scholarly journals 888 An integrative approach to optimize a synthetic EphA2/CD137 agonist: balancing potency, physiochemical properties, and pharmacokinetics to achieve robust anti-tumor activity

2021 ◽  
Vol 9 (Suppl 3) ◽  
pp. A931-A931
Author(s):  
Punit Upadhyaya ◽  
Gemma Mudd ◽  
Kristen Hurov ◽  
Johanna Lahdenranta ◽  
Elizabeth Repash ◽  
...  
Keyword(s):  
T Cell ◽  
Tumor Cell ◽  
Immune Cell ◽  
List Type ◽  
Continuous Exposure ◽  
Target Engagement ◽  
In Vivo Models ◽  
Anti Tumor Activity

BackgroundCD137 (4-1BB) is a resurging target in immunotherapy after the first generation of monoclonal antibodies were limited by hepatotoxicity1 or lack of efficacy.2 A new generation of CD137 agonists are now in clinical development but they exclusively utilize large molecules derived from recombinant technology and are associated with long circulating terminal half-lives.3–6 Unlike checkpoint inhibition where complete saturation of the receptors drives the reversal of immunosuppression, intermittent target engagement that reflects the physiological context of T cell co-stimulation may be more appropriate for a CD137 agonist.7 Bicyclic peptides or Bicycles are a class of small (MW~2kDa), highly constrained peptides characterized by formation of two loops cyclized around a symmetric scaffold. To develop a differentiated tumor antigen dependent CD137 agonist for treating EphA2 expressing solid tumors, we integrated structure activity relationship (SAR) data from biochemical binding studies and in-vitro and in-vivo models to understand the relationship between exposure, target engagement and preclinical efficacy.MethodsOver 150 different EphA2/CD137 tumor-targeted immune cell agonists (Bicycle TICAs) were synthesized by linking Bicycle® binders to EphA2 to those binding CD137.8 The molecules were assessed in vitro using a CD137 reporter assay and by measuring cytokine production from primary human PBMC in tumor cell co-cultures. The pharmacokinetics were evaluated in rodents using Phoenix WinNonlin. The in vivo activity was determined in syngeneic mouse tumor models by measuring tumor growth kinetics and using tumor immune cell and transcriptional profiling by IHC and NanoString.ResultsEvaluation of the Bicycle TICAs in co-culture assays with EphA2-expressing tumor cell lines and Jurkat reporter cells overexpressing CD137 or human PBMCs demonstrated that constructs bearing two CD137 binding Bicycles to one EphA2 binding Bicycle (1:2 format) were more potent than the 1:1 format.8 Several Bicycle TICAs with amino acid substitutions to the EphA2 binding Bicycle maintained sub-nanomolar potency in-vitro and exhibited a plasma terminal half-life (t1/2) in rodents ranging from 0.4 and 4.0 h. Modifications that conferred aqueous solubility of greater than 10 mg/mL were considered suitable for further development. Treatment of MC38 tumors in immunocompetent mice with this series of molecules demonstrated that low MW Bicycle TICAs with sub-nanomolar potency and a t½ of ~1 h in mouse maintained target coverages necessary to produce robust modulation of the tumor immune microenvironment and tumor regression.ConclusionsA differentiated EphA2-dependent CD137 agonist was developed that exploits intermittent rather than continuous exposure for robust anti-tumor activity.ReferencesSegal NH, Logan TF, Hodi FS, et al. Results from an integrated safety analysis of urelumab, an agonist anti-CD137 monoclonal antibody. Clin Cancer Res 2017;23(8):1929–1936.Segal NH, Aiwu RH, Toshihiko D, et al. Phase I study of single-agent utomilumab (PF-05082566), a 4-1BB/CD137 agonist, in patients with advanced cancer. Clin Cancer Res 2018;24(8):1816–1823.Chester C, Sanmamed MF, Wang J, Melero I. Immunotherapy targeting 4-1BB: mechanistic rationale, clinical results, and future strategies. Blood 2018;131(1):49–57.Hinner MJ, Aiba RSB, Jaquin TJ, et al. Tumor-localized costimulatory T-cell engagement by the 4-1BB/HER2 bispecific antibody-anticalin fusion PRS-343. Clin Cancer Res 2019;25(19):5878–5889.Claus C, Ferrara, C, Xu W, et al. Tumor-targeted 4-1BB agonists for combination with T cell bispecific antibodies as off-the-shelf therapy. Sci Transl Med 2019;11(496):eaav5989.Eskiocak U, Guzman W, Wolf B, et al. Differentiated agonistic antibody targeting CD137 eradicates large tumors without hepatotoxicity. JCI Insight 2020;5(5):e133647.Mayes PA, Hance KW, Hoos A. The promise and challenges of immune agonist antibody development in cancer. Nat Rev Drug Discov 2018;17:509–27.Upadhyaya P, Lahdenranta J, Hurov K, et al. Anticancer immunity induced by a synthetic tumor-targeted CD137 agonist. J Immunother Cancer 2021;9:e001762.

96 Decitabine gene modulation sensitizes human non–small cell lung cancer (NSCLC) to NY-ESO-1 TCR immunotherapy (letetresgene autoleucel; GSK3377794) in vivo

2020 ◽  
Vol 8 (Suppl 3) ◽  
pp. A107-A107
Author(s):  
Dmitry Pankov ◽  
Ioanna Eleftheriadou ◽  
Anna Domogala ◽  
Sara Brett ◽  
Lea Patasic ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Tumor Cell ◽  
Human Leukocyte Antigen ◽  
Human Leukocyte ◽  
List Type ◽  
Leukocyte Antigen ◽  
Engineered T Cells

BackgroundNY-ESO-1–specific T cells (letetresgene autoleucel [lete-cel] GSK3377794) are autologous CD4+ and CD8+ T cells transduced to express a high-affinity T-cell receptor (TCR) capable of recognizing NY-ESO-1 and LAGE-1a antigens in complex with human leukocyte antigen (HLA)-A*02. NY-ESO-1 (CTAG1B) and LAGE-1a (CTAG2) are tumor-associated antigens (TAA) that share the SLLMWITQC peptide bound to human leukocyte antigen HLA-A*02 and are expressed in various cancers. Emerging evidence suggests that TCR-engineered T cells targeting NY-ESO-1 hold promise for patients with solid tumors.1 Approximately 75% of synovial sarcomas can over-express NY-ESO-1 vs 12% of NSCLC,2 however, NSCLC expression of NY-ESO-1/LAGE1-a may have therapeutic potential.3 A separate study using engineered T cells targeting NY-ESO-1 has shown a partial response in a patient with advanced lung adenocarcinoma.4 Decitabine (DAC) is a hypomethylating agent and potent inducer of TAA, including NY-ESO-1.5 We have reported in vitro use of DAC to selectively modulate TAA expression in TAA low-expressing tumor cell lines in order to enhance lete-cel therapy.3 The aim of this study was to assess enhancement of combination therapy with lete-cel and DAC in an in vivo NSCLC model.MethodsNOD scid gamma (NSG) mice were injected subcutaneously with the human NSCLC tumor cell line NCI-H1703. Upon engraftment, tumor-bearing mice were treated with a 5-day course of DAC or vehicle control followed by 2 days of rest. Lete-cel was infused on Day 8. RNA was isolated from tumor formalin-fixed paraffin-embedded blocks, and levels of NY-ESO-1 and LAGE-1a transcript were measured by RT-qPCR. Expression pattern of the NY-ESO-1 protein was assessed via immunohistochemistry. Efficacy was defined by changes in tumor volume and systemic IFN-γ secretion.ResultsConsistent with our previous in vitro studies, DAC treatment in vivo resulted in induction of NY-ESO-1 and LAGE-1a in NSCLC tumors. Lete-cel in combination with DAC significantly enhanced antitumor efficacy in vivo compared with lete-cel alone. This was associated with increased interferon-γ secretion. Mice that received DAC treatment only did not show statistically significant tumor reduction compared with untreated mice.Ethics ApprovalAll animal studies were ethically reviewed and carried out in accordance with Animals (Scientific Procedures) Act 1986 and the GSK Policy on the Care, Welfare and Treatment of Animals. Human biological samples were sourced ethically and their research use was in accord with the terms of the informed consents under an Institutional Review Board/Ethics Committee approved protocol.ConclusionsGSK is currently enrolling a Phase Ib/IIa, multi-arm, open-label pilot study (NCT03709706) of lete-cel as a monotherapy or in combination with pembrolizumab in HLA-A*02–positive patients with NSCLC whose tumors express NY-ESO-1/LAGE-1a. This work may support rationale for the use of DAC in combination with lete-cel to improve adoptive T-cell therapy by increasing levels of target antigens and antitumor effect in NSCLC.AcknowledgementsFunding: GSKReferencesD’Angelo SP, Melchiori L, Merchant MS, et al. Cancer Discov 2018;8:944–957.Kerkar SP, Wang Z-F, Lasota J, et al. J Immunother 2016;39:181–187.Eleftheriadou I, Brett S, Domogala A, et al. Ann Oncol 2019:30(Suppl 5):v475–v532.Xia Y, Tian X, Wang J, et al. Oncol Lett 2018;16:6998–7007.Schrump DS, Fischette MR, Nguyen DM, et al. Clin Cancer Res 2006;12:5777–5785.

468 Enhancers and repressors of immunotherapy: translational perspectives on gene-mediated cytotoxic immunotherapy in glioblastoma

2020 ◽  
Vol 8 (Suppl 3) ◽  
pp. A498-A498
Author(s):  
Sean Lawler ◽  
Marilin Koch ◽  
Mikolay Zdioruk ◽  
Estuardo Aguilar-Cordova ◽  
Laura Aguilar ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
T Cell ◽  
Tumor Cell ◽  
Immune Cell ◽  
Cell Killing ◽  
Future Application ◽  
Cytotoxicity Assays

AcknowledgementsThis was supported by NCI P01CA069246 (Chiocca)ConclusionsOur data suggest that dexamethasone may decrease the efficacy of immunotherapy for glioma through impaired T cell function: this emphasizes the need in identifying alternatives to dexamethasone to prevent attenuated responses in immunotherapies. The combination of GMCI with ATRi however points to additional therapeutic benefit through enhanced cytotoxic efficacy, improved immunogenicity in vitro and increased long-term survival in vivo, making it a promising future approach for the treatment of glioblastoma.ResultsCytotoxicity assays showed that dexamethasone has a slight impact on GMCI in vitro. In T-cell-functional assays, we observed a significantly impaired tumor cell killing. Immune cell response assays revealed a reduced immune cell proliferation after co-culture with supernatant from dexamethasone or combination treated glioblastoma cells. In vivo, while treatment with GMCI alone resulted in longer median symptom-free survival (39.5d) versus no treatment (23d), the combination of GMCI and dexamethasone resulted in the significant reduction of this effect (29d vs 39.5d ; p = 0.0184).The combination of ATRi with GMCI proved to be synergistic in cytotoxicity assays. Flow cytometry revealed a significant increase in DSB-associated H2AX foci as well as an improved immune profile by downregulation of GMCI-induced PD-L1 expression. In vivo, the combination with ATRi led to an increase in long-term surviving animals (66.7%) compared to GMCI (50%) and proved to be highly significant compared to the untreated control (p=0.0022).MethodsWe investigated the effects of ATR-inhibition and dexamethasone on GMCI in vitro using cytotoxicity, flow cytometry and T-cell-killing assays in glioblastoma cell lines. The impact of dexamethasone and ATRi in vivo was assessed in an orthotopic syngeneic murine glioblastoma model. Tumor immune infiltrates were analyzed with flow cytometry.BackgroundGene-mediated cytotoxic immunotherapy (GMCI) is a local tumor immunotherapy that uses aglatimagene besadenovec (a non-replicating serotype 5 adenovirus, expressing HSV1 thymidine kinase) with the prodrug ganciclovir to induce DNA double strand breaks (DSB), leading to immunogenic tumor cell death and intratumoral immune cell invasion. Here we investigate potential repressors and enhancers of GMCI’s effectiveness. GMCI is currently in clinical trials in combination with immune checkpoint blockade in glioblastoma. Thus we set out to identify potential areas to improve this approach for future application. Dexamethasone is used in symptomatic treatment of glioma patients, although it is known to cause immune suppression. However, the influence of dexamethasone on the efficacy of GMCI has not been explored. In contrast, DNA damage response inhibitors like the ATR inhibitor (ATRi) AZD6738 might not only amend the cytotoxic but also the immunogenic profile of GMCI, rendering it an attractive combination partner.

scholarly journals Single-cell transcriptomics reveals the effect of PD-L1/TGF-β blockade on the tumor microenvironment

BMC Biology ◽  
2021 ◽  
Vol 19 (1) ◽  
Author(s):  
Yoong Wearn Lim ◽  
Garry L. Coles ◽  
Savreet K. Sandhu ◽  
David S. Johnson ◽  
Adam S. Adler ◽  
...  
Keyword(s):  
T Cell ◽  
Tumor Microenvironment ◽  
Single Cell ◽  
Immune Cell ◽  
Single Cells ◽  
Cell Infiltration ◽  
In Vivo Models ◽  
T Cell Infiltration ◽  
Anti Tumor Activity

Abstract Background The anti-tumor activity of anti-PD-1/PD-L1 therapies correlates with T cell infiltration in tumors. Thus, a major goal in oncology is to find strategies that enhance T cell infiltration and efficacy of anti-PD-1/PD-L1 therapy. TGF-β has been shown to contribute to T cell exclusion, and anti-TGF-β improves anti-PD-L1 efficacy in vivo. However, TGF-β inhibition has frequently been shown to induce toxicity in the clinic, and the clinical efficacy of combination PD-L1 and TGF-β blockade has not yet been proven. To identify strategies to overcome resistance to PD-L1 blockade, the transcriptional programs associated with PD-L1 and/or TGF-β blockade in the tumor microenvironment should be further elucidated. Results We used single-cell RNA sequencing in a mouse model to characterize the transcriptomic effects of PD-L1 and/or TGF-β blockade on nearly 30,000 single cells in the tumor and surrounding microenvironment. Combination treatment led to upregulation of immune response genes, including multiple chemokine genes such as CCL5, in macrophages, and downregulation of extracellular matrix genes in fibroblasts. Analysis of publicly available tumor transcriptome profiles showed that the chemokine CCL5 was strongly associated with immune cell infiltration in various human cancers. Further investigation with in vivo models showed that intratumorally administered CCL5 enhanced cytotoxic lymphocytes and the anti-tumor activity of anti-PD-L1. Conclusions Taken together, our data could be leveraged translationally to complement or find alternatives to anti-PD-L1 plus anti-TGF-β combination therapy, for example through companion biomarkers, and/or to identify novel targets that could be modulated to overcome resistance.

561 DuoBody®-PD-L1×4–1BB (GEN1046) induces superior immune-cell activation, cytokine production and cytotoxicity by combining PD-L1 blockade with conditional 4–1BB co-stimulation

2020 ◽  
Vol 8 (Suppl 3) ◽  
pp. A595-A595
Author(s):  
Alexander Muik ◽  
Isil Altintas ◽  
Rachelle Kosoff ◽  
Friederike Gieseke ◽  
Kristina Schödel ◽  
...  
Keyword(s):  
Cell Proliferation ◽  
T Cells ◽  
T Cell ◽  
Cd8 T Cells ◽  
Cell Activation ◽  
Cytokine Production ◽  
Immune Cell ◽  
Anti Tumor Activity

BackgroundCheckpoint inhibitors targeting the PD-1/PD-L1 axis (CPI) have changed the treatment paradigm and prognosis for patients with advanced solid tumors; however, many patients experience limited benefit due to treatment resistance. 4-1BB co-stimulation can activate cytotoxic T-cell- and NK-cell-mediated anti-tumor immunity and has been shown to synergize with CPI in preclinical models. DuoBody-PD­L1×4-1BB is a first-in-class, Fc-silenced, bispecific next-generation checkpoint immunotherapy that activates T cells through PD-L1 blockade and simultaneous PD-L1-dependent 4-1BB co-stimulation. Here we present preclinical evidence for the mechanism of action of DuoBody-PD-L1×4-1BB, and proof-of-concept using mouse-reactive mbsAb-PD-L1×4-1BB in vivo.MethodsRNA sequencing analyses was performed on primary human CD8+ T cells that were co-cultured with PD-L1+ monocytes in the presence of anti-CD3/anti-CD28 and test compounds. T-cell proliferation and cytokine production were analyzed in primary human T-cell and mixed lymphocyte reaction (MLR) assays in vitro, and using patient-derived tumor-infiltrating lymphocytes (TILs). Cytotoxic activity was assessed in co-cultures of CLDN6+PD-L1+ MDA-MB-231 tumor cells and CLDN6-TCR+CD8+ T cells. Anti-tumor activity of mbsAb-PD-L1×4-1BB was tested in vivo using the CT26 mouse tumor model. Immunophenotyping of the tumor microenvironment (TME), tumor-draining lymph nodes (tdLNs) and peripheral blood was performed by flow cytometry.ResultsDuoBody-PD-L1×4-1BB significantly induced expression of genes associated with immune cell proliferation, migration and cytokine production in activated CD8+ T cells, which were not altered by CPI. DuoBody-PD-L1×4-1BB dose-dependently enhanced expansion of human TILs ex vivo. DuoBody-PD-L1×4-1BB dose-dependently enhanced T-cell proliferation and pro-inflammatory cytokine production in vitro (e.g. IFNγ and TNFα; in polyclonal and antigen-specific T-cell proliferation assays and MLR), which was dependent on crosslinking to PD-L1+ cells and superior to CPI or the combination of Fc-silenced PD-L1- and 4-1BB-specific antibodies. DuoBody-PD-L1x4-1BB induced upregulation of degranulation marker CD107a and granzyme B in CD8+ T cells, resulting in antigen-specific T-cell-mediated cytotoxicity of MDA-MB-231 tumor cells in vitro, superior to CPI. In mice bearing subcutaneous CT26 tumors, a model that was insensitive to PD-L1 blockade, mbsAb-PD-L1×4-1BB elicited tumor rejection in the majority of the mice at active dose levels and significantly improved survival. Dose-dependent anti-tumor activity was associated with expansion of tumor antigen-specific T cells in the blood and enhanced immune-cell activation in tdLNs and TME.ConclusionsCombining PD-L1 blockade with conditional 4-1BB co-stimulation using bispecific antibodies induced T-cell activation, expansion, and cytotoxic activity in vitro and potent anti-tumor activity in vivo superior to CPI. DuoBody-PD-L1×4-1BB is currently being evaluated in patients with advanced solid tumors in a first-in-human trial (NCT03917381).Ethics ApprovalAll mice studies were performed by BioNTech SE at its research facilities in Germany, and the mice were housed in accordance with German federal and state policies on animal research. All experiments were approved by the regulatory authorities for animal welfare in Germany. The use of tumor tissue resections was approved by BioNTech SE‘s Ethics Board, approval number 837.309.12 (8410-F).

scholarly journals A Novel CD19-Anti-CD20 Bridging Protein Prevents and Reverses CD19-Negative Relapse from CAR19 T Cell Treatment In Vivo

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 252-252 ◽  
Author(s):  
Paul Rennert ◽  
Lihe Su ◽  
Fay Dufort ◽  
Alyssa Birt ◽  
Tom Sanford ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
B Cells ◽  
Tumor Cell ◽  
In Vivo Models ◽  
Car T ◽  
Equity Ownership ◽  
Anti Cd20

Introduction CAR T cells that recognize the antigen CD19 (CAR19s) have achieved remarkable success in otherwise untreatable B cell malignancies including refractory and relapsed ALL and NHL. However, clinical data from diverse CAR19 trials, and real-world experience with the approved CAR19 therapeutics (tisagenlecleucel and axicabtagene ciloleucel), highlight a critical issue, that of patient relapse due to the loss of expression of the target antigen (CD19) or the antigenic epitope. Antigen loss relapse rate of up to 50% have been reported across indications (adult ALL, pediatric ALL, adult NHL) irrespective of the specific CAR19 used. Attempts to treat patients who have relapsed from CAR19 treatment include provision of a CAR T cell to a second antigen, for example CD22. Such attempts have met with limited success, further, many patients cannot tolerate a second regimen of apheresis, consolidation, lymphodepletion and CAR T infusion. Importantly, many of the patients relapsing with CD19-negative malignancies still have detectable levels of CAR19 T cells in circulation, since the CAR19s persist in the presence of normal B cells being produced by the bone marrow (these B cells are CD19-positive). Therefore, a technology that reactivates the patient-resident CAR19s to attack the relapsing tumor cell would be a highly attractive alternative to subsequent CAR T therapy. Here we present this technology and illustrate its' ability to prevent relapses and importantly, to reverse relapses in vivo. Experimental Procedures A stabilized form of the CD19 extracellular domain (ECD) was cloned in frame with an anti-CD20 scFv and an anti-albumin VHH, to create a monomeric CD19-ECD-anti-CD20 bridging protein with extended circulating half-life characteristics. The protein was purified from a mammalian cell expression system. Protein stability, binding affinities, and cytotoxic activity were analyzed in vitro. We utilized CD19-positive, CD20-positive and double positive cell lines to assess single and dual antigen activity. We utilized patient derived CD20-positive/CD19-negative cells to demonstrate translational relevance. Finally, we used single and dual flank in vivo models to assess the potency of the bridging protein in the relapse setting and in the prevention setting. Results and Discussion The CD19-anti-CD20 bridging protein was shown to be expressed at high levels, readily purified and highly stable (no aggregation or clipping, thermostable, and stable in media/serum at 37oC for extended periods). The purified bridging protein directed CAR19 cytotoxicity against CD19-negative/CD20-positive cells with superb potency (IC50 = 23pM = 1.6 ng/ml). CAR19 T cells that were previously activated by a CD19-positive tumor cell could subsequently be activated by a CD19-negative tumor cell in the presence of the CD19-anti-CD20 bridging protein. In vitro, a CAR19 T cells found and eliminated CD19-negative cells "hidden" in a population of dual-positive cells in a mixing experiment but only if the bridging protein was present, otherwise, the CD19-negative cells invariably escaped from CAR19 T cells. The activity of the CD19-anti-CD20 bridging protein extended to CD19-negative/CD20-positive patient-derived cells tested in vitro. In vivo, using a dual flank model, CAR19 T cells plus the injected bridging protein controlled both CD19-positive/CD20-positive and CD19-negative/CD20-positive tumors, while CAR19 alone did not impact the latter tumor. In a relapse setting the growth of a mixture of CD19-positive and CD19-negative cells was merely delayed by CAR19 T cells alone but was eradicated when CAR19 cells were given along with the CD19-anti-CD20 bridging protein injected systemically. Importantly, CAR19 cells that had "lost" control over the mixed population could be restimulated to eliminate the CD19-negative population when the CD19-anti-CD20 bridging protein was added after those cells have begun to escape the initial (CAR19-only) treatment in vivo. These results have led to the identification of a development candidate for the treatment of CD19-negative relapse from CAR19 treatment. The GMP production campaign is underway. The first-in-human trial will enroll patients relapsing from CAR19 therapy with CD19-negative malignancy, in whom CAR19 T cells are shown to still be present. Disclosures Rennert: Aleta Biotherapeutics: Employment, Equity Ownership. Su:Aleta Biotherapeutics: Employment. Dufort:Aleta Biotherapeutics: Employment. Birt:Aleta Biotherapeutics: Employment. Sanford:Aleta Biotherapeutics: Employment. Wu:Aleta Biotherapeutics: Employment. Ambrose:Aleta Biotherapeutics: Employment. Lobb:Aleta Biotherapeutics: Consultancy, Equity Ownership.

scholarly journals Preclinical Characterization of an ANTI-CD38/CD3 T CELL-Redirecting Bispecific Antibody

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 4463-4463
Author(s):  
Xiao He ◽  
Yanliang Zhang ◽  
Yun Wei Lai ◽  
Stephanie Baguley ◽  
Yan Li ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Tumor Cell ◽  
T Cell Activation ◽  
Cytokine Release ◽  
Employment Equity ◽  
Equity Ownership ◽  
Anti Tumor Activity

Introduction: Multiple Myeloma (MM) and Non-Hodgkin Lymphoma (NHL) are hematologic malignancies that remain difficult to treat. While autologous CAR-T cell therapies have shown promise in treating these diseases, these therapies are not without issues, including lack of response in many patients, lengthy time to produce CAR-T cells, occasional production failures, as well as high manufacturing costs. As an alternative approach, protein-based T cell engaging and redirecting bispecific antibodies (BsAbs) have been developed. We have generated anti-CD38/CD3 BsAbs to redirect T cells against CD38, a clinically validated antigen in MM and studied their ability to elicit target-dependent tumor cell lysis. The lead molecule is a humanized, stability-engineered CD3-engaging and CD38 antigen affinity-optimized BsAb with reduced effector function to mitigate antigen-independent T cell toxicity. Preclinical data demonstrate potent anti-tumor activity in vitro assays and in vivo studies against CD38-expressing lymphoma and MM cell lines. Methods: Anti-CD38/CD3 BsAbs were generated by CH3 Fc domain interface engineering for heterodimerization of a CD38-targeting Fab arm and anti-CD3-scFv-Fc fusion chain with hinge mutations for reduced FcR affinities. Novel bispecific molecules that bind to CD38 with various affinities/binding kinetics were evaluated in a series of in vitro and in vivo studies, including target-specific redirected T cell cytotoxicity (RTCC) against cancer cell lines. T cell response profiles, and cytokine release. The lead CD38/CD3 BsAb was selected and further evaluated for its ability to inhibit tumor growth and prolong survival in a disseminated luciferase-expressing Raji xenograft mouse model co-implanted with primary human peripheral blood mononuclear cells (hPBMC). Results: Our lead CD38/CD3 BsAb possesses the desired CD38 and CD3, affinities resulting in effective tumor antigen and T cell engagement for RTCC. The CD38/CD3 BsAb induced potent T cell-dependent lysis of CD38-positive cancer cells in vitro, with the CD38 antigen density positively correlating with the cytotoxicity potency. Antigen dependent and dose-dependent T cell activation and cytokine release were studied in vitro, with the level of T cell activation and cytokine release being indicative of the anti-tumor potency but not necessarily anti-CD3 affinity. In an in vivo study, we evaluated the impact of CD38 affinity of the BsAb on anti-tumor activity of the BsAbs. The data showed that a balanced CD38 vs CD3 affinity was shown to be preferred for T cell stimulation and prolonged anti-tumor activity. In preclinical cytotoxicity assays against a cancer cell line panel using hPBMC from healthy donors, our lead CD38/CD3 BsAb was benchmarked against daratumumab, a marketed anti-CD38 antibody for MM, and demonstrated more potent tumor cell killing. These data suggest a more robust anti-tumor activity exerted by the CD38/CD3 BsAb through RTCC than daratumumab through antibody-dependent cellular cytotoxicity (ADCC). In Raji tumor cell-bearing NSG mice implanted with previously unstimulated hPBMCs, our CD38/CD3 BsAb induced tumor growth inhibition and prolonged survival compared to control BsAb or hPBMCs-only treated animals. Conclusions: Our preclinical data demonstrate that our lead CD38/CD3 BsAb recruits T cells against CD38-positive tumor MM and lymphoma cells in a potent target and dose-dependent manner in preclinical studies. These preclinical characterizations support the rationale for clinical investigation of the lead BsAb in selected CD38-positive malignancies. Disclosures He: Sorrento Therapeutics, Inc.: Employment, Equity Ownership, Patents & Royalties. Zhang:Sorrento Therapeutics, Inc.: Employment, Equity Ownership, Patents & Royalties. Lai:Sorrento Therapeutics, Inc.: Employment, Equity Ownership, Patents & Royalties. Baguley:Sorrento Therapeutics, Inc.: Employment, Equity Ownership. Li:Sorrento Therapeutics, Inc.: Employment, Equity Ownership. Cao:Sorrento Therapeutics, Inc.: Employment, Equity Ownership, Patents & Royalties. Yan:Sorrento Therapeutics, Inc.: Employment, Equity Ownership. Takeshita:Sorrento Therapeutics, Inc.: Employment, Equity Ownership. Zeldis:Sorrento Therapeutics Inc: Employment, Equity Ownership. Ji:Sorrento Therapeutics Inc: Employment, Equity Ownership, Patents & Royalties; Celularity, Inc.: Equity Ownership, Membership on an entity's Board of Directors or advisory committees. Kaufmann:Sorrento Therapeutics, Inc.: Employment, Equity Ownership, Patents & Royalties.

scholarly journals 265 CD47xPD-L1 bispecific antibodies for cancer therapy

2021 ◽  
Vol 9 (Suppl 3) ◽  
pp. A287-A287
Author(s):  
Xavier Chauchet ◽  
Elise Pernarrieta ◽  
Nicolas Bosson ◽  
Sébastien Calloud ◽  
Louis Hellequin ◽  
...  
Keyword(s):  
T Cell ◽  
T Cell Activation ◽  
Cell Activation ◽  
Xenograft Model ◽  
Bispecific Antibodies ◽  
List Type ◽  
Binding Assays ◽  
Anti Tumor Activity

BackgroundPD-1/PD-L1 blockade can significantly improve survival across many types of cancer, but only in a minority of patients. To broaden its therapeutic efficacy, several combination partners are now being evaluated together with PD-1/PD-L1 blockade. Agents blocking CD47/SIRPα innate immune checkpoint are one such example, and co-targeting PD-1/PD-L1 and CD47 with monoclonal antibody (mAb) combinations showed increased antitumor responses in preclinical studies. However, CD47 mAbs are hindered by ubiquitous CD47 expression leading to rapid target-mediated clearance and safety concerns. Consequently, dual-targeting CD47xPD-L1 bispecific antibodies (bsAbs) enabling preferential inhibition of CD47 on PD-L1-positive cells are being tested as an alternative approach. We compare here two distinct bsAbs, based on a common PD-L1 antibody arm, with differing FcgR-enabling effector functions and CD47-binding arm affinities.MethodsAn array of fully human bsAbs associating a high affinity PD-L1 arm to CD47 arms with varying affinities were generated using the κλ-body platform.1 CD47xPD-L1 bsAbs of human IgG1 isotype (CD47 low affinities) or IgG4 isotype (CD47 high affinities) were screened in various binding assays (including to red blood cells (RBC)) and in receptor-blocking assays, and then tested for their Fc-mediated killing and T-cell activation activity (SEA-stimulated PBMC assay). Selected molecules were evaluated in vivo.ResultsBoth bsAb approaches demonstrated strong blockade of PD-1/PD-L1 interaction and significantly enhanced T-cell activation in vitro. CD47lowxPD-L1 IgG1 bsAbs did not bind to RBC and showed PD-L1-guided inhibition of CD47. ADCP and ADCC experiments with a panel of tumor cell lines expressing various target levels showed superior killing activity with CD47lowxPD-L1 IgG1 bsAbs as compared to the anti-PD-L1 IgG1 mAb, avelumab. On the other hand, CD47highxPD-L1 IgG4 bsAbs showed residual RBC binding and PD-L1-independent blocking of CD47/SIRPα. These CD47high IgG4 bsAbs were able to enhance the anti-tumor activity of anti-tumor-associated antigen (TAA) mAbs in vitro (phagocytosis), and in vivo (Raji lymphoma xenograft model). In addition, anti-tumor activity of mouse CD47xPD-L1 bsAbs in a syngeneic MC38 colon carcinoma model was demonstrated.ConclusionsWith the objective of finding the optimal CD47xPD-L1 bsAb design, two approaches targeting CD47 and PD-L1 inhibition were tested. Both the CD47lowxPD-L1 IgG1 bsAbs and CD47highxPD-L1 IgG4 bsAbs were able to mediate enhanced antitumor responses, the former as a standalone treatment, the latter in conjunction with an anti-TAA mAb. To further characterize the CD47lowxPD-L1 and CD47highxPD-L1 bsAbs, lead candidates will be tested in PK and tolerability studies in non-human primates.ReferencesFischer N, Elson G, Magistrelli G, Dheilly E, Fouque N, Laurendon A, et al. Exploiting light chains for the scalable generation and platform purification of native human bispecific IgG. Nat Commun 2015 May;6(1):6113.

scholarly journals 700 EphA2/CD137 Bicycle tumor-targeted immune cell agonists (TICAsTM) induce tumor regressions, immunogenic memory, and reprogramming of the tumor immune microenvironment

2020 ◽  
Vol 8 (Suppl 3) ◽  
pp. A742-A742
Author(s):  
Kristen Hurov ◽  
Johanna lahdenranta ◽  
Gemma Mudd ◽  
Punit Upadhyaya ◽  
Elizabeth Repash ◽  
...  
Keyword(s):  
T Cell ◽  
Tumor Cell ◽  
Immune Cell ◽  
Immune Microenvironment ◽  
New Class ◽  
Tumor Immune Microenvironment ◽  
Nf Kappab ◽  
Animal Care ◽  
Anti Tumor Activity

BackgroundDespite compelling preclinical data, agonistic anti-CD137 antibodies have been hampered by failure to delineate hepatotoxicity from efficacy in clinical studies.1 2 A new generation of both systemic and targeted CD137 agonists that are now entering clinical development rely on biologic agents with suboptimal properties for CD137 agonism due to their relatively large sizes and long circulating half-lives.3–5 These properties may limit their tissue penetration and cause sustained agonism resulting in overstimulation and activation-induced cell death of lymphocytes due to continuous exposure.BCY12491 is a tumor-targeted immune cell agonist (TICATM) that exemplifies a new class of fully synthetic immunomodulators with constrained bicyclic peptides (Bicycles®) targeting a tumor antigen and a co-stimulatory molecule. We developed this new class of synthetic molecules with antibody-like affinities and target selectivity to circumvent the beforementioned barriers to optimal targeted CD137 agonistic therapeutics. BCY12491 (EphA2/CD137 TICA) is designed to deliver a highly potent CD137 agonist to EphA2 overexpressing tumor tissue with an intermittent dosing schedule maximizing anti-tumor activity while circumventing the need for continuous systemic exposure.MethodsBCY12491 bioactivity was assessed in vitro using a CD137 reporter assay and by measuring cytokine production from primary human PBMC/tumor cell co-cultures. BCY12491 in vivo activity was determined in huCD137-syngeneic tumor models by measuring tumor growth kinetics and using tumor immune cell and transcriptional profiling by FACS, IHC, and Nanostring.ResultsBCY12491 engages EphA2 and CD137 with high affinity resulting in picomolar potency in co-culture assays consisting of EphA2-expressing tumor cell lines and CD137-expressing Jurkat NF-kappaB-luciferase reporter cells. Moreover, BCY12491 caused EphA2-dependent CD137 agonism in primary human PBMCs co-cultured with tumor cells with varied levels of EphA2 expression. Treatment of MC38 tumors in immunocompetent mice with BCY12491 leads to a profound reprogramming of the tumor immune microenvironment including increased T cell infiltration and stimulation of NF-kappaB signaling, costimulatory signaling, cytotoxicity and cytokine/chemokine signaling functional pathways. BCY12491 treatment leads to MC38 tumor regressions, complete responses, and immunogenic memory without continuous drug exposure in the periphery. This anti-tumor activity is dependent on CD8+ T cells, but not on NK 1.1+ cells.ConclusionsBCY12491 is a potent EphA2-dependent CD137 agonist with optimal target binding, pharmacologic, and pharmacokinetic properties that enable anti-tumor TME remodeling and complete responses in vivo with intermittent dosing. This work unleashes a new and tractable avenue to testing a novel class of therapeutic CD137 agonists in humans for the treatment of cancer.Ethics ApprovalThe care and use of animals were reviewed and approved by the Institutional Animal Care and Use Committee (IACUC) of WuXi AppTec and conducted in accordance with the regulations of the Association for Assessment and Accreditation of Laboratory Animal Care (AAALAC).ReferencesSegal NH, Logan TF, Hodi FS, et al. Results from an integrated safety analysis of urelumab, an agonist anti-CD137 monoclonal antibody. Clin Cancer Res 2017;23(8):1929–1936.Chester C, Sanmamed MF, Wang J, Melero I. Immunotherapy targeting 4-1BB: mechanistic rationale, clinical results, and future strategies. Blood 2018;131(1): 49–57.Hinner MJ, Aiba RSB, Jaquin TJ, et al. Tumor-Localized Costimulatory T-Cell Engagement by the 4-1BB/HER2 Bispecific Antibody-Anticalin Fusion PRS-343. Clin Cancer Res. 2019;25(19):5878–5889.Claus C, Ferrara, C, Xu W, et al. Tumor-targeted 4-1BB agonists for combination with T cell bispecific antibodies as off-the-shelf therapy. Sci Transl Med 2019;11(496): eaav5989.Eskiocak U, Guzman W, Wolf B, et al. Differentiated agonistic antibody targeting CD137 eradicates large tumors without hepatotoxicity. JCI Insight 2020;5(5):e133647.

591 Comparison of two oHSV Vectors for the treatment of glioblastoma

2020 ◽  
Vol 8 (Suppl 3) ◽  
pp. A626-A626
Author(s):  
Joseph Jackson ◽  
Bonnie Hall ◽  
Lisa Bailey ◽  
E Chiocca ◽  
Justus Cohen
Keyword(s):  
Tumor Microenvironment ◽  
Immune Cell ◽  
Polymorphonuclear Cells ◽  
List Type ◽  
Cell Composition ◽  
Animal Survival ◽  
Strain Derivative ◽  
Anti Tumor Activity

BackgroundGlioblastoma multiforme (GBM) is the most common human brain cancer. Despite a well-established standard of care, the 5-year mortality rate of GBM patients is 95%, highlighting the need for innovative therapeutic interventions. A variety of oncolytic viruses, including those derived from herpes simplex virus (oHSV), have been designed for GBM therapy, but early-phase clinical trials have reported few complete responses and no evidence of durable anti-tumor immunity. Potential reasons for the lack of efficacy are limited vector potency (i.e., virulence) and the presence of a highly immunosuppressive tumor microenvironment (TME) comprised of few activated lymphocytes, large numbers of immunosuppressive myeloid cells (macrophages, myeloid derived suppressor cells [MDSCs], microglia), and an agglomerate of immunosuppressive cytokines (IL-10, VEGF, MIF, etc.).1 Herein we explore these obstacles by comparing the anti-tumor activity two different oHSV designs, an HSV-1 KOS strain derivative designated KG4:T124, and an F strain derivative designated rQNestin34.5v.1 (a similar oHSV, rQNestin34.5v.2, is currently in a phase I clinical trial for GBM).2MethodsUsing the murine syngeneic GBM models, GL261N4 and CT2A, we compared the anti-tumor activity of KG4:T124 and rQNestin34.5v1. In vitro, we evaluated the viral entry, replication capacity, and cytotoxicity of both oHSVs. In vivo, we measured the impact of both vectors on tumor progression, TME immune cell composition, and animal survival.ResultsVirus entry into cancer cells of KG4:T124 or rQNestin34.5v1 was relatively similar, but rQNestin34.5v.1 replicated more effectively and generally induced greater viral mediated cytotoxicity. In syngeneic mice, rQNestin34.5v.1 reduced orthotopic GL261N4 tumor burden and enhanced animal survival compared to KG4:T124. However, preliminary data indicate that multiple injections of KG4:T124 but not rQNestin34.5 enhance GL261N4 survival outcome. Neither oHSV impacted survival outcomes in the more pernicious CT2A model. Analysis, of either the GL261N4 or CT2A TME two days post virus administration revealed that both viruses had reduced microglia cell frequency, induced the influx of tumor associated macrophages and polymorphonuclear cells, but did not alter the frequency of monocytic MDSCs, natural killer cells, CD8+ or CD4+ T-cells.ConclusionsrQNestin34.5 had greater oncolytic activity In vivo and in vitro, but did not benefit from multiple oHSV injections. Both viruses induced similar changes in the TME immune cell composition. However, the presence of vital adaptive immune cell types within the TME was not observed at 2 days post oHSV treatment.AcknowledgementsResearch reported in this abstract was supported by the National Institutes of Health grants P01CA163205 (EAC & JCG) and 5T32CA082084-19 (JWJ).ReferencesMuller S, Kohanbash G, Liu SJ, Alvarado B, Carrera D, Bhaduri A, Watchmaker PB, Yagnik G, Di Lullo E, Malatesta M, Amankulor NM, Kriegstein AR, Lim DA, Aghi M, Okada H, Diaz A. Single-cell profiling of human gliomas reveals macrophage ontogeny as a basis for regional differences in macrophage activation in the tumor microenvironment. Genome Biol. 2017;18(1):234. Epub 2017/12/22. doi: 10.1186/s13059-017-1362-4. PubMed PMID: 29262845; PMCID: PMC5738907.Chiocca EA, Nakashima H, Kasai K, Fernandez SA, Oglesbee M. Preclinical Toxicology of rQNestin34.5v.2: An Oncolytic Herpes Virus with Transcriptional Regulation of the ICP34.5 Neurovirulence Gene. Mol Ther Methods Clin Dev 2020;17:871–93. Epub 2020/05/07. doi: 10.1016/j.omtm.2020.03.028. PubMed PMID: 32373649; PMCID: PMC7195500.

102 Cord-blood derived NK cells, and CAR-T cells, an attractive improved immunotherapy treatment to be considered for hematological malignancies

2020 ◽  
Vol 8 (Suppl 3) ◽  
pp. A113-A113
Author(s):  
Mireia Bachiller García ◽  
Lorena Pérez-Amill ◽  
Anthony Battram ◽  
Alvaro Urbano-Ispizua ◽  
Beatriz Martín-Antonio
Keyword(s):  
T Cells ◽  
Tumor Cell ◽  
Cord Blood ◽  
Immune Cells ◽  
In Vivo Models ◽  
Cell Clusters ◽  
Cytotoxicity Assays ◽  
Anti Tumor Activity

BackgroundMultiple myeloma (MM) remains an incurable hematological malignancy where a proportion of patients relapse or become refractory to current treatments. Administration of autologous T cells modified with a chimeric antigen receptor (CAR) against B cell maturation antigen (BCMA) has achieved high percentages of complete responses. Unfortunately, the lack of persistence of CART-BCMA cells in the patient leads to relapses. On the other side, cord-blood derived natural killer cells (CB-NK) is an off-the-shelf cellular immunotherapy option to treat cancer patients with high potential due to their anti-tumor activity. However, clinical results in patients up to date have been sub-optimal. Whereas CB-NK are innate immune cells and their anti-tumor activity is developed in a few hours, CART cells are adaptive immune cells and their activity develops at later time points. Moreover, we previously described that CB-NK secrete inflammatory proteins that promote the early formation of tumor-immune cell clusters bringing cells into close contact and thus, facilitating the anti-tumor activity of T cells. Therefore, we hypothesized that the addition of a small number of CB-NK to CART cells would improve the anti-tumor activity and increase the persistence of CART cells.MethodsT cells transduced with a humanized CAR against BCMA and CB-NK were employed at 1:0.5 (CART:CB-NK) ratio. Cytotoxicity assays, activation markers and immune-tumor cell cluster formation were evaluated by flow cytometry and fluorescence microscopy. In vivo models were performed in NSG mice.ResultsThe addition of CB-NK to CART cells demonstrated higher anti-MM efficacy at low E:T ratios during the first 24h and in long-term cytotoxicity assays, where the addition of CB-NK to CART cells achieved complete removal of tumor cells. Analysis of activation marker CD69 and CD107a degranulation from 4h to 24h of co-culturing proved differences only at 4h, where CD69 and CD107a in CART cells were increased when CB-NK were present. Moreover, CB-NK accelerated an increased formation of CART-tumor cell clusters facilitating the removal of MM cells. Of note, CB-NK addition did not increase total TNFα and IFNγ production. Finally, an in vivo model of advanced MM with consecutive challenge to MM cells evidenced that the addition of CB-NK achieved the highest efficacy of the treatment.ConclusionsOur results suggest that the addition of ‘off-the-shelf’ CB-NK to CART cells leads to a faster and earlier immune response of CART cells with higher long-term maintenance of the anti-tumor response, suggesting this combinatorial therapy as an attractive immunotherapy option for MM patients.

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