scholarly journals 714 PD1 x TGFβR2 bispecifics selectively block TGFβR2 on PD1-positive T cells, promote T cell activation, and elicit an anti-tumor response in solid tumors

2020 ◽  
Vol 8 (Suppl 3) ◽  
pp. A756-A756
Author(s):  
Gregory Moore ◽  
Suzanne Schubbert ◽  
Christine Bonzon ◽  
Kendra Avery ◽  
Rumana Rashid ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Solid Tumors ◽  
T Cell Activation ◽  
Tumor Response ◽  
Nsg Mice ◽  
Pd1 Blockade ◽  
Anti Tumor Activity

BackgroundTGFβ production by solid tumors and their microenvironment is a major mechanism used by tumors to avoid immunosurveillance. Blockade of TGFβ has been shown to promote an anti-tumor response; however, systemic blockade of TGFβ has also been associated with toxicity. We hypothesized that a PD1 x TGFβR2 bispecific antibody could selectively block the suppressive activity of TGFβ on tumor T cells and enhance their anti-tumor activity while avoiding the toxicity associated with systemic blockade.MethodsWe engineered bispecific antibodies that simultaneously engage PD1 and TGFβR2 using Xencor’s XmAb platform. The anti-TGFβR2 arm was tuned for optimal activity by introducing affinity-modulating amino acid substitutions. The activity of PD1 x TGFβR2 bispecifics was evaluated in vitro using a signaling assay to measure phosphorylated SMAD (pSMAD) by flow cytometry with exogenous TGFβ in unactivated and activated PBMC. In vivo activity was evaluated by monitoring the engraftment of human PBMC in NSG mice (huPBMC-NSG). Anti-tumor activity was assessed in huPBMC-NSG mice engrafted with established human cancer cell lines. Antibodies against other T cell targets were also incorporated into TGFβR2 bispecifics, and similarly evaluated in vitro and in vivo.ResultsPD1 x TGFβR2 bispecifics were confirmed to bind PD1 and block binding of TGFβ to TGFβR2. In vitro, we found that T cells from activated, serum-deprived PBMC exhibited robust induction of pSMAD in response to TGFβ, and PD1 x TGFβR2 bispecifics selectively inhibited pSMAD induction in PD1-positive T cells as demonstrated by over a 100-fold potency increase compared to an untargeted anti-TGFβR2 control. Additionally, we saw an enhancement of potency when evaluating blocking activity in activated (PD1-high) vs. unactivated (PD1-low) T cells. Similar selectivity was measured when comparing inhibition of pSMAD induction for activated T cells versus other PD1-negative, TGFβ-responsive immune cells. Intriguingly, TGFβR2 bispecifics incorporating antibodies against other T cell targets allowed for the targeting of a broader population of T cells while still conferring potent selectivity against target-negative cells. In vivo, treatment of huPBMC-NSG mice with TGFβR2 bispecifics promoted superior T cell engraftment and combined additively with PD1 blockade. Furthermore, TGFβR2 bispecific treatment of huPBMC-NSG mice containing established MDA-MB-231 triple-negative breast cancer tumors promoted an anti-tumor response that was also augmented with PD1 blockade.ConclusionsMultiple PD1 x TGFβR2 bispecifics were engineered to selectively block TGFβR2 on PD1-positive T cells and evaluated in vitro and in vivo. Compelling activity, including additivity with PD1 blockade, suggests that clinical development is warranted for the treatment of human malignancies.

scholarly journals 872 PD1 x TGFbR2 and CD5 x TGFbR2 bispecifics selectively block TGFbR2 on target-positive T cells, promote T cell activation, and elicit an anti-tumor response in solid tumors

2021 ◽  
Vol 9 (Suppl 3) ◽  
pp. A913-A913
Author(s):  
Gregory Moore ◽  
Suzanne Schubbert ◽  
Christine Bonzon ◽  
Kendra Avery ◽  
Rumana Rashid ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Solid Tumors ◽  
T Cell Activation ◽  
Tumor Response ◽  
Bispecific Antibodies ◽  
Nsg Mice ◽  
Anti Tumor Activity

BackgroundTGFbeta production by solid tumors and their microenvironment is a major mechanism used by tumors to avoid immunosurveillance. Blockade of TGFbeta has been shown to promote an anti-tumor response; however, systemic blockade of TGFbeta has also been associated with toxicity. We hypothesized that a T cell-targeted TGFbR2 bispecific antibody could selectively block the suppressive activity of TGFbeta on T cells and enhance their anti-tumor activity while avoiding toxicity associated with systemic blockade.MethodsWe engineered bispecific antibodies that simultaneously engage PD1 (activated) or CD5 (pan T) and block TGFbR2 using Xencor’s XmAb® platform. The anti-TGFbR2 arm was tuned for optimal activity by introducing affinity-modulating amino acid substitutions. The activity of TGFbR2 bispecifics was evaluated in vitro using a signaling assay to measure phosphorylated SMAD (pSMAD) by flow cytometry with exogenous TGFbeta in unactivated and activated PBMC. In vivo activity was evaluated by monitoring the engraftment of human PBMC in NSG mice (huPBMC-NSG). Anti-tumor activity was assessed in huPBMC-NSG mice engrafted with established human cancer cell lines.ResultsTGFbR2 bispecifics were confirmed to bind PD1 or CD5 and block binding of TGFbeta to TGFbR2. In vitro, we found that T cells from serum-deprived PBMC exhibited robust induction of pSMAD in response to TGFbeta, and TGFbR2 bispecifics selectively inhibited pSMAD induction in target-positive T cells as demonstrated by over a 100-fold potency increase compared to an untargeted anti-TGFbR2 control. Additionally, we saw an enhancement of potency when evaluating activity in target-high T cells versus target-low or -negative immune cells. Intriguingly, CD5-targeted TGFbR2 bispecifics allowed for the targeting of a broader population of T cells compared to PD1-targeting while still conferring potent selectivity against target-negative cells. In vivo, treatment of huPBMC-NSG mice with TGFbR2 bispecifics promoted superior T cell engraftment. Furthermore, TGFbR2 bispecific treatment of huPBMC-NSG mice containing established MDA-MB-231 triple-negative breast cancer tumors promoted an anti-tumor response that was augmented with PD1 blockade.ConclusionsPD1 x TGFbR2 and CD5 x TGFbR2 bispecific antibodies were engineered to selectively block TGFbR2 on target-positive T cells and evaluated in vitro and in vivo. These observations are compelling and suggest that development of these bispecifics is warranted for the treatment of human malignancies.

scholarly journals 697 Tumor-targeted CD28 costimulatory bispecific antibodies enhance T cell activation in solid tumors

2020 ◽  
Vol 8 (Suppl 3) ◽  
pp. A739-A739
Author(s):  
Michael Hedvat ◽  
Veronica Zeng ◽  
Juan Diaz ◽  
Christine Bonzon ◽  
Kendra Avery ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Solid Tumors ◽  
Cancer Cell ◽  
T Cell Activation ◽  
Cell Activation ◽  
Bispecific Antibodies ◽  
Anti Tumor Activity

BackgroundT cells in the tumor micro-environment require TCR/MHC engagement and co-stimulatory receptor engagement to achieve complete activation. Solid tumors often lack expression of CD28 ligands, so we hypothesized that activation of CD28 signaling could be beneficial in solid tumors. We designed tumor-associated-antigen (TAA) x CD28 bispecific antibodies that conditionally costimulate CD28 only in the presence of TAA and TCR engagement. Clinical application of this class of antibodies has potential to enhance activity of either anti-PD(L)1 antibodies or TAA x CD3 T cell engagers.MethodsWe designed a stability and affinity optimized anti-CD28 antibody that can be paired with TAA of choice to engage CD28 monovalently using Xencor’s XmAb 2+1 and 1+1 platforms. In vitro T cell activation with these bispecifics was measured by T cell proliferation, cytokine production, and cytotoxicity, in co-cultures of human cancer cell lines mixed with primary human CD3-stimulated T cells. In vitro activity was validated in a CMV recall assay measuring CMV+ T cell proliferation of CMV+ PBMC co-cultured with cancer cell lines ectopically treated with pp65-derived NLV-peptide. In vivo anti-tumor and T cell proliferative activity of B7H3 x CD28 bispecific antibodies were determined in tumor-bearing huPBMC-NSG mice treated simultaneously with TAA x CD3 bispecific antibody. In vivo activity of PDL1 x CD28 antibodies was determined with hCD28 KI mice inoculated with MC38 tumors expressing hPDL1-antigen. Finally, safety and tolerability of B7H3 x CD28 and PDL1 x CD28 was determined in cynomolgus monkeys.ResultsB7H3 x CD28 and PDL1 x CD28 antibodies enhanced T cell degranulation, cytokine secretion, and cancer cell cytotoxicity in concert with CD3 stimulation only in the presence of target antigen. B7H3 x CD28, alone or in combination with anti-PD1 antibody, enhanced proliferation of CMV+ T cells recognizing cancer cells loaded with pp65-derived NLV peptide. PDL1 x CD28 also enhanced CMV+ cell expansion but did not synergize with anti-PD1 antibody treatment. B7H3 x CD28 significantly enhanced in vivo anti-tumor activity of TAA x CD3 antibodies while also promoting greater T cell expansion. In hCD28 mice inoculated with MC38 tumors expressing hPDL1, PDL1 x CD28 antibody inhibited tumor growth greater than an anti-PDL1 antibody alone. B7H3 x CD28 and PDL1 x CD28 were well tolerated in cynomolgus monkeys.ConclusionsB7H3 x CD28 and PDL1 x CD28 bispecific antibodies show promising anti-tumor activity and warrant further development.

scholarly journals 137 Development of anti-Mucin1 CAR-T against solid tumors

2021 ◽  
Vol 9 (Suppl 3) ◽  
pp. A146-A146
Author(s):  
Jihyun Lee ◽  
Areum Park ◽  
Jungwon Choi ◽  
Dae Gwan Yi ◽  
Hee Jung Yang ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Solid Tumors ◽  
Cell Therapies ◽  
Car T Cells ◽  
Car T Cell ◽  
Car T ◽  
Anti Tumor Activity

BackgroundChimeric antigen receptor (CAR) -T cell therapies have proven to be effective against various liquid tumors. However, the development of CAR-T against solid tumors has been challenging due to insufficient efficacy and potential on-target off-tumor toxicities caused by low expression of tumor antigens on normal tissues. Testing various affinities of CARs has demonstrated that lower affinity CARs maintain its anti-tumor effect while minimizing safety concerns (1). In order to develop a CAR-T against solid tumors expressing Mucin1, we have screened for Mucin1 binding antibodies and tested their anti-tumor effect in vitro and in vivo. The potential of on-target off-tumor toxicity was also measured in vitro.MethodsAnti-Mucin1 human single chain variable fragments (scFv) were obtained via screening against a scFv display library. Anti-Mucin1 scFvs were incorporated into CARs and in vitro, in vivo functions against various tumor cells expressing Mucin1 were tested. For in vivo studies, tumor bearing NOG mice (HCC1954 cells) received anti-Mucin1 CAR-T cells. Therapeutic efficacy was evaluated by measuring tumor volumes. Potential on-target off-tumor toxicity against Mucin1 on normal cells was tested by investigating the killing effect of anti-Mucin1 CAR-T against cancer cell line (HCC70) and non-tumorigenic breast epithelial cell line (MCF-10A) in co-culture systemsResultsIn vitro activity of anti-Mucin1 CAR-T cells that displayed a range of affinities for Mucin1 (27nM to 320nM) showed similar cytokine secretion levels and cytotoxicity against Mucin-1 expressing tumor cell lines (HCC70 and T47D). Robust anti-tumor activity was also demonstrated in vivo against large tumors (400~500 mm3) with relatively small numbers of CAR-T cells (0.5 x 106 CAR-T cells per mouse). In vivo expansion of CAR-T cells were observed in all scFv-CAR-T cases and accompanied by close to complete regression of tumors within 25 days post CAR-T cell injection. Of the 4 scFv CAR-Ts, 2H08 (with a Kd of 94nM) was tested for activity against normal breast epithelial cells. When 2H08-CAR-T was cocultured with a mixture of HCC70 and MCF-10A cells, they preferentially killed only the Mucin1 overexpressing HCC70 cells leaving MCF-10 cells intact.ConclusionsOur study demonstrates anti-tumor activity of a novel scFv-derived CAR-T recognizing Mucin1 and its effectiveness in large pre-established tumors in vivo. We also demonstrate that 2H08-CAR-T can distinguish between target overexpressing cancer cells and normal epithelial cells, which suggests that by toning down the affinity of CAR against antigen one can improve the safety profile of solid tumor antigen targeting CAR-T cell therapies.ReferenceCastellarin M, Sands C, Da T, Scholler J, Graham K, Buza E, Fraietta J, Zhao Y, June C. A rational mouse model to detect on-target, off-tumor CAR T cell toxicity. JCI Insight 2020; 5:e136012Ethics ApprovalAll experiments were done under protocols approved by the Institutional Animal Care and Use Committee (IACUC) (Study#LGME21-011).ConsentWritten informed consent was obtained from the patient for publication of this abstract and any accompanying images. A copy of the written consent is available for review by the Editor of this journal.

The immunological impact of the RAF inhibitor BMS908662: Preclinical and early clinical experience in combination with CTLA-4 blockade.

2012 ◽  
Vol 30 (15_suppl) ◽  
pp. 2521-2521 ◽  
Author(s):  
Margaret Callahan ◽  
Gregg Masters ◽  
Jessica Katz ◽  
Valerie Russell ◽  
Ruth Ann Roman ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Combination Therapy ◽  
T Cell Activation ◽  
Cell Activation ◽  
Mapk Pathway ◽  
Preclinical Studies ◽  
Anti Tumor Activity

2521 Background: Two new approaches to treat advanced melanoma have transformed the standard of care: the CTLA-4 blocking antibody, ipilimumab, and the targeted inhibitor of mutated BRAF, vemurafenib. These agents are mechanistically unique and combination therapy is a promising next step. We evaluated the combination of BMS908662 (662), a pan RAF inhibitor, with CTLA-4 blockade in preclinical studies and report first-in-human clinical experience with this combination. Methods: 1) We tested the impact of 662 on T cells in vitro, using cultured human T cells, and in vivo, using OT-1 transgenic mice. T cell activation and MAPK pathway signaling were assessed in parallel. 2) Preclinical studies measuring the anti-tumor activity of combination therapy were performed in CT-26 and SA1N tumor models. 3) Three pts with BRAF mutant stage IV melanoma were treated at MSKCC on CA206005, an IRB-approved protocol, receiving ipilimumab (3 mg/kg) and 662 (25 mg bid) (NCT01245556). Two pts consented to an IRB-approved protocol permitting immune monitoring. Results: 1) In vitro studies demonstrate that 662 can potentiate T cell activation after stimulation. This corresponds with increased MAPK pathway signaling, consistent with paradoxical activation of the MAPK pathway in wild type cells, a class effect of RAF inhibitors. In vivo, enhanced expansion of OT-1 cells after ovalbumin challenge was seen in mice treated with 662. T cell expansion was greatest in mice treated with a combination of CTLA-4 blockade and 662 (p<0.05). 2) Both preclinical models demonstrate superior anti-tumor activity with combination therapy compared to monotherapy (p<0.05). 3) All pts treated on protocol CA206005 tolerated combination therapy. New keratoacanthomas and SCCs, likely related to 662, were identified. One pt has an ongoing response at 10 mos (-85%), one had stable disease for 24 wks (-19%) and a third had disease progression. Enhanced MAPK signaling in PBMCs after treatment with 662 was detected ex vivo. Conclusions: RAF inhibitors may potentiate T cell activation in vitro and in vivo, offering one explanation for the enhanced anti-tumor activity seen in combination with CTLA-4 blockade in pre-clinical models.

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 SIRPγ-CD47 Interaction Positively Regulates the Activation of Human T Cells in Situation of Chronic Stimulation

2021 ◽  
Vol 12 ◽  
Author(s):  
Safa Dehmani ◽  
Véronique Nerrière-Daguin ◽  
Mélanie Néel ◽  
Nathan Elain-Duret ◽  
Jean-Marie Heslan ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
T Cell Activation ◽  
Cell Activation ◽  
Cell Interaction ◽  
Activated T Cells ◽  
Chronic Stimulation ◽  
Nsg Mice

A numerous number of positive and negative signals via various molecules modulate T-cell activation. Within the various transmembrane proteins, SIRPγ is of interest since it is not expressed in rodents. SIRPγ interaction with CD47 is reevaluated in this study. Indeed, we show that the anti-SIRPγ mAb clone LSB2.20 previously used by others has not been appropriately characterized. We reveal that the anti-SIRPα clone KWAR23 is a Pan anti-SIRP mAb which efficiently blocks SIRPα and SIRPγ interactions with CD47. We show that SIRPγ expression on T cells varies with their differentiation and while being expressed on Tregs, is not implicated in their suppressive functions. SIRPγ spatial reorganization at the immune synapse is independent of its interaction with CD47. In vitro SIRPα-γ/CD47 blockade with KWAR23 impairs IFN-γ secretion by chronically activated T cells. In vivo in a xeno-GvHD model in NSG mice, the SIRPγ/CD47 blockade with the KWAR23 significantly delays the onset of the xeno-GvHD and deeply impairs human chimerism. In conclusion, we have shown that T-cell interaction with CD47 is of importance notably in chronic stimulation.

scholarly journals 704 Preclinical mechanism of action and pharmacodynamic biomarker studies of DuoBody®-CD3x5T4 in vitro and in vivo in solid cancer models

2020 ◽  
Vol 8 (Suppl 3) ◽  
pp. A746-A746
Author(s):  
Kristel Kemper ◽  
Ellis Gielen ◽  
Mischa Houtkamp ◽  
Peter Boross ◽  
Saskia Burm ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Tumor Cells ◽  
T Cell Activation ◽  
Cell Activation ◽  
T Cell Subsets ◽  
Animal Protection ◽  
Anti Tumor Activity

BackgroundThe tumor-associated antigen 5T4 is expressed across a wide range of solid cancers. DuoBody-CD3x5T4 is a bispecific antibody (bsAb) that crosslinks CD3 on T cells with 5T4 on tumor cells, thereby inducing T-cell activation and T-cell mediated cytotoxicity in 5T4-expressing tumor cells. Here, we tested the capacity of DuoBody-CD3x5T4 to engage different T-cell subsets in vitro and investigated the mechanism of action (MoA) in vivo by combining preclinical efficacy studies with exploratory pharmacodynamic (PD) biomarker analysesMethodsImmunohistochemistry was performed on patient-derived tumor tissue-microarrays using a commercial 5T4 monoclonal antibody (EPR5529). The capacity of DuoBody-CD3x5T4 to engage naïve and memory T-cell subsets was assessed in co-cultures of T cells and 5T4-positive tumor cells, using T-cell activation and T-cell mediated cytotoxicity as readouts. Anti-tumor activity in vivo as well as peripheral and intratumoral PD biomarkers were investigated in humanized mice bearing 5T4-expressing cell line-derived xenograft (CDX) or patient-derived xenograft (PDX) tumor models.ResultsHigh prevalence of 5T4 expression (in >86% of biopsies) was observed in NSCLC, SCCHN, TNBC, bladder, esophageal, prostate and uterine cancer. In co-cultures of 5T4+ tumor cells and T cells in vitro, DuoBody-CD3x5T4 induced dose-dependent cytotoxicity, associated with T-cell activation, proliferation, and cytokine, perforin and granzyme production. Crosslinking of T cells with 5T4-expressing tumor cells was essential as no cytotoxicity was observed in CRISPR-Cas9-generated 5T4-knockout tumor cells or with control bsAbs targeting only CD3 or 5T4. Importantly, naïve and memory CD4+ or CD8+ T-cell subsets had equal capacity to mediate DuoBody-CD3x5T4-induced cytotoxicity, although naïve T-cell subsets showed slower kinetics. DuoBody-CD3x5T4 (0.5–20 mg/kg) demonstrated anti-tumor activity in 5T4+ breast and prostate cancer CDX and lung cancer PDX models in humanized mice. Treatment with DuoBody-CD3x5T4 was associated with intratumoral and peripheral T-cell activation as well as elevated cytokine levels, including IFNγ, IL-6 and IL-8, in peripheral blood.ConclusionsDuoBody-CD3x5T4 induced T-cell mediated cytotoxicity in 5T4-expressing tumor cells, associated with T-cell activation and cytokine production in vitro. DuoBody-CD3x5T4 efficiently engaged naïve and memory T cells within both CD4+ and CD8+ T-cell populations to induce T-cell mediated cytotoxicity in 5T4+ tumor cells. In humanized CDX and PDX mouse models, DuoBody-CD3x5T4 showed anti-tumor activity, in addition to PD biomarkers associated with T-cell activation in the tumor and periphery. Currently, DuoBody-CD3x5T4 is being investigated in a first-in-human clinical trial for the treatment of solid tumors (NCT04424641), in which exploratory biomarker analyses to study the clinical MoA and PD are included.Ethics ApprovalThe CDX animal experiments performed are in compliance with the Dutch animal protection law (WoD) translated from the directives (2010/63/EU) and are approved by the Ethical committee of Utrecht. For the PDX models, all patients had given written informed consent, and the animal experiments were carried out in accordance with the German Animal Protection Law (LaGeSoBerlin, A0452/08). The studies were approved by the local Institutional Review Board of Charite University Medicine, Germany.

627 The DLL3-targeted half-life extended bispecific T cell engager (HLE BiTE®) immune-oncology therapy AMG 757 has potent antitumor activity in neuroendocrine cancer

2020 ◽  
Vol 8 (Suppl 3) ◽  
pp. A663-A663
Author(s):  
Keegan Cooke ◽  
Juan Estrada ◽  
Jinghui Zhan ◽  
Jonathan Werner ◽  
Fei Lee ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Tumor Growth ◽  
Mouse Models ◽  
T Cell Activation ◽  
Phase 1 ◽  
Phase 1 Study ◽  
Human T Cells

BackgroundNeuroendocrine tumors (NET), including small cell lung cancer (SCLC), have poor prognosis and limited therapeutic options. AMG 757 is an HLE BiTE® immune therapy designed to redirect T cell cytotoxicity to NET cells by binding to Delta-like ligand 3 (DLL3) expressed on the tumor cell surface and CD3 on T cells.MethodsWe evaluated activity of AMG 757 in NET cells in vitro and in mouse models of neuroendocrine cancer in vivo. In vitro, co-cultures of NET cells and human T cells were treated with AMG 757 in a concentration range and T cell activation, cytokine production, and tumor cell killing were assessed. In vivo, AMG 757 antitumor efficacy was evaluated in xenograft NET and in orthotopic models designed to mimic primary and metastatic SCLC lesions. NSG mice bearing established NET were administered human T cells and then treated once weekly with AMG 757 or control HLE BiTE molecule; tumor growth inhibition was assessed. Pharmacodynamic effects of AMG 757 in tumors were also evaluated in SCLC models following a single administration of human T cells and AMG 757 or control HLE BiTE molecule.ResultsAMG 757 induced T cell activation, cytokine production, and potent T cell redirected killing of DLL3-expressing SCLC, neuroendocrine prostate cancer, and other DLL3-expressing NET cell lines in vitro. AMG 757-mediated redirected lysis was specific for DLL3-expressing cells. In patient-derived xenograft and orthotopic models of SCLC, single-dose AMG 757 effectively engaged human T cells administered systemically, leading to a significant increase in the number of human CD4+ and CD8+ T cells in primary and metastatic tumor lesions. Weekly administration of AMG 757 induced significant tumor growth inhibition of SCLC (figure 1) and other NET, including complete regression of established tumors and clearance of metastatic lesions. These findings warranted evaluation of AMG 757 (NCT03319940); the phase 1 study includes dose exploration (monotherapy and in combination with pembrolizumab) and dose expansion (monotherapy) in patients with SCLC (figure 2). A study of AMG 757 in patients with neuroendocrine prostate cancer is under development based on emerging data from the ongoing phase 1 study.Abstract 627 Figure 1AMG 757 Significantly reduced tumor growth in orthotopic SCLC mouse modelsAbstract 627 Figure 2AMG 757 Phase 1 study designConclusionsAMG 757 engages and activates T cells to kill DLL3-expressing SCLC and other NET cells in vitro and induces significant antitumor activity against established xenograft tumors in mouse models. These preclinical data support evaluation of AMG 757 in clinical studies of patients with NET.Ethics ApprovalAll in vivo work was conducted under IACUC-approved protocol #2009-00046.

scholarly journals TRPM2 Is Not Required for T-Cell Activation and Differentiation

2022 ◽  
Vol 12 ◽  
Author(s):  
Niels C. Lory ◽  
Mikolaj Nawrocki ◽  
Martina Corazza ◽  
Joanna Schmid ◽  
Valéa Schumacher ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
T Cell Activation ◽  
Cell Activation ◽  
Transient Receptor Potential ◽  
Cell Function ◽  
Intestinal Inflammation ◽  
Activation Markers

Antigen recognition by the T-cell receptor induces a cytosolic Ca2+ signal that is crucial for T-cell function. The Ca2+ channel TRPM2 (transient receptor potential cation channel subfamily M member 2) has been shown to facilitate influx of extracellular Ca2+ through the plasma membrane of T cells. Therefore, it was suggested that TRPM2 is involved in T-cell activation and differentiation. However, these results are largely derived from in vitro studies using T-cell lines and non-physiologic means of TRPM2 activation. Thus, the relevance of TRPM2-mediated Ca2+ signaling in T cells remains unclear. Here, we use TRPM2-deficient mice to investigate the function of TRPM2 in T-cell activation and differentiation. In response to TCR stimulation in vitro, Trpm2-/- and WT CD4+ and CD8+ T cells similarly upregulated the early activation markers NUR77, IRF4, and CD69. We also observed regular proliferation of Trpm2-/- CD8+ T cells and unimpaired differentiation of CD4+ T cells into Th1, Th17, and Treg cells under specific polarizing conditions. In vivo, Trpm2-/- and WT CD8+ T cells showed equal specific responses to Listeria monocytogenes after infection of WT and Trpm2-/- mice and after transfer of WT and Trpm2-/- CD8+ T cells into infected recipients. CD4+ T-cell responses were investigated in the model of anti-CD3 mAb-induced intestinal inflammation, which allows analysis of Th1, Th17, Treg, and Tr1-cell differentiation. Here again, we detected similar responses of WT and Trpm2-/- CD4+ T cells. In conclusion, our results argue against a major function of TRPM2 in T-cell activation and differentiation.

scholarly journals 702 TJ-CD4B (ABL111), a Claudin18.2-targeted 4-1BB tumor engager induces potent tumor-dependent immune response without dose-limiting toxicity in preclinical studies

2021 ◽  
Vol 9 (Suppl 3) ◽  
pp. A730-A730
Author(s):  
Wenqing Jiang ◽  
Zhengyi Wang ◽  
Zhen Sheng ◽  
Jaeho Jung ◽  
Taylor Guo
Keyword(s):  
T Cells ◽  
T Cell ◽  
T Cell Activation ◽  
Cd8 T Cells ◽  
Gene Expression Analysis ◽  
Cell Activation ◽  
Clinical Development ◽  
Cynomolgus Monkeys ◽  
Anti Tumor Activity

Background4-1BB (CD137) is a co-stimulatory receptor that stimulates the function of multiple immune cells. Its ability to induce potent anti-tumor activity makes 4-1BB an attractive target for immuno-oncology. However, clinical development of a monospecific 4-1BB agonistic antibody has been hampered by dose-limiting hepatic toxicities. To minimize systemic toxicities, we have developed a novel Claudin18.2 (CLDN18.2) x 4-1BB bispecific antibody, TJ-CD4B (ABL111) that stimulates 4-1BB pathway only when it engages with Claudin 18.2, a tumor-associated antigen specifically expressed in gastrointestinal cancers. TJ-CD4B (ABL111) is now being evaluated in patients with advanced solid tumors in a first-in-human trial (NCT04900818).MethodsTJ-CD4B (ABL111) was evaluated in vivo using the human 4-1BB knock-in mice bearing CLDN18.2 expressing MC38 tumor cells. Pharmacodynamic effects upon treatment were characterized in tumor tissue and blood. Immunophenotyping of the tumor microenvironment (TME) and peripheral blood was performed by flow cytometry. Soluble biomarkers were measured using Luminex-based multiplex assay. In-depth gene expression analysis was performed on primary human CD8+ T cells that were co-cultured with CLDN18.2 expressing cells in the presence of anti-CD3 using NanoString nCounter®. Pharmacokinetic (PK) and toxicity study were performed in cynomolgus monkeys.ResultsTJ-CD4B (ABL111) elicited complete tumor regression in 13 out of 18 MC38 tumor bearing mice given at a dose above 2 mg/kg. Dose-dependent anti-tumor activity was associated with enhanced T cell activation in TME and expansion of memory T cells in the peripheral blood. Increased CD8+ T cells number and proliferation were observed in both tumor nest and surrounding stroma while the level of soluble 4-1BB in the serum was also elevated in response to the treatment. In vitro gene expression analysis by Nanostring revealed TJ-CD4B(ABL111) effectively activated immune pathways characterized by IFN?-signaling and T cell inflammation. Preclinically, TJ-CD4B was well tolerated at the repeated doses up to 100 mg/kg/wk in cynomolgus monkeys without the adverse influence on the liver function which is generally affected by 4-1BB activation. Besides, no cytokine release or immune activation was observed in the periphery.ConclusionsTJ-CD4B (ABL111) is a novel CLDN18.2 dependent 4-1BB bispecific agonist antibody that induced T cell activation and memory response in tumor with CLDN18.2 expression, leading to a strong anti-tumor activity in vivo. TJ-CD4B did not induce systemic immune response nor hepatic toxicity due to the CLDN18.2 dependent 4-1BB stimulation. These data warrant the current clinical development in phase I trial to validate the safety properties and tumor specific responses.

Sign in / Sign up

Export Citation Format

Share Document