toxicity levels were comparable in both arms; however, pts in arm 2 had slightly lower grade 3 febrile neutropenia (57% vs. 71%) and shorter hospitalization (median 16 days vs. 18 days) than pts in Arm 1. While no correlation was observed between the phenotype of the TIL product and clinical response, both PR pts received high numbers of TIL (38.6 × 10^9 and 93.7 × 10^9; mean: 32.6 × 10^9 cells), with a high CD8+/CD4+ T cell ratio (284.7 and 2.7; median: 2.7). IL-2 dose did not affect circulating Treg frequency, phenotype, or proliferation by flow cytometry 3 weeks post TIL infusion. IL-2 dose was not associated with CD4+ non-Treg and CD8+ T cell phenotype or proliferation. Addition of anti-PD1 reduced expression of checkpoints but did not boost T cell proliferation. Conclusion: Our treatment strategy did not yield any distinct difference between low and high dose IL-2, suggesting the potential of low-dose IL-2 as an alternative. Anti-PD1 did not have any appreciable effect on immune states potentially due to the cohorts being comprised of a heavily pre-treated heterogenous pt population. Citation Format: Merve Hasanov, Simin Kiany, Marie-Andrée Forget, Roland L. Bassett, Michael A. Davies, Adi Diab, Jeffrey E. Gershenwald, Isabella C. Glitza, Jeffrey E. Lee, Anthony Lucci, Jennifer L. McQuade, Sapna P. Patel, Merrick I. Ross, Hussein A. Tawbi, Jennifer Wargo, Michael K. Wong, Chantale Bernatchez, Patrick Hwu, Cara Haymaker, Rodabe N. Amaria. Phase II study of pembrolizumab in conjunction with lymphodepletion, TIL ACT, and high- or low-dose IL-2 in patients with metastatic melanoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3605.

Abstract 3999: Augmentation of antitumor effects by CAR-T cells secreting anti-PD-1 single-chain antibody fused with IL-21

Abstract Background: Chimeric antigen receptor (CAR) T cell therapy has shown remarkable responses in hematologic malignancies. However, many patients suffer from limited response and tumor relapse due to lack of persisting CAR-T cells and immune escape. Delivery of immunomodulatory cytokines is one of the strategies to improve immune function; however, the systemic effect of cytokines can be detrimental. Methods: To prevent the toxic effects of cytokines, we proposed the concept of targeting cytokines to tumor-specific T cells in vivo. To this end, we developed CAR-T cells secreting a fusion protein (PD-1Ab21) composed of anti-PD-1 single chain antibody and IL-21, which target IL-21 to CAR-T cells to promote their proliferation and differentiation into memory cells while blocking the PD-1 signal of CAR-T cells. The secretion and functions of PD-1Ab21 were identified. The proliferation and differentiation of CAR-T cells secreting PD-1Ab21 (PD-1Ab21-CAR-T) were compared in vitro with that of the conventional CAR-T cells. The therapeutic effects of CAR-T cells secreting PD-1Ab21 were studied in immunodeficient mice engrafted with human Burkitt lymphoma Daudi, multiple myeloma cell U266 and Her2/neu+ breast cancer cell BT474. Results: The results showed that the culture supernatant of PD-1Ab21-CAR-T cells, like anti-PD-1 antibody, blocked the binding of PD-1 to PD-L1 and stimulated STAT3 phosphorylation in human T lymphocytic leukemia cell Hut78 with the same activity as IL-21. In the conventional culture milieu with high dose of IL-2, the proliferation of the PD-1Ab21-CAR-T cells was not different from that of conventional CAR-T cells. While cultured with low dose of IL-2 that could not support the proliferation of conventional CAR-T cells, the PD-1Ab21-CAR-T cells still showed significant proliferation. A major fraction of the PD-1Ab21-CAR-T products was shown to co-express CCR7, CD62L and CD45RA, and express high level of TCF-1, indicating a memory stem-like phenotype. While the most of conventional CAR-T cells showed effector phenotype defined as CD45RA+CCR7-. Furthermore, adoptive transfer of PD-1Ab21-CAR-T cells exhibited significantly improved CAR-T cell expansion and resulted in superior tumor eradication in the xenograft models of human B-cell malignancy, multiple myeloma and Her2/neu+ breast cancer. The enhanced cellular function by secreting PD-1Ab21 was mediated through an autocrine and/or paracrine manners. Conclusion: We developed an enhanced CAR-T cells. Our data provide preclinical evidence to support the translation of this design for an improved CAR-T cell-mediated anti-tumor response. Citation Format: Ying Li, Jitao Zhao, Xiangrui Cheng, Hang Li, Ran Tian, Qianqian He, Shengdian Wang. Augmentation of antitumor effects by CAR-T cells secreting anti-PD-1 single-chain antibody fused with IL-21 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3999.

Assessing the effectiveness of Interleukin-2 therapy in experimental type 1 diabetes

AimMuch focus of immunotherapy for type 1 diabetes (T1D) has been devoted on selectively boosting regulatory T (Treg) cells using low dose IL-2 due to their constitutive expression of IL-2Rα, CD25. However, several clinical trials using a low dose of IL-2 only showed a limited improvement of metabolic control. It can therefore be hypothesized that further decreasing IL-2 dosage may increase the selective responsiveness of Treg cells.MethodsWe induced experimental T1D using multiple low dose streptozotocin (STZ) injections and treated the mice with an ultra-low dose IL-2 (uIL-2, approximately 7-fold lower than low dose). Immune response was studied using multicolor flow cytometry.ResultsWe found that uIL-2 did not protect STZ mice from developing hyperglycemia. It did neither increase Treg cell proportions, nor did it correct the phenotypic shift of Treg cells seen in T1D. It only partially decreased the proportion of IFN-γ+ T cells. Likewise, uIL-2 also did not protect the dysfunction of regulatory B (Breg) cells. Strikingly, when administered in combination with an anti-inflammatory cytokine IL-35, uIL-2 abrogated IL-35’s protective effect. Low dose IL-2, on the other hand, protected half of the STZ mice from developing hyperglycemia. No difference was found in the Treg and Breg response, and it only tended to decrease CD80 expression in macrophages and dendritic cells.ConclusionIn conclusion, further decreasing IL-2 dosage may not be a suitable approach for T1D therapy, and the limited success suggests that an alternative low dose IL-2 therapy strategy or other immunotherapies should be considered.

Efficacy and safety of autologous tumor-infiltrating lymphocytes in recurrent or refractory ovarian cancer, colorectal cancer, and pancreatic ductal adenocarcinoma

BackgroundTumor-infiltrating lymphocyte (TIL) therapy has shown efficacy in metastatic melanoma, non-small cell lung cancer, and other solid tumors. Our preclinical work demonstrated more robust CD8 predominant TIL production when agonistic...

Final results of a phase I trial of HERV-E TCR transduced T cells for the treatment of HLA-A*11 patients with metastatic clear cell renal cell carcinoma (mccRCC).

435 Background: Human endogenous retrovirus type E (HERV-E) is specifically expressed in ccRCC providing a safe target for T cell-based therapies. We investigated T cells transduced with a TCR targeting HERV-E (HERV-E T cells) for the treatment of mccRCC. Methods: This first-in-human study assessed the safety & efficacy of escalating doses of HERV-E T cells and manufacturing/clinical endpoints correlative analysis. HLA-A*11+ mccRCC patients (pts) were treated with a conditioning regimen, infusion of HERV-E T cells & IL-2. (NCT03354390). Results: Nineteen of185 pts tested were found to express HLA-A*11. 17 HLA-A*11 + pts enrolled on the study: 3 pts on each DL1-3 & 6 pts on DL4. 2 pts did not receive HERV-E T cells given disease progression during manufacturing period. Median age was 57 years. 86% received ≥ 3 prior systemic treatment (range 1-8). The manufacturing failure rate after first apheresis was 12% (n=2); both met target dose after second apheresis and repeat in vitro expansion. All HERV-E T cell products met release criteria for infusion including INF-γ production in response to HERV-E/HLA-A11-expressing tumor cells. Median HERV-E vector copy number (VCN) was 1.9. No dose-limiting toxicities (DLT), off-target toxicities or treatment-related deaths occurred. Pt#17 is on DLT monitoring period. 7 pts completed the planned 14 doses of IL-2 and all received at least 8 doses. Reasons for IL-2 discontinuation: hemodynamic (57%), cardiovascular (28%), pulmonary (28%), renal (14%) criteria & pts decision (14%). The best response was partial response in 7% & stable disease at least 8 weeks in 29% pts. HERV-E mRNA expression was detected in 5 primary & 9 metastatic specimens. HERV-E T cells were measurable in circulation post-dosing, with peak concentrations in the peripheral blood mononuclear compartment on day(D)+7. [DL1: 0.3 %, DL2: 1.2%, DL3: 0.5%, DL4: 12.3%]. Median HERV-E T cell VCN showed no correlation with HERV-E T cells peak concentration on D+4 & D+7. Conclusions: Proof of concept that HERV-T cells can induce tumor regression without evidence of causing off-target toxicities has been established by this trial. Infused HERV-E T cells were detectable transiently in vivo and induce effector cytokine production. Our initial results support the further development of HERV-E-directed therapies that focus on methods to improve in vivo persistence of TCR engineered T-cells and to target HERV-E antigens expressed on more commonly expressed HLA alleles.[Table: see text]

Interleukin-7 improves the fitness of regulatory T cells for adoptive transfer.

Adoptive regulatory T-cell (Treg) transfer has emerged as a promising therapeutic strategy for regulating immune responses in organ transplantation, graft versus host disease, and autoimmunity, including Type 1 diabetes. Traditionally, Treg for adoptive therapy have been sorted and expanded in vitro using high doses of IL-2, demonstrating stability and suppressive capabilities. However, limitations in their long-term survival post-infusion into patients have been observed. To address this challenge, we investigated a novel expansion protocol incorporating interleukin-7 (IL-7) alongside the traditional method utilizing IL-2 (referred to as IL-7 method, IL-7M). Our study revealed that naïve Treg express significant levels of CD127 and display robust responsiveness to IL-7, characterized by STAT-5 phosphorylation. Expanding naïve Treg with the IL-7M protocol led to a substantial enrichment of CD45RA+ CD62L+ CD95+ Treg but showing a reduction in the final cell yield and suppressive function. Moreover, Treg expanded with the IL-7M exhibited preserved telomere length and demonstrated enhanced resistance to cytokine withdrawal and fas-mediated apoptosis. When transferred into NSG mice IL-7M-Treg persisted longer and reduced the expansion of T cells, but did not significantly reduce the severity of xenoGvHD. In conclusion, our data demonstrate the feasibility of expanding naïve Treg in the presence of IL-7 to generate a Treg product enriched in poorly differentiated CD45RA+ cells with enhanced survival capabilities.

Insulin-like Growth Factor-1 Synergizes with IL-2 to Induce Homeostatic Proliferation of Regulatory T Cells.

IL-2 has been proposed to restore tolerance via regulatory T cell (Treg) expansion in autoimmunity, yet off-target effects necessitate identification of a combinatorial approach allowing for lower IL-2 dosing. We recently reported reduced levels of immunoregulatory insulin-like growth factor-1 (IGF1) during type 1 diabetes progression. Thus, we hypothesized that IGF1 would synergize with IL-2 to expand Tregs. We observed IGF1 receptor was elevated on murine memory and human naive Treg subsets. IL-2 and IGF1 promoted PI3K/Akt signaling in Tregs, inducing thymically-derived Treg expansion beyond either agent alone in NOD mice. Increased populations of murine Tregs of naive or memory, as well as CD5lo polyclonal or CD5hi likely self-reactive, status were also observed. Expansion was attributed to increased IL-2Rγ subunit expression on murine Tregs exposed to IL-2 and IGF1 as compared with IL-2 or IGF1 alone. Assessing translational capacity, incubation of naive human CD4+ T cells with IL-2 and IGF1 enhanced thymically-derived Treg proliferation invitro, without the need for TCR ligation. We then demonstrated that IGF1 and IL-2 or IL-7, which is also IL-2Rγ-chain dependent, can be used to induce proliferation of genetically engineered naive human Tregs or T conventional cells, respectively. These data support the potential use of IGF1 in combination with common γ-chain cytokines to drive homeostatic T cell expansion, both invitro and invivo, for cellular therapeutics and ex vivo gene editing.

Harnessing the power of tumor-infiltrating lymphocytes: A first-in-human study of GT201 as monotherapy in advanced solid tumors.

2551 Background: Genetically engineered TIL has shown features such as prolonged memory that can improve therapeutic efficacy in tumor mouse models. GT201 is an engineered TIL that expresses a membrane-bound IL-15 (mbIL-15), and preclinical studies have demonstrated enhanced antitumor reactivity. A phase 1 study was initiated to evaluate the preliminary safety of GT201 and to identify Optimal Biological Dose (OBD) suitable for monotherapy expansion. Methods: The dose-escalation study (using TITE-BOIN design) intended to enroll patients (pts) with advanced treatment-refractory solid tumors. Once the OBD is identified, a monotherapy expansion phase will be opened for patients with various solid tumors. Enrolled patients were preconditioned with a nonmyeloablative (NMA) lymphodepletion regimen and treated by autologous infusion of the GMP-grade G201 cell product, followed with supportive low dose IL-2 administration. Results: As of January 17, 2023, the dose-escalation study enrolled 4 pts, all of whom were female with a median age of 52.6 years. The median number of infused TIL cells was 5.3E+09. All pts experienced TRAEs, with the majority being grade 1 or 2. The most common grade 3/4 TRAEs were lymphopenia (100%), leukopenia (25%) and neutropenia (25%), which were related to the NMA regimen. No dose-limiting toxicities (DLTs) or TIL-related grade ≥3 TRAEs were observed. Median time on study was 17 wks (6–30 wks). One pt with highly treatment-refractory cervical cancer experienced a confirmed partial response (PR) after 16 wks that was durable for 30 wks (RECIST 1.1). Two pts (cervical cancer and melanoma) experienced disease stabilization, while another malignant melanoma pt experienced progressive disease. Despite varying doses of TILs (5.0E+09 to 1.40E+10) and IL-2 (up to 3.0E+05 IU/kg), all patients receiving GT201 therapy showed robust T cell expansion post-infusion, with higher doses of IL-2 leading to more rapid TIL expansion. GT201 TILs could be detected in patients after one month post-infusion, indicating promising activation and long-term persistence of the infused TILs. Based on safety, tolerability, PKs, and preliminary efficacy, a TIL dose of 1.0E+10 and an IL-2 dose of 3.0E+05IU/kg was selected as the OBD for the study. Conclusions: The results of the first-in-human study of GT201, a mIL-15 expressed on TILs, demonstrate promising preliminary findings as a monotherapy in pts with advanced cancers. The administration of GT201 was associated with robust T cell expansion, as well as prolonged persistence of infused TILs. Results of mbIL-15 expressed TIL demonstrates that GT201 has good tolerability with encouraging preliminary antitumor activity as a monotherapy. Further investigations, including correlative biomarker analyses and updated safety and clinical outcome data, will be presented at the meeting. Clinical trial information: NCT05430360 .

A phase 1/2 study of AU-007, a monoclonal antibody (mAb) that binds to IL-2 and inhibits CD25 binding, in patients with advanced solid tumors: Interim results.

e14507 Background: AU-007 is a computationally designed mAb that binds to IL-2 on its CD25 binding epitope. AU-007 bound IL-2 (A/IL-2) cannot bind to trimeric (CD25, CD122, CD132) IL-2 receptors (IL-2R) on Tregs, vascular endothelium, or eosinophils, but IL-2’s binding to dimeric IL-2Rs (CD122, CD132) on T effector and NK cells is unhindered. AU-007 thus redirects endogenous or exogenous IL-2 (aldesleukin) towards T effector and NK cell activation, while diminishing immunosuppressive Treg activation and vascular leak. Unique in the IL-2 field, AU-007 can redirect endogenous IL-2 generated from A/IL-2 driven T effector cell expansion in vivo, converting a Treg-mediated autoinhibitory loop into an immune stimulating loop. A/IL-2 is expected to prolong the 85-minute T1/2 of IL-2, allowing endogenous IL-2 (as A/IL-2) or low dose aldesleukin to initiate an anti-tumor response. Methods: This first-in-human Phase 1 study consists of 3 dose escalation arms, each starting with one 1+2 cohort followed by 3+3 cohorts. Arm 1A evaluates escalating doses of monotherapy AU-007 (intravenous, every 2 weeks [Q2W]). Arm 1B evaluates AU-007 (Q2W) plus 1 loading dose of subcutaneous, low dose aldesleukin with the 1st AU-007 dose. The AU-007 dose will be fixed with escalating aldesleukin doses in sequential cohorts. Arm 1C evaluates AU-007 plus escalating doses of concomitant subcutaneous, low dose aldesleukin, both Q2W. The dose-limiting toxicity (DLT) evaluation period is the first 28 days of the 1st cycle. Tumor assessments by computed tomography scan occur with each 8-week cycle. The AU-007 and aldesleukin dose and schedule for Phase 2 expansion will be based on safety, efficacy, pharmacokinetics (PK), and pharmacodynamics (PD). Results: As of 01 February 2023, 8 patients have been enrolled into the first 3 Cohorts of Arm 1A (0.5, 1.5, and 4.5 mg/kg AU-007). AU-007 was well tolerated with no DLTs and all treatment-related adverse events were Grade 1. These occurred in 3 patients, 1 at each dose level. The 4th Cohort of Arm 1A (9 mg/kg AU-007) and the 1st Cohort of Arm 1B (4.5 mg/kg AU-007 + 1 aldesleukin dose of 15,000 IU/kg) are now being evaluated. Three of 4 tumor evaluable patients had a best response of stable disease and 2 continue treatment. Two patients discontinued with objective progression and one with clinical progression. Initial PK data (0.5 and 1.5 mg/kg) demonstrate dose proportional AU-007 serum concentrations with typical characteristics of an IgG1 human mAb. All 7 patients with available PD data demonstrate overall decreasing Tregs (% change and absolute) and eosinophils, which both express the trimeric IL-2 receptor. Conclusions: AU-007 monotherapy at doses up to 4.5 mg/kg Q2W is safe and well tolerated, with initial signs of immune modulation consistent with AU-007’s mechanism of action. These findings warrant continued escalation and combination with aldesleukin. Clinical trial information: NCT05267626 .