anti-LAG3 Antibodies Research Articles

Abstract 7534: Tobemstomig, a novel bispecific checkpoint inhibitory antibody to preferentially block PD-1 and LAG-3 on CD8 TILs over Tregs

Abstract Checkpoint inhibitors targeting PD-1 have shown unprecedented clinical efficacy in several cancer indications and therefore have, revolutionized the standard of care. However, despite this advancement, only ~20-30% of the patients derive durable benefit from such a treatment. One of the suggested reasons for this limited success is the expression/activation of compensatory inhibitory pathways such as LAG-3 on tumor-reactive T cells. Therefore, it is envisioned that simultaneous antagonism of PD-1 and LAG-3 receptors would overcome this adaptive resistance mechanism and allow a more profound reinvigoration of tumor-reactive T cells. LAG-3 is constitutively expressed by regulatory T cells (on Tregs) and it has been reported that its blockade increases Treg suppressive function which could potentially off-sets the benefit of blocking LAG-3 on tumor-reactive CD8 T cells.We therefore developed a 1+1 PD1-LAG3 bispecific antibody, Tobemstomig, with a unique PD-1 binder recognizing a glycoepitope and more than 20 fold higher affinity than the LAG-3 binder which recognizes a novel and differentiated binding epitope (E3). This difference in affinities allows for an avidity driven selectivity gain to PD-1 and LAG-3 double positive T cells over and the following advantages over monospecific and other bispecific aPD-1/-L1 and aLAG-3 antibodies:1.improved targeting to tumor-reactive T cells rather than Tregs due to the selectivity gain and different expression patterns of PD-1 and LAG-3 on these two T cell types, 2.reduced internalization, 3.resistance to drug-shaving by macrophages due to Fc -silencing via P329G LALA,4.increased in vitro T cell effector functions even in the presence of Tregs, 5.in-vivo targeting and expansion of CD8 TILs, including stem-like T cells, and increase in T cell effector functions and6.superior in vivo tumor control/eradication compared to combination of monospecific anti-PD1 and anti-LAG3 antibodies,in several mouse models including Panc02 in syngeneic huPD-1xhuLAG3 dtg mice and BXPC3, WSU, OCI-Ly18 and BC004 in humanized mice Tobemstomig has completed dose escalation and is currently tested in a composite proof of concept Ph2 trial in several solid tumor indications. Citation Format: Laura Codarri-Deak, Patrick Aaron Weber, Valeria Nicolini, Christian Klein, Pablo Umana, Stefan Seeber, Mario Perro, Stephane Leclair. Tobemstomig, a novel bispecific checkpoint inhibitory antibody to preferentially block PD-1 and LAG-3 on CD8 TILs over Tregs [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 7534.

Beyond PD(L)-1 Blockade in Microsatellite-Instable Cancers: Current Landscape of Immune Co-Inhibitory Receptor Targeting.

High microsatellite instability (MSI-H) derives from genomic hypermutability due to deficient mismatch repair function. Colorectal (CRC) and endometrial cancers (EC) are the tumor types that more often present MSI-H. Anti-PD(L)-1 antibodies have been demonstrated to be agnostically effective in patients with MSI-H cancer, but 50-60% of them do not respond to single-agent treatment, highlighting the necessity of expanding their treatment opportunities. Ipilimumab (anti-CTLA4) is the only immune checkpoint inhibitor (ICI) non-targeting PD(L)-1 that has been approved so far by the FDA for MSI-H cancer, namely, CRC in combination with nivolumab. Anti-TIM3 antibody LY3321367 showed interesting clinical activity in combination with anti-PDL-1 antibody in patients with MSI-H cancer not previously treated with anti-PD(L)-1. In contrast, no clinical evidence is available for anti-LAG3, anti-TIGIT, anti-BTLA, anti-ICOS and anti-IDO1 antibodies in MSI-H cancers, but clinical trials are ongoing. Other immunotherapeutic strategies under study for MSI-H cancers include vaccines, systemic immunomodulators, STING agonists, PKM2 activators, T-cell immunotherapy, LAIR-1 immunosuppression reversal, IL5 superagonists, oncolytic viruses and IL12 partial agonists. In conclusion, several combination therapies of ICIs and novel strategies are emerging and may revolutionize the treatment paradigm of MSI-H patients in the future. A huge effort will be necessary to find reliable immune biomarkers to personalize therapeutical decisions.

Abstract A114: Naturally occurring LAG3-blocking recombinant antibodies as a novel class of checkpoint inhibitors

Abstract LAG3 is a promising cancer immunotherapeutic target due to its negative regulatory role on T cells, NK cells, and B cells. Using our proprietary technology [US patent US9810694; 2017], we have isolated, sequenced, and pre-examined the potential immunological capability of fully human, naturally occurring anti-LAG3 antibodies. The best candidate, monoclonal antibody IG2 with molecular weight of 150 kDa and affinity of 4.4 nM, a complete recombinant reproduction of the antibody isolated from the human donor, was selected from 6 candidates for future research. In vitro allogeneic mixed lymphocyte reaction (MLR) assays using human T cells were performed to evaluate lymphocyte responses to stimulation with IG2 (5 μg/ml) or regular RPMI media alone. LAG3 blockade systematically resulted in an enhancement of CD3+ T cell proliferation by 45%. The main impact was on proliferation of CD4+ T cells with 50% increase. The count of CD8+ T cells also increased by 36%. Another assay evaluated IFNγ expression in NK92 cells treated with anti-LAG3 antibody. NK92 cells were starved without IL-2 for 24 hours in complete NK cell media and then cultivated with IG2 (5 μg/ml and 10 μg/ml) or media for 24 hours. Even short-term cultivation led to an increase in IFNg expression by 50%. Increasing the dose of the antibody did not affect the level of IFNγ expression. We did not observe any antibody dependent cellular cytotoxicity (ADCC) in co-cultures of NK92 cells with 786-O (renal), H358 (lung) or Hct116 (colon) cancer cell lines. Several studies demonstrate that NK cells can stimulate the expression of pro-imflammatory/pro-tumorigenic cytokines, IL-6 and IL-8, by tumor cells. Therefore, we evaluated the effects of IG2 on those genes’ expression induced by NK cells. Clear cell RCC cells (786-O) and NSCLC (H358) cell lines were co-cultured with NK92 cells for 2 hours and overnight with IG2 (1 mcg/ml) or media only. Co-cultivation of NK cells, cancer cells, and IG2 did not result in robust up-regulation of IL-6 and IL-8 by tumor cells. In order to evaluate the direct impact of the IG2 antibody on tumor cells, we incubated 786-O renal carcinoma cells with escalating concentrations of anti-LAG3 antibodies for 72 hours followed by Cell Titer Blue analysis when compared to un-treated cells (0 ug/ml). As expected, there was no direct cytotoxic effect of the antibody on tumor cells. Finally, assuming that novel naturally occurring antibodies may lead to fewer adverse events, we evaluated the preliminary toxicity in vivo. Eighteen mice were randomized in a 1:1 ratio to IG2 antibody group (1 mg/kg, intravenously, twice a week, 6 weeks) or to the untreated control group. The use of the antibody at this dose did not lead to the development of toxicity and a change in the weight of the animals compared to the control group. The results of the first studies demonstrate the potential activity and safety of a recombinant anti-LAG3 antibody reproduced from a human donor. Citation Format: Ilya Tsimafeyeu, Petr Makhov, Gennady Bratslavsky. Naturally occurring LAG3-blocking recombinant antibodies as a novel class of checkpoint inhibitors [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr A114.

Melanoma-the therapeutic considerations in the clinical practice.

The incidence of melanoma is increasing and prolonged exposure to ultraviolet (UV) radiation remains the main risk factor. Public health measures have been vital in tackling the increased incidence and prevalence of melanoma. The management of melanoma has been revolutionised with the approval of new immunotherapy treatments (anti PD-1, CTLA-4 and LAG-3 antibodies) and targeted therapies (BRAF and MEK inhibitors). With some of these therapies becoming the standard of care in the management of advanced disease, it is likely we will see their use increase in the adjuvant and neoadjuvant setting. Recently, literature has demonstrated the benefits patients could derive from the combination of immune checkpoint inhibitors (ICIs) due to the promising results on its efficacy when compared to monotherapy. However, greater clarity on its use is needed in more unique presentations such as BRAF-wild type melanoma, where the lack of driver mutations makes disease management more challenging. Surgical resection remains an integral part of the management of earlier stages of the disease with a consequent decrease in reliance on other forms of therapy such as chemotherapy and radiotherapy. Finally, we evaluated the novel emerging experimental approaches to treatment such as adoptive T cell therapy, novel oncolytic treatments and cancer vaccines. We discussed how their use could improve patients' prognosis, enhance treatment efficacy, and potentially achieve cure.

CryoEM structure of a therapeutic antibody (favezelimab) bound to human LAG3 determined using a bivalent Fab as fiducial marker

Lymphocyte activation gene 3 protein (LAG3) is an inhibitory receptor that is upregulated on exhausted Tcells in tumors. LAG3 is a major target for cancer immunotherapy with many anti-LAG3 antibodies in clinical trials. However, there is no structural information on the epitopes recognized by these antibodies. We determined the single-particle cryoEM structure of a therapeutic antibody (favezelimab) bound to LAG3 to 3.5Å resolution, revealing that favezelimab targets the LAG3-binding site for MHC class II, its canonical ligand. The small size of the complex between the conventional (monovalent) Fab of favezelimab and LAG3 (∼100kDa) presented a challenge for cryoEM. Accordingly, we engineered a bivalent version of Fab favezelimab that doubled the size of the Fab-LAG3 complex and conferred a highly identifiable shape to the complex that facilitated particle selection and orientation for image processing. This study establishes bivalent Fabs as new fiducial markers for cryoEM analysis of small proteins.

LAG3 Regulates T Cell Activation and Plaque Infiltration in Atherosclerotic Mice

BackgroundThe immune checkpoint receptor lymphocyte-activation gene 3 (LAG3) is a new target for immune checkpoint blockade (ICB), but the effects of LAG3 on atherosclerosis are not known. ObjectivesThe aim of the study was to evaluate the role of LAG3 on plaque inflammation using murine hypercholesterolemic models of atherosclerosis. MethodsTo study the role of LAG3 in atherosclerosis, we investigated both bone marrow chimeras lacking LAG3 in hematopoietic cells as well as global Lag3-/- knockout mice. Effects of anti-LAG3 monoclonal antibody monotherapy and combination therapy with anti-programmed cell death protein 1 (PD-1) were tested in hypercholesterolemic low-density lipoprotein receptor knockout (Ldlr-/-) mice and evaluated by histology and flow cytometry. ResultsLAG3-deficiency or treatment with blocking anti-LAG3 monoclonal antibodies led to increased levels of both interferon gamma–producing T helper 1 cells and effector/memory T cells, balanced by increased levels of regulatory T cells. Plaque size was affected by neither LAG3 deficiency nor LAG3 blockade, although density of T cells in plaques was 2-fold increased by loss of LAG3. Combination therapy of anti-PD-1 and anti-LAG3 had an additive effect on T cell activation and cytokine production and promoted plaque infiltration of T cells. ConclusionsLoss of LAG3 function promoted T cell activation and accumulation in plaques while not affecting plaque burden. Our report supports further clinical studies investigating cardiovascular risk in patients treated with anti-LAG3 ICB.

Enhanced antitumor immunity through sequential targeting of PI3Kδ and LAG3

BackgroundDespite striking successes, immunotherapies aimed at increasing cancer-specific T cell responses are unsuccessful in most patients with cancer. Inactivating regulatory T cells (Treg) by inhibiting the PI3Kδ signaling enzyme has...

A phase Ia/Ib, dose-escalation/expansion study of BI 907828 in combination with BI 754091 and BI 754111 in patients (pts) with advanced solid tumors.

TPS3660 Background: Preclinical data show that the combination of a murine double minute 2–tumor protein 53 (MDM2–TP53) antagonist with anti-PD-1 and anti-LAG3 antibodies produces an anti-tumor effect in multiple tumor types. This Phase Ia/Ib study aims to determine the safety, recommended dose for expansion (RDE), and preliminary efficacy of BI 907828, a MDM2–TP53 antagonist, with BI 754091, an anti-PD-1 antibody, and BI 754111, an anti-LAG-3 antibody, in a variety of TP53 wild-type cancers. Methods: In Phase Ia (dose escalation), ~30 pts with a confirmed diagnosis of any unresectable, advanced/metastatic solid tumor, irrespective of TP53 mutation status, will be enrolled. Pts will receive one dose of BI 907828 every 21 days (Q3W), at a starting dose of 10 mg orally, plus BI 754091 and BI 754111 (240 mg and 600 mg, respectively, Q3W, intravenously). Dose escalation will be guided by a Bayesian Logistic Regression Model with overdose control. The primary endpoint is the maximum-tolerated dose of BI 907828 based on dose-limiting toxicities (DLTs) during the first treatment cycle. Secondary endpoints include pharmacokinetics and DLTs in the treatment period (to determine the RDE). In Phase Ib (dose expansion), pts with previously treated, unresectable, advanced/metastatic TP53 wild-type tumors with ≥1 measurable target lesion will be enrolled into four expansion cohorts (1: NSCLC; 2: melanoma; 3: well-differentiated/dedifferentiated liposarcoma or undifferentiated pleomorphic sarcoma; 4: hepatocellular carcinoma). The RDE of BI 907828 will be administered with fixed doses of BI754091 and BI 754111 (Q3W). In the NSCLC cohort only, pts will be randomized to one of three arms: RDE of BI 907828 + 240 mg BI 754091 + 600 mg BI 754111 (arm A, 32 pts); 240 mg BI 754091 + 600 mg BI 754111 (arm B, 32 pts); RDE of BI 907828 + 240 mg BI 754091 (arm C, 16 pts). The primary endpoint is objective response (OR, per RECIST 1.1). Secondary endpoints include OR (per iRECIST), disease control (per RECIST 1.1 and iRECIST), progression-free survival (PFS), PFS rate at 12 and 24 weeks (cohort 3), and safety. Phase Ib will include at least 140 evaluable pts (80 pts in cohort 1 and 20 pts each in cohorts 2–4). Clinical trial information: NCT03964233 .

Abstract C047: A phase Ia/Ib, dose-escalation/expansion study of BI 907828, an MDM2-p53 antagonist, in combination with BI 754091, an anti-PD-1 antibody, and BI 754111, an anti-LAG-3 antibody, in patients with advanced solid tumors

Abstract The murine double minute 2 (MDM2) oncogene is the primary negative regulator of the tumor suppressor, tumor protein 53 (TP53). TP53 is either mutated or deleted in human cancers to ablate its pro-apoptotic functions, or its wild-type function is inhibited by high MDM2 levels, contributing to tumor cell proliferation and cancer growth. BI 907828 is a potent MDM2-p53 antagonist that has shown potent anti-tumor activity in preclinical studies. Increasing evidence suggests TP53 also plays a role in the regulation of immune responses. Preclinical data show that the combination of an MDM2-p53 antagonist with anti-PD-1 and anti-LAG3 antibodies produces a synergistic anti-tumor effect in multiple tumor types. This Phase Ia/Ib study aims to determine the safety, recommended phase 2 dose (RP2D), and preliminary anti-tumor activity of BI 907828 in combination with BI 754091, an anti-PD-1 antibody, and BI 754111, an anti-LAG-3 antibody, in a variety of TP53 wild-type cancers. In the Phase Ia (dose escalation) part of the study, patients with a confirmed diagnosis of unresectable, advanced and/or metastatic solid tumors (of any type) irrespective of TP53 mutation status will be enrolled. Patients will receive one dose of BI 907828 every 21 days, at a starting dose of 10 mg orally, in combination with BI 754091 and BI 754111 (fixed doses of 240 mg and 600 mg respectively, intravenously) every 21 days. Dose escalation will be guided by a Bayesian Logistic Regression Model with overdose control. The primary endpoint is to determine the maximum-tolerated dose (MTD) of BI 907828 based on dose-limiting toxicities (DLTs) during the first treatment cycle. Secondary endpoints include pharmacokinetics and safety. Phase Ia will include approximately 30 evaluable patients. In the Phase Ib (dose expansion) part of the study, patients with previously treated, unresectable, advanced or metastatic tumors that are TP53 wild-type and have at least one measurable target lesion will be enrolled into four expansion cohorts (1: non-small cell lung cancer [NSCLC]; 2: melanoma; 3: well-differentiated/dedifferentiated liposarcoma or undifferentiated pleomorphic sarcoma; 4: hepatocellular carcinoma). The recommended dose for expansion (RDE; established in Phase Ia) of BI 907828 will be administered in combination with fixed doses of BI 754091 and BI 754111 every 21 days. In the NSCLC cohort only, patients will be randomized to one of three arms: RDE of BI 907828 in combination with 240 mg fixed dose BI 754091 and 600 mg fixed dose of BI 754111 every 21 days (arm A, 32 patients); 240 mg fixed dose of BI 754091 in combination with 600 mg fixed dose of BI 754111 every 21 days (arm B, 32 patients); RDE of BI 907828 in combination with 240 mg fixed dose of BI 754091 every 21 days (arm C, 16 patients). The primary endpoint is objective response (OR; investigator-assessed, per RECIST 1.1). Secondary endpoints include OR (per iRECIST), disease control (investigator assessed; per RECIST 1.1 and iRECIST), progression-free survival (PFS); PFS rate at 12 and 24 weeks (cohort 3 only) and safety. Phase Ib will include at least 140 evaluable patients (80 patients in cohort 1 and 20 patients each in cohorts 2, 3 and 4). Citation Format: Anthony Tolcher, Navid Hafez, Noboru Yamamoto, Jaehong Park, Rolf Grempler, Anthony Lucarelli, Markus P Vallaster, Bushi Wang, David Hyman. A phase Ia/Ib, dose-escalation/expansion study of BI 907828, an MDM2-p53 antagonist, in combination with BI 754091, an anti-PD-1 antibody, and BI 754111, an anti-LAG-3 antibody, in patients with advanced solid tumors [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; 2019 Oct 26-30; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2019;18(12 Suppl):Abstract nr C047. doi:10.1158/1535-7163.TARG-19-C047

499P - Identification of first-in-class, naturally occurring LAG3 checkpoint inhibitor

BackgroundDespite the impressive impact of CTLA4 and PD-1/L1 cancer immunotherapy, a large proportion of patients with many tumor types fail to respond. Lymphocyte activation gene 3 (LAG3) is the third checkpoint receptor that plays an important role in the pathogenesis of cancer. We have previously described a novel methodology in the identification of therapeutic antibodies (US Patent #9810694). Here we report the discovery of first-in-class, naturally occurring LAG3 checkpoint inhibitor in cancer patients. MethodsAnti-LAG3 antibody presence was evaluated in 35 individuals: 11 healthy donors (controls) and 24 cancer patients in 3 different laboratories blinded to clinical information. Surface plasmon resonance analysis was done using the optical biosensor Biacore T200 where LAG3 protein was covalently immobilized on the optical chip and biosensor signals from human serum samples were analyzed. Western Blotting used recombinant LAG3 loaded on a 10% SDS PAGE followed by blotting with plasma samples followed by biotinylated anti-human Ig and IrDye 800 streptavidin. Plasma anti-LAG3 IP utilized recombinant LAG3 crosslinked to MagResyn Carboxyl beads followed by incubation with plasma, followed by a biotinylated anti-human Ig and IrDye streptavidin. Affinity purification protocol with elution of antigen-specific antibody with pH gradient was developed. REmAb™ Protein Sequencing with WILD™ analysis (Rapid Novor) is performed. ResultsNo anti-LAG3 antibodies were detected in the control group. Among three assays there was a complete correlation for presence of anti-LAG3 antibody in 5 patients. Three of these patients had sufficient plasma quantity and anti-LAG3 concentration (21 – 33 µg/ml) to allow for the antibody purification. Immunoglobulin isotypes were IgG and IgM. The ELISA results of purified antibody samples showed a high quantity of LAG3-specific antibody (0.22mg, 0.52mg, 0.3mg in each sample, respectively). The results of the characterization of novel LAG3 checkpoint inhibitor will be presented at the congress. ConclusionsTo our knowledge, this is the first report of checkpoint antibody presence in humans. Further study with cloning and evaluation of therapeutic properties of novel anti-LAG3 antibody in xenograft models will be performed. Legal entity responsible for the studyThe authors. FundingILGEN Inc. DisclosureI. Tsimafeyeu: Research grant / Funding (self), Shareholder / Stockholder / Stock options, Full / Part-time employment: ILGEN Inc. G. Bratslavsky: Shareholder / Stockholder / Stock options, Officer / Board of Directors: ILGEN Inc.

Abstract 4115: Naturally circulating anti-LAG3 antibodies exist in cancer patients

Abstract To date there was no evidence of existence of the naturally circulating antibodies to checkpoint receptors in humans. LAG3 checkpoint is crucial in modulation of immune response that is altered in humans with inflammatory and malignant conditions. We have previously described novel methodology in identification of diagnostic and therapeutic antibodies (US Patent #9810694). Here we report identification of novel potentially therapeutic fully human anti-LAG3 antibodies in cancer patients. Anti-LAG3 antibody presence was evaluated in 25 individuals: 11 healthy donors (controls) and 14 cancer patients in two different laboratories blinded to clinical information. Surface plasmon resonance analysis was done using the optical biosensor BiacoreT200 where LAG3 protein was covalently immobilized on the optical chip and biosensor signals from human serum samples were analyzed. Western Blotting used recombinant LAG3 loaded on a 10% SDS PAGE followed by blotting with plasma samples followed by biotinylated anti-human IgA/IgG/IgM and IrDye 800 streptavidin. Plasma anti-LAG3 IP utilized recombinant LAG3 crosslinked to MagResyn Carboxyl beads followed by incubation with plasma, followed by a biotinylated anti human IgA/IgG/IgMand IrDye streptavidin. No anti-LAG3 antibodies were detected in control group. Among three assays there was complete correlation for presence of anti-LAG3 antibody in 3 cancer patients. One of these patients had sufficient plasma quantity and anti-LAG3 concentration (21 - 33 µg/ml) that should allow for the antibody purification and de novo sequencing. The novel fully human anti-LAG3 antibody will be identified, purified, and the sequence will be determined.To our knowledge this is the first report of checkpoint antibody presence in humans. Further study with cloning and evaluation of therapeutic properties of novel fully human anti-LAG3 antibody in xenograft models will be performed. Citation Format: Ilya Tsimafeyeu, Pavel Ershov, Leonid Kaluzhskiy, Jean-Philippe Couture, Hugo Gagnon, Vladimir Kuznetsov, Aleksei Kalachev, Alexis Ivanov, Gennady Bratslavsky. Naturally circulating anti-LAG3 antibodies exist in cancer patients [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4115.

Abstract A33: Tumor derived T cell clones for evaluation of check point inhibitor therapeutics

Abstract Improving clinical outcomes using immunotherapeutics in cancer treatments will depend on a variety of factors including the composition and phenotype of effector T cell in tumors, the inhibitory factors from myeloid, tumor and stromal compartments of the tumor and selection of efficacious combination of therapeutic immunomodulatory agents that either neutralize immune checkpoint inhibitors or activate T cell costimulatory pathways. To address the issue of selecting optimal combinations of immune modulating agents we developed an in-vitro culture system for allo-stimulating and expanding tumor infiltrating lymphocytes (TILS) from tumor digests with an MHC Class II alloantigen expressing cell line. We selected allo-specific CD4 and CD8 clones from expanded TILS with an exhausted phenotype confirmed by cell surface expression of PD-1, LAG3 and TIGIT. T cell clones were incubated with the alloantigen expressing cell lines transduced with different combinations of immodulatory ligands and compared the effect of single agent versus combinations of checkpoint inhibitory monoclonal antibodies on T cell proliferation and cytokine production. Taqman and flow cytometry analysis was performed on several clones following allo-stimulation. A decrease in TIL clonality with repeated allo-stimulation was confirmed by TcR beta chain sequencing. In-vitro IFN gamma and Granzyme B production was enhanced with anti-PD-1 antibodies in combination with either anti-LAG3 or anti-TIGIT antibodies. This in-vitro system has the potential of screening combinations of immodulatory antibodies in a variety of TILS from different tumor types and can improve our understanding of new checkpoint inhibitor combinations. Citation Format: Robert L. Shields, Sabine Le Saux, Sophie Li, Manjiri Sathe, Terri McClanahan, Rene De Waal Malefyt. Tumor derived T cell clones for evaluation of check point inhibitor therapeutics [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2017 Oct 1-4; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2018;6(9 Suppl):Abstract nr A33.

Abstract 1665: MuScreenTM : A well-characterized syngeneic model platform for rapid in vivo screening

Abstract Background: Syngeneic tumor models, many shelved for decades, have been revived as effective models for immunotherapy, accompanying the clinical success of the immuno-checkpoint inhibitors (e.g. anti-CTLA4, anti-PD1, anti-PD-L1 antibodies). In vitro cell-based screens that are frequently used in oncology to quickly identify responsive cells and assess PD effects, often fail in immuno-oncology, due to immunotherapeutics targeting the complex host immune system. Alternatively, an in vivo screen with a panel of models addresses many of these questions, e.g. PD and efficacy, but may be cost prohibitive. Material and methods: Leveraging in-house detailed profiling data on our syngeneic models, including efficacy benchmarking with anti PD-1, PD-L1, CTLA-4, OX-40, GITR , LAG3 and TIM3 antibodies, RNAseq data on tumor samples, and FACS analysis on both baseline and post-treatment tumor samples, we created a new in vivo screening tool for immune-oncology: MuScreen. MuScreen includes up to 20 well-characterized syngeneic models in a 3 month screening run. Both PD and efficacy may be determined in the run, allowing researchers to make decisions based on results observed from a large dataset. To address the cost issue, test agents from multiple clients are pooled together for each run (sharing vehicle and other common groups) providing a significant reduction in the number of animals used and the associated costs. Results: CrownBio has established the largest collection of syngeneic models with well-characterized immunotherapy data. With the three MuScreen runs, we have generated new data on common IO agents (e.g. aPD-1 antibody) and combinations treatments, with FACS analysis, IHC, and efficacy data. Based on these data, a bioinformatics analyses were further explored to identify the makers/pathways that could be correlated with the efficacy or PD effect of the tested IO treatment. Conclusions: MuScreen, the first in vivo screening tool for cancer immunotherapeutics, provides detailed response data on a panel of syngeneic models, and may help on biomarker discovery in a cost and time efficient manner. Citation Format: Lan Zhang, Binchen Mao, Qian Shi. MuScreenTM : A well-characterized syngeneic model platform for rapid in vivo screening [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1665. doi:10.1158/1538-7445.AM2017-1665

Abstract PR03: Combinatorial blockade of PD-1, CTLA-4, and LAG-3 pathways inhibits murine ovarian tumor growth.

Abstract We have previously shown that tumor-associated or infiltrating T cells (TALs or TILs) often co-express immune inhibitory receptors lymphocyte activation gene-3 (LAG-3) and programmed cell death 1 (PD-1) in human and murine ovarian tumor microenvironment (TME). Dual antibody blockade or genetic knockout of LAG3 and PD1 significantly enhanced T effector function and delayed ovarian tumor growth. The relative contribution of other immune inhibitory receptors to immune suppression in the ovarian TME, and whether they co-regulate each other are currently unknown. To address this, we analyzed the expression of multiple immune checkpoint proteins and their cognate ligands in ovarian tumor-bearing wild-type (WT), LAG3KO and PD1KO mice. We found that PD-1, cytotoxic T lymphocyte antigen 4 (CTLA-4), LAG-3, T-cell immunoglobulin domain and mucin domain 3 (TIM-3) and CD160 were elevated in TILs from the WT mice. Interestingly, compared with TILs of WT mice, the levels of LAG-3, TIM-3, and CTLA-4 were further significantly elevated in TILs from the tumor-bearing PD1KO mice; while the levels of PD-1, CTLA-4, and CD160 were further increased in the tumor-bearing LAG3KO mice. These results suggest that PD-1 and LAG-3 regulate the expression of multiple inhibitory receptors in the ovarian TME. Furthermore, combinatorial blockade with anti-LAG3 and anti-CTLA4 antibodies in the tumor-bearing PD1KO mice resulted in 30% tumor-rejection as compared to 10% by anti-CTLA4 alone or only delayed tumor growth by anti-LAG3. These data provide a strong basis for rational clinical testing of combinatorial blockade of the PD-1, LAG-3 and CTLA-4 immune inhibitory pathways for ovarian cancer immunotherapy. This abstract is also presented as Poster A51. Citation Format: Ruea-Yea Huang, AJ McGray, Kunle Odunsi. Combinatorial blockade of PD-1, CTLA-4, and LAG-3 pathways inhibits murine ovarian tumor growth. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Ovarian Cancer Research: Exploiting Vulnerabilities; Oct 17-20, 2015; Orlando, FL. Philadelphia (PA): AACR; Clin Cancer Res 2016;22(2 Suppl):Abstract nr PR03.