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 Prophylactic Itacitinib (INCB039110) for the Prevention of Cytokine Release Syndrome Induced By Chimeric Antigen Receptor T-Cells (CAR-T-cells) Therapy

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 1934-1934 ◽  
Author(s):  
Eduardo Huarte ◽  
Roddy S O'Connor ◽  
Melissa Parker ◽  
Taisheng Huang ◽  
Michael C. Milone ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cytokine Release ◽  
Employment Equity ◽  
Car T Cells ◽  
Car T Cell ◽  
Car T ◽  
Equity Ownership

Background: T-cells engineered to express a chimeric antigen receptor (CAR-T-cells) are a promising cancer immunotherapy. Such targeted therapies have shown long-term relapse survival in patients with B cell leukemia and lymphoma. However, cytokine release syndrome (CRS) represents a serious, potentially life-threatening, side effect often associated with CAR-T cells therapy. The Janus kinase (JAK) tyrosine kinase family is pivotal for the downstream signaling of inflammatory cytokines, including interleukins (ILs), interferons (IFNs), and multiple growth factors. CRS manifests as a rapid (hyper)immune reaction driven by excessive inflammatory cytokine release, including IFN-g and IL-6. Itacitinib is a potent, selective JAK1 inhibitor which is being clinically evaluated in several inflammatory diseases. Aims: To evaluate in vitro and in vivo the potential of itacitinib to modulate CRS without impairing CAR-T cell anti-tumor activity. Materials and Methods: In vitro proliferation and cytotoxic activity of T cells and CAR-T cells was measured in the presence of increasing concentrations of itacitinib or tocilizumab (anti-IL-6R). To evaluate itacitinib effects in vivo, we conducted experiments involving adoptive transfer of human CD19-CAR-T-cells in immunodeficient animals (NSG) bearing CD19 expressing NAMALWA human lymphoma cells. The effect of itacitinib on cytokine production was studied on CD19-CAR-T-cells expanded in the presence of itacitinib or tocilizumab. Finally, to study whether itacitinib was able to reduce CRS symptoms in an in vivo setting, naïve mice were stimulated with Concanavalin-A (ConA), a potent T-cell mitogen capable of inducing broad inflammatory cytokine releases and proliferation. Results: In vitro, itacitinib at IC50 relevant concentrations did not significantly inhibit proliferation or anti-tumor killing capacity of human CAR-T-cells. Itacitinib and tocilizumab (anti-IL-6R) demonstrated a similar effect on CAR T-cell cytotoxic activity profile. In vivo, CD19-CAR-T-cells adoptively transferred into CD19+ tumor bearing immunodeficient animals were unaffected by oral itacitinib treatment. In an in vitro model, itacitinib was more effective than tocilizumab in reducing CRS-related cytokines produced by CD19-CAR-T-cells. Furthermore, in the in vivo immune hyperactivity (ConA) model, itacitinib reduced serum levels of CRS-related cytokines in a dose-dependent manner. Conclusion: Itacitinib at IC50 and clinically relevant concentrations did not adversely impair the in vitro or in vivo anti-tumor activity of CAR-T cells. Using CAR-T and T cell in vitro and in vivo systems, we demonstrate that itacitinib significantly reduces CRS-associated cytokines in a dose dependent manner. Together, the data suggest that itacitinib may have potential as a prophylactic agent for the prevention of CAR-T cell induced CRS. Disclosures Huarte: Incyte corporation: Employment, Equity Ownership. Parker:Incyte corporation: Employment, Equity Ownership. Huang:Incyte corporation: Employment, Equity Ownership. Milone:Novartis: Patents & Royalties: patents related to tisagenlecleucel (CTL019) and CART-BCMA; Novartis: Research Funding. Smith:Incyte corporation: Employment, Equity Ownership.

scholarly journals Development of a Genetically-Engineered Allogeneic Anti-CD38 T Cell Therapy Utilizing a Novel Antigen Receptor Structure

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 4444-4444
Author(s):  
Bei Bei Ding ◽  
John Dixon Gray ◽  
Irina Krapf ◽  
Yanliang Zhang ◽  
Nan Zhang ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
T Cells ◽  
T Cell ◽  
Cell Therapy ◽  
Employment Equity ◽  
T Cell Therapy ◽  
Equity Ownership ◽  
Anti Tumor Activity

Background: Autologous Chimeric Antigen Receptor (CAR) T cell therapy has shown great promise as a treatment modality for a variety of hematological malignancies. But autologous cell therapies still face several practical hurdles, including reliance on patient immune cells and manufacturing difficulties. Sorrento has pioneered an allogeneic T cell therapy approach utilizing genetic engineering of donor-derived T cells to express a Dimeric Antigen Receptor (DAR). The first DAR-T cell therapy being developed is targeted against CD38, a clinically-validated antigen in multiple myeloma. Preclinical data demonstrate potent anti-tumor activity in both in vitro assays and in vivo studies against CD38-expressing lymphoma and multiple myeloma (MM) cell lines. Methods: Anti-CD38 DAR-T cells were generated through genetic engineering of T cells derived from healthy donors inserting the anti-CD38 DAR construct into the TRAC gene locus resulting in loss of endogenous TCR expression while expressing the DAR. Three distinct DAR constructs were utilized differing only in the intracellular signaling components, namely CD28/CD3zeta, 4-1BB/CD3zeta and CD28/4-1BB/CD3zeta. The CD38 DAR-T were expanded and purified for subsequent preclinical studies. Using in vitro assays, the 3 different CD38 DAR-T cells were evaluated against multiple myeloma and lymphoma cell lines for specific cytotoxicity as well as stimulus-induced cytokine secretion and cell expansion. The in vivo anti-tumor activity was assessed using luciferase-expressing RPMI8226 cells in NSG mice in a model of disseminated disease. A single dose of anti-CD38 DAR-T cells or relevant control cells was administered and tumor burden was assessed weekly using bioluminescence imaging. Results: An anti-CD38 DAR gene was efficiently integrated into TRAC locus of T cells from healthy donors by one step knock out/knock in (KOKI) methodology with high efficiency (~40-80% CD38 DAR expression and ~90% TCR knock out). Following a CD3-depletion step, the TCR-positive T cells were less 1%. When co-cultured with CD38-positive tumor cells, anti-CD38 DAR T cells killed as effectively as retroviral anti-CD38 CAR-T cells with similar cytokine secretion profiles while no cytotoxicity was observed against CD38-negative cancer cells. Moreover, in vivo DAR-T cells showed better killing activity against multiple myeloma cell lines than CAR-T cell with anti-CD38 4-1BB/CD3zeta DAR demonstrating the best anti-tumor activity in an NSG mouse model. The anti-CD38 DAR-T cells with 41BB/CD3 zeta internal signals have been selected for clinical development. Conclusions: All tested anti-CD38 DAR-T cells exhibited potent in vitro and in vivo anti-tumor activity. Direct comparison of three different cytoplasmic signaling compositions of the DAR allowed for selection of the most potent construct, namely the anti-CD38 DAR utilizing 4-1BB and CD3zeta signaling domains. Based on these data, further development of CD38 DAR-T therapy for hematological malignancies is warranted. GMP manufacturing of the allogeneic anti-CD38 DAR-T cells has been initiated. Disclosures Ding: Sorrento Therapeutics, Inc.: Employment, Equity Ownership, Patents & Royalties. Gray:Sorrento Therapeutics, Inc.: Employment, Equity Ownership, Patents & Royalties. Krapf:Sorrento Therapeutics, Inc.: Employment, Equity Ownership. Zhang:Sorrento Therapeutics, Inc.: Employment, Equity Ownership, Patents & Royalties. Zhang:Sorrento Therapeutics, Inc.: Employment, Equity Ownership. Deng:Sorrento Therapeutics, Inc.: Employment, Equity Ownership. Wei:Sorrento Therapeutics, Inc.: Employment, Equity Ownership. Knight:Sorrento Therapeutics, Inc.: Employment, Equity Ownership. Zeldis:Sorrento Therapeutics Inc: Employment, Equity Ownership. Kaufmann:Sorrento Therapeutics, Inc.: Employment, Equity Ownership, Patents & Royalties. Ji:Sorrento Therapeutics Inc: Employment, Equity Ownership, Patents & Royalties; Celularity, Inc.: Equity Ownership, Membership on an entity's Board of Directors or advisory committees. Guo:Sorrento Therapeutics, Inc.: Employment, Equity Ownership, Patents & Royalties.

TNB-486, a Novel Fully Human Bispecific CD19 x CD3 Antibody That Kills CD19-Positive Tumor Cells with Minimal Cytokine Secretion

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 4070-4070
Author(s):  
Harbani Malik ◽  
Ben Buelow ◽  
Udaya Rangaswamy ◽  
Aarti Balasubramani ◽  
Andrew Boudreau ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
T Cells ◽  
T Cell ◽  
B Cell ◽  
Tumor Cells ◽  
Cytokine Secretion ◽  
Employment Equity ◽  
Equity Ownership

Introduction The restricted expression of CD19 in the B-cell lineage makes it an attractive target for the therapeutic treatment of B-cell malignancies. Many monoclonal antibodies and antibody drug conjugates targeting CD19 have been developed, including bispecific T-cell redirecting antibodies (T-BsAbs). In addition, anti-CD19 chimeric antigen receptor T-cells (CAR-T) have been approved to treat leukemia and lymphoma. However, despite the impressive depth of responses achieved by T-cell redirecting approaches such as T-BsAbs and CAR-T cells, toxicity from over-activation of T-cells remains a substantial limitation for this type of therapy, in particular neurotoxicity. In designing TNB-486, a novel CD19 x CD3 T-BsAb, we endeavored to retain activity against CD19-positive tumor cells while limiting the cytokine secretion thought to underlie toxicity from T-cell redirecting therapies. Utilizing TeneoSeek, a next generation sequencing (NGS)-based discovery pipeline that leverages in silico analysis of heavy chain only/fixed light chain antibody (HCA/Flic, respectively) sequences to enrich for antigen specific antibodies, we made a high affinity αCD19 HCA and a library of αCD3 Flic antibodies that showed a >2 log range of EC50s for T cell activation in vitro. Of note, the library contained a low-activating αCD3 that induced minimal cytokine secretion even at concentrations that mediated saturating T-cell dependent lysis of lymphoma cells (when paired with an αCD19 HCA). We characterized the relative efficacy and potential therapeutic window of this unique molecule, TNB-486, in vitro and in vivo and compared it to two strongly activating bispecific CD19 x CD3 antibodies similar to those currently available and in clinical development. Methods Affinity measurements of the αCD19 moiety were made via Biacore (protein) and flow cytometry (cell surface). Stability measurements were made by subjecting the molecule to thermal stress and the %aggregation was measured by Size Exclusion Chromatography. T-cell activation was measured via flow cytometry (CD69 and CD25 expression) and cytokine was measured by ELISA (IL-2, IL-6, IL-10, INF-ɣ, and TNFα) in vitro. Lysis of B-cell tumor cell lines (Raji, RI-1, and Nalm6) was measured via flow cytometry in vitro. In vivo, NOG mice were engrafted subcutaneously with NALM-6 or SUDHL-10 cells and intravenously with human peripheral blood mononuclear cells (huPBMC), and the mice treated with multiple doses of TNB-486 or negative or positive control antibody. Tumor burden was evaluated via caliper measurement. Pharmacodynamic/Pharmacokinetic (PK/PD) studies were performed in NOG mice. A pharmacokinetic (PK) study was performed in BALB/c mice, and a tolerability and PK study are ongoing in cynomolgus monkeys. Results TNB-486 bound to cell surface CD19 with single digit nanomolar affinity (~3nM). EC50s for cytotoxicity were in the single-digit nanomolar range for TNB-486, and sub-nanomolar for the strongly activating controls; TNB-486 maximum achievable lysis was identical to the positive controls. TNB-486 induced significantly less cytokine release for all cytokines tested compared to the positive controls even at doses saturating for tumor lysis. No off-target activation was observed in the absence of CD19 expressing target cells. In vivo, TNB-486 eradicated all CD19-positive tumors tested (NALM-6 and SUDHL10) at doses as little as 1µg administered every four days after tumors had reached ~200mm3. TNB-486 showed a PK profile consistent with other IgG molecules in mice (T1/2 ~6 days in mice). Conclusions TNB-486 induced comparable lysis of CD19-positive tumor cells as the strongly activating control bispecific antibodies while inducing significantly reduced cytokine secretion, even at doses saturating for tumor lysis in vitro. In vivo TNB-486 eradicated all tested CD19 positive tumor cell lines in established tumor models. No off-target binding was observed. In summary, TNB-486 shows promise as a lymphoma therapeutic differentiated from T-cell targeted therapies currently in the clinic and in clinical trials. Disclosures Malik: Teneobio, Inc.: Employment, Equity Ownership. Buelow:Teneobio, Inc.: Employment, Equity Ownership. Rangaswamy:Teneobio, Inc.: Employment, Equity Ownership. Balasubramani:Teneobio, Inc.: Employment, Equity Ownership. Boudreau:Teneobio, Inc.: Employment, Equity Ownership. Dang:Teneobio, Inc.: Employment, Equity Ownership. Davison:Teneobio, Inc.: Employment, Equity Ownership. Force Aldred:Teneobio, Inc.: Equity Ownership. Iyer:Teneobio, Inc.: Employment, Equity Ownership. Jorgensen:Teneobio, Inc.: Employment, Equity Ownership. Pham:Teneobio, Inc.: Employment, Equity Ownership. Prabhakar:Teneobio, Inc.: Employment, Equity Ownership. Schellenberger:Teneobio, Inc.: Employment, Equity Ownership. Ugamraj:Teneobio, Inc.: Employment, Equity Ownership. Trinklein:Teneobio, Inc.: Employment, Equity Ownership. Van Schooten:Teneobio, Inc.: Employment, Equity Ownership.

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 Preclinical Evaluation of Human Placental-Derived Allogeneic CD19 CAR-T Cells Against B Cell Malignancies

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 3222-3222
Author(s):  
Kathy Karasiewicz ◽  
Shuyang He ◽  
Mary Ng ◽  
Kristina Tess ◽  
Weifang Ling ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cell Receptor ◽  
Employment Equity ◽  
Car T Cells ◽  
Car T ◽  
Equity Ownership ◽  
Robust Process

Celularity, Inc. is developing a CD19 CAR-T Cell therapy using an allogeneic platform derived from postpartum human placental cells. T cells isolated from placenta/ umbilical cord blood and genetically modified to express CD19 chimeric antigen receptor (CAR), termed Placental-derived (P-) CD19 CAR T cells, are in development for the treatment of B cell malignancies. Unlike adult peripheral blood mononuclear cell (PBMC)-derived T cells, P-T cells are mostly naïve (CD45RA+) and can be readily expanded while maintaining an earlier differentiation phenotype such as greater expression of naïve/ memory markers, lower expression of effector/ exhaustion markers, allowing for greater proliferative potential of these cells ex vivo. These cells are also known to have greater immune tolerance to HLA mismatch and display impaired allogeneic activation, contributing to lower incidences of severe graft-verse-host disease (GvHD) (Barker, et. al. Blood, 2001; Chen, et al. Biology of Blood and Marrow Transplantation, 2006), making them an attractive cell population for use as an allogeneic, adoptive cell therapy. A robust process for the isolation, transduction, and expansion of placental-derived T cells to generate "off-the-shelf" allogeneic P-CD19 CAR T cells was developed. Twenty-One day expanded, non-modified P-T cells (N=3) were compared to adult PBMCs for their allo-reactivity in a Xenogeneic GvHD model in NCG mice. P-T cells did not induce xeno-GvHD whereas PBMCs did, as evidenced by significant weight loss and death of all mice (N=5) by Day 28 post infusion. Despite expanded P-T cells demonstrating lack of in vivo GvHD, current manufacture of P-CD19 CAR T cells does include a CRISPR-mediated T-cell receptor a constant (TRAC) knockout (KO) step as an additional risk-mitigation strategy to circumvent any potential GvHD stemming from expression of endogenous T cell receptor. CD19 CAR transduction using a retrovirus provided by Sorrento Therapeutics, Inc., followed by TRAC knockout with CRISPR results in both high efficiency of CD19 CAR expression (~30% CD19 Fc+) and TCR KO (>96% CD3-/ TCR a/b-). In vitro, the functional activity of P-CD19 CAR-TRAC KO T cells against CD19+ Burkitt's Lymphoma (Daudi) and Acute lymphoblastic Leukemia (NALM6) cell lines was assessed in cytotoxicity and cytokine release assays. P-CD19 CAR T cells specifically lyse CD19+ Daudi/ Nalm6 targets in both 4-hour endpoint FACS and ACEA kinetic cytotoxicity assays, and in most cases at levels equivalent to or greater than PBMC-derived CD19 CAR T cells. When P-CD19 CAR T cells were co-cultured with CD19+ Daudi/ Nalm6 target cells for 24-hours, they secreted pro-inflammatory cytokines and effector proteins in an antigen-specific manner. In vivo, the anti-tumor activity of P-CD19 CAR T cells was assessed using a disseminated lymphoma xenograft model in NSG mice. Luciferase expressing Daudi cells (3×106) were intravenously (IV) injected on Day 0, followed by IV injection of P-CD19 CAR T cells (14×106) on Day 7. Bioluminescence Imaging (BLI) and survival were used as primary study endpoints. P- CD19 CAR T cells were well tolerated and safe. P-CD19 CAR T cells significantly reduced tumor burden, and improved survival. Four weeks after treatment, the vehicle group had a 100% mortality rate, while all animals from P-CD19 CAR T-treated group (N=5) remained alive without clinical symptoms including weight loss or changes in their fur. In summary, Celularity has defined a robust process for the generation and expansion of CD19 CAR T cells from human placenta. These cells exhibit potent anti-tumor activity both in vitro and in vivo with little evidence of acute GvHD induction, highlighting their potential as an allogeneic, adoptive cell therapeutic agent. Future in vivo GvHD studies will include assessment of both CD19 CAR and TRAC KO genetically modified P-T cells. Disclosures Karasiewicz: Celgene: Equity Ownership; Celularity, Inc.: Employment, Equity Ownership, Patents & Royalties: Patent Inventor. He:Celularity Inc: Employment. Ng:Celularity, Inc.: Employment. Tess:Celularity, Inc.: Employment. Ling:Celularity Inc: Employment. Kaufmann:Sorrento Therapeutics, Inc.: Employment, Equity Ownership, Patents & Royalties. Zeldis:Sorrento Therapeutics Inc: Employment, Equity Ownership. Ji:Celularity, Inc.: Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Sorrento Therapeutics Inc: Employment, Equity Ownership, Patents & Royalties. Hariri:Celularity Inc: Employment. Zhang:Celularity Inc: Employment.

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 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 844 A trispecific ROR1 x CD3 T cell engager mediates in vitro tumor cell killing and in vivo tumor eradication

2021 ◽  
Vol 9 (Suppl 3) ◽  
pp. A885-A885
Author(s):  
Bithi Chatterjee ◽  
Daniel Snell ◽  
Daniel Snell ◽  
Christian Hess ◽  
Matthias Brock ◽  
...  
Keyword(s):  
T Cell ◽  
Tumor Cell ◽  
T Cell Activation ◽  
Cell Activation ◽  
T Cell Proliferation ◽  
Cytokine Release ◽  
Mantle Cell ◽  
Tumor Cell Killing

BackgroundReceptor tyrosine kinase-like orphan receptor 1 (ROR1) is expressed on a variety of difficult to treat solid and hematological malignancies. Several therapeutic concepts targeting ROR1 are currently in clinical studies, including antibody-drug conjugates (ADCs), chimeric antigen receptor engineered T cells, as well as a bispecific T cell engager. In contrast to ADCs, T cell engagers have the capacity to induce tumor cell depletion irrespective of tumor cell mitotic activity. For the therapy of ROR1 expressing tumors, we engineered a T cell engager with prolonged half-life to support convenient administration schemes.MethodsNM32-2668, a ROR1-targeting T cell engager with prolonged serum half-life was engineered by joining three humanized rabbit antibody variable region (Fv) fragments specific for ROR1, CD3ɛ, and serum albumin, into our tri-specific scMATCHTM3 format. Each Fv fragment was stabilized using the ʎ-capTM technology. NM32-2668 was tested in assays for specific tumor lysis, induction of T cell proliferation, and cytokine release. These studies were performed using human T cells co-cultured with tumor cell lines and human tumor samples expressing various levels of ROR1. In vivo xenograft mouse studies were conducted using a human mantle cell lymphoma model in NCG mice engrafted with human PBMCs.ResultsHere we report the design and the promising preclinical activity of the scMATCHTM3 ROR1/CD3/hSA T cell engager NM32-2668 in vitro and in vivo. Importantly, we demonstrate potent and specific cytotoxic activity in the sub-nanomolar range on tumor cell lines expressing different levels of ROR1. NM32-2668 also mediates ROR1 dependent T cell activation and cytokine release. We observe robust tumor cell killing activity of NM32-2668 over an extended time period and at multiple ratios of effectors to targets in a real time imaging-based cytotoxicity assay. This molecule also mediates T cell proliferation in response to target cell binding. NM32-2668 mediates in vitro lysis of CLL patient tumor cells, T cell activation, and cytokine release, with minimal IL-6 involvement. In an in vivo mantle cell lymphoma model (Jeko-1) engrafted with human PBMCs, we observe tumor regression and eradication.ConclusionsCollectively, these data demonstrate robust anti-tumor efficacy by NM32-2668, a scMATCHTM3 ROR1/CD3/hSA. Our results demonstrate that NM32-2668 promotes ROR1 dependent T cell activation and proliferation, as well as T cell-mediated tumor cell lysis. The activity of NM32-2668 has the potential to provide significant benefit to patients with ROR1+ malignancies on a convenient dosing schedule. We intend to rapidly progress NM32-2668 to clinical development.

scholarly journals Development of an Allogeneic Anti-Bcma T Cell Therapy Utilizing a Novel Dimeric Antigen Receptor (DAR) Structure

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 1942-1942 ◽  
Author(s):  
Bei Bei Ding ◽  
John Dixon Gray ◽  
Nan Zhang ◽  
Yanliang Zhang ◽  
Xia Cao ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
T Cells ◽  
T Cell ◽  
Cell Therapy ◽  
Antigen Receptor ◽  
Employment Equity ◽  
T Cell Therapy ◽  
Equity Ownership ◽  
Anti Tumor Activity

Background: Multiple myeloma remains an incurable malignancy of plasma cells. Adoptive transfer of chimeric antigen receptor (CAR)-expressing T cells is a promising new therapy for hematologic malignancies. B-cell maturation antigen (BCMA) is a protein that is selectively expressed by B-lineage cells, including Multiple Myeloma (MM) cells, and represents a suitable target for T cell therapy. We have developed an allogeneic T cell therapy approach utilizing genetic engineering of donor-derived T cells to express an anti-BCMA Dimeric Antigen Receptor (DAR) using a proprietary non-viral vector Knock out/knock in (KOKI) technology. Preclinical data demonstrate potent anti-tumor activity both vitro and in vivo against BCMA-expressing MM cell lines. Methods: Anti-BCMA DAR-T cells were generated through genetic engineering of T cells derived from healthy donors by inserting the anti-BCMA DAR construct into the TRAC gene locus, resulting in loss of endogenous TCR expression while expressing the DAR. Distinct DAR constructs were utilized differing only in their intracellular signaling components, namely combinations of 4-1BB, CD28, and CD3zeta. The anti-BCMA DAR-T cells were expanded and purified for subsequent preclinical studies. Using in vitro assays, the different anti-BCMA DAR-T cells were evaluated against multiple myeloma cell lines for specific cytotoxicity as well as stimulus-induced cytokine secretion and cell expansion. The in vivo anti-tumor activity was assessed using luciferase-expressing RPMI8226 cells in NSG mice in a model of disseminated disease. A single dose of anti-BCMA DAR-T cells or relevant control cells was administered, and tumor burden was assessed weekly using bioluminescence imaging. Results: After purification, the anti-BCMA DAR T cells population contained less than 1% TCR-expressing ab T cells. The DAR-positive T cell population was between 20-50%. All anti-BCMA DAR-T cells exhibited BCMA-specific activation, including cytokine production, proliferation, cytotoxicity, and in vivo tumor eradication. The DAR-T cells using a third generation signaling configuration containing components from 4-1BB, CD28 and CD3zeta signaling domains performed best overall. Conclusions: All tested anti-BCMA DAR-T cells exhibited effective anti-tumor activity. Direct comparison of different cytoplasmic signaling compositions of the DAR allowed for selection of the most potent construct, namely the anti-BCMA DAR utilizing a 3rd generation signaling domain configuration. Based on these data, further development of anti-BCMA DAR-T therapy for hematological malignancies is warranted. These allogeneic abTCR-negative anti-BCMA DAR-T cells have been selected for clinical development. Disclosures Ding: Sorrento Therapeutics, Inc.: Employment, Equity Ownership, Patents & Royalties. Gray:Sorrento Therapeutics, Inc.: Employment, Equity Ownership, Patents & Royalties. Zhang:Sorrento Therapeutics, Inc.: Employment, Equity Ownership. Zhang:Sorrento Therapeutics, Inc.: Employment, Equity Ownership, Patents & Royalties. Cao:Sorrento Therapeutics, Inc.: Employment, Equity Ownership, Patents & Royalties. Krapf:Sorrento Therapeutics, Inc.: Employment, Equity Ownership. Deng:Sorrento Therapeutics, Inc.: Employment, Equity Ownership. Wei:Sorrento Therapeutics, Inc.: Employment, Equity Ownership. Zeldis:Sorrento Therapeutics Inc: Employment, Equity Ownership. Knight:Sorrento Therapeutics, Inc.: Employment, Equity Ownership. Kaufmann:Sorrento Therapeutics, Inc.: Employment, Equity Ownership, Patents & Royalties. Ji:Sorrento Therapeutics Inc: Employment, Equity Ownership, Patents & Royalties; Celularity, Inc.: Equity Ownership, Membership on an entity's Board of Directors or advisory committees. Guo:Sorrento Therapeutics, Inc.: Employment, Equity Ownership, Patents & Royalties.

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.

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