A Novel Full Length Anti-FLT3 CD3 Bispecific Antibody for the Treatment of Acute Myeloid Leukemia

Abstract FLT3 is a receptor tyrosine kinase expressed on the surface of acute myeloid leukemia (AML) patient blasts. FLT3 is the most frequently mutated gene in AML patients, and these mutations are associated with poor prognosis. Despite the development of small molecule inhibitors of FLT3 function and neutralizing FLT3 antibodies, there remains a need for antibodies that target the broad AML patient population with improved efficacy and safety. We chose to use Pfizer's proprietary full length humanized CD3 bispecific IgG molecule platform. The first step in the development was finding a suitable targeting epitope on FLT3 as not all epitopes result in optimal T cell activation in the context of CD3 binding. Through a combination of in vitro and in vivo studies, FLT3 antibodies targeting extracellular domain 4 of FLT3 were found to be more effective at AML cell depletion than other domains in the full-length bispecific IgG format, significantly outperforming the antibodies targeting the most membrane proximal region of domain 5. The final candidate antibody was engineered to have picomolar affinity for recombinant human FLT3 (<50 pM) to ensure that low levels of FLT3 present on AML (<5,000 per cell) could efficiently trigger T cell cytotoxicity. Anti-tumor activity of the final molecule was further characterized in vitro and in vivo. Three AML cell lines, Eol-1, Molm-13 and MV-411 express high, medium and low levels of FLT3, respectively, and were targeted by activated healthy donor T cells in the presence of the FLT3 bispecific with an efficiency that correlated with FLT3 surface density (EC50 range was 0.5 pM to 40 pM at E:T ratios of 1:1). Ability to target primary AML cells was confirmed in assays with primary AML blasts and autologous patient T cells (EC50s 7 - 15 nM, for E:T ratios of up to 1:50). In orthotopic xenograft models with the same set of cell lines, all three cell lines were efficiently eliminated by activated T cells in the presence of a single dose of FLT3 bispecific (dose range 10 to 100 μg/kg), confirming the anti-tumor activity of the bispecific antibody in vivo. The safety of Pfizer's FLT3 bispecific antibody was also studied in preclinical studies. We first analyzed gene expression of FLT3 and three other commonly considered AML targets (CD33, CD123, and CLL-1) in publicly available gene expression databases. FLT3 had the highest differential expression between AML and healthy tissues with blood and brain showing expression of low levels of FLT3 RNA. In tissue cross-reactivity studies, we detected little to no binding of the candidate FLT3 antibody to human brain tissue sections. In blood, the highest expression of FLT3 was in hematopoietic stem cells and progenitors, dendritic cells and monocytes consistent with previous publications and known roles of FLT3 in hematopoiesis and dendritic cell homeostasis. Of note, expression of FLT3 in monocytes and whole blood was the lowest compared to three other AML targets. To address any potential toxicities of FLT3 bispecific, we performed exploratory studies in cynomolgus monkeys. Affinity of FLT3 bispecific for cynomolgus FLT3 was ~20x lower than for human FLT3 (~1 nM), as determined in binding assays with cells expressing human and cynomolgus FLT3. No major clinical signs or toxicological findings were observed up to the highest dose level tested (3 mg/kg) following two administrations at weekly intervals. On target activity was demonstrated by nearly complete elimination of FLT3+ dendritic cells in the blood of treated monkeys two days after administration. In addition, we detected elimination of FLT3+ CD34+ stem cells in the bone marrow of treated monkeys two days after the second dose. Remarkably, both dendritic cells and FLT3+ CD34+ stem cells rebounded to baseline levels observed in control animals when analyzed at study termination, two weeks following the second dose. Consistent with minimal expression of FLT3 on healthy tissues and the presence of FLT3+ on rare blood subsets, cytokine increases typically associated with CD3 bispecific administration were minimal (only 6 to 17 and 46 to 89 fold over baseline for IFN-γ and IL-6, respectively, at the highest dose tested). In conclusion, the robust anti-tumor activity of Pfizer's FLT3 bispecific antibody combined with good tolerability in cynomolgus monkeys, reversible hematological toxicity and absence of non-hematological toxicity support its further clinical development in AML. Disclosures Djuretic: Pfizer Inc.: Employment. Krishnamoorthy:Pfizer: Employment. Sommer:Allogene Therapeutics: Employment, Equity Ownership, Patents & Royalties. Dettling:Maverick Therapeutics: Employment. Poulsen:Allogene Therapeutics: Employment, Equity Ownership. Chen:Pfizer: Employment. Hu:Pfizer: Employment. Given Chunyk:Pfizer: Employment. Lindquist:Pfizer: Employment. Potluri:Pfizer: Employment. Rickert:Pfizer: Employment. Sasu:Allogene Therapeutics: Employment, Equity Ownership, Patents & Royalties. Chaparro-Riggers:Pfizer Inc.: Employment, Patents & Royalties. Yeung:Pfizer: Employment, Patents & Royalties.

Download Full-text

Preclinical Development of an Anti-CD38 Antibody-Drug Conjugate for Treatment of Hematological Malignancies

Blood ◽

10.1182/blood-2019-132062 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 5621-5621 ◽

Cited By ~ 1

Author(s):

Lingna Li ◽

Wenyong Tong ◽

Megan Lau ◽

Katherine Fells ◽

Tong Zhu ◽

...

Keyword(s):

Multiple Myeloma ◽

Cell Lines ◽

Cell Binding ◽

Antibody Drug Conjugate ◽

Employment Equity ◽

Cd38 Expression ◽

Equity Ownership ◽

Anti Tumor Activity ◽

Antibody Drug

CD38 is a validated target for the treatment of multiple myeloma (MM). Daratumumab (Darzalex®), an anti-CD38 monoclonal antibody (mAb), has shown great clinical efficacy and has been approved for multiple myeloma treatment. However, both primary refractoriness and development of resistance to daratumumab therapy have been reported. Based on the therapeutic benefits of this CD38 antibody, we developed a CD38-targeting antibody-drug conjugate (ADC), employing a fully human anti-CD38 antibody STI-6129, identified from Sorrento's G-MAB® antibody library, and proprietary linker-toxin technology. The toxin payload is duostatin 5.2 (Duo.5.2), a microtubule inhibitor, conjugated to STI-6129 via a non-polyethylene glycol linker resulting in our lead ADC CD38-077. Cell binding studies showed that it specifically binds to CD38-positive tumor cells but not CD38-negative cell lines. The cell binding was proportional to the CD38 expression level on the cell surface. The ADC was internalized into CD38-positive cells at a rate comparable to that of the unconjugated antibody, indicating that conjugation did not change the binding characteristics of STI-6129 to its antigen. In cytotoxicity studies, CD38-077 exhibited a CD38-dependent cytotoxic activity against a panel of CD38-expressing tumor cell lines and was more potent in cells with high CD38 expression. The cytotoxic effect of CD38-077 was also examined against human PBMC cells, as it has been reported that certain types of the immune cells express CD38. The result indicated that normal PBMC cells were generally insensitive to the ADC up to 1 µM following 120 hr exposure. We investigated the anti-tumor activity of CD38-077 in xenograft animal models of Burkitt's lymphoma and two different multiple myeloma (MM) cell lines. The studies evaluated different dose levels and dosing regimens, including single dose and multiple doses at various intervals. The data showed that the ADC has a broad, potent and CD38-dependent in vivo efficacy in all three xenograft tumor models examined. In a pharmacokinetic study in naïve mice, CD38-077 was found to be stable, with T1/2 of about 7-11 days, comparable to that of the unconjugated STI-6129 antibody. In summary, CD38-077 exhibits strong anti-tumor activity in vitro and in vivo. The ADC showed specific activity towards CD38-expressing tumors but was less active against CD38-expressing normal PBMC cells, which express relatively low levels of CD38 level and where internalization was not detectable. These results warrant further development exploration of CD38-077. Disclosures Li: Concortis Biotherapeutics: Employment, Equity Ownership. Lau:Levena Biopharma: Employment, Equity Ownership. Fells:Sorrento Therapeutics, Inc.: Employment, Equity Ownership. Zhu:Levena Biopharma: Employment, Equity Ownership, Patents & Royalties. Sun:Levena Biopharma: Employment, Equity Ownership. Kovacs:Levena Biopharma: Employment, Equity Ownership. Khasanov:Levena Biopharma: Employment, Equity Ownership. Yan:Levena Biopharma: Employment, Equity Ownership. Deng:Levena Biopharma: Employment, Equity Ownership. Takeshita: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. Li:Levena Biopharma: Employment, Equity Ownership, Patents & Royalties; Sorrento Therapeutics, Inc.: Employment, Equity Ownership, Patents & Royalties. Zhang:Concortis Biotherapeutics: Employment, Equity Ownership, Patents & Royalties.

Download Full-text

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

Blood ◽

10.1182/blood-2019-131892 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 1942-1942 ◽

Cited By ~ 1

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.

Download Full-text

CTX-8573, an Innate-Cell Engager Targeting BCMA, is a Highly Potent Multispecific Antibody for the Treatment of Multiple Myeloma

Blood ◽

10.1182/blood-2019-128749 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 3182-3182 ◽

Cited By ~ 2

Author(s):

Jennifer Watkins-Yoon ◽

Wilson Guzman ◽

Amanda Oliphant ◽

Sara Haserlat ◽

Alan Leung ◽

...

Keyword(s):

T Cells ◽

Cell Lines ◽

Cell Activation ◽

Γδ T Cells ◽

Therapeutic Window ◽

Target Cells ◽

Employment Equity ◽

Equity Ownership

Introduction: Current therapies for multiple myeloma (MM), such as immunomodulatory agents, proteasome inhibitors, stem-cell transplantation, and monoclonal antibodies against tumor-associated antigens have greatly improved patient survival. However, MM remains an incurable disease as most patients will eventually relapse. Recent advances in targeted T-cell therapies have shown promise in clinical trials but the adaptive immune system may be insufficient to eradicate all MM clones. In contrast, treatments harnessing the innate immune system have been relatively underdeveloped in MM despite evidence suggesting a role of innate immunity in the efficacy of existing therapies. Innate or innate-like cells, such as NK and γδ T cells, have the potential to display strong anti-tumor activity, and strategies aimed to improve or re-direct their cytotoxicity represent a new opportunity for cancer immunotherapies and a complementary approach to existing therapies. Here we describe the preclinical characterization of CTX-8573, a novel multispecific antibody that targets B-cell maturation antigen (BCMA) on tumor cells and promotes potent cytotoxicity by NK and γδ T cells through engagement of the activating receptors NKp30 and CD16a. Method: Bispecific constructs were generated by appending two common-light chain compatible anti-NKp30 Fab fragments to the C-terminus of an anti-BCMA IgG1 antibody containing an afucosylated Fc for enhanced CD16a engagement. To test the effects of targeting NKp30 alone, variants were expressed with an aglycosylated Fc to eliminate CD16a binding. In-vitro assays were performed with primary NK or γδ T cells to determine innate-cell activation, cytokine production, proliferation, and target-cell cytotoxicity against tumor cell lines with a range of BCMA expression levels. In-vivo efficacy studies were performed in multiple humanized mouse models and pharmacokinetics and safety were evaluated in Cynomolgus monkeys. Results: CTX-8573 is highly expressed in CHO cells with minimal aggregation and displays stability, solubility, and binding to BCMA and NKp30 equivalent to the parental monoclonal antibodies. By engaging NKp30 and CD16a, CTX-8573 promotes potent cytotoxicity of BCMA expressing target cells by NK and γδ T cells with >100 fold reduced EC50 compared to the corresponding BCMA monoclonal antibody control. CTX-8573 also demonstrates robust killing of low BCMA expressing cell lines including RPMI-8226 where monoclonal BCMA antibodies lack activity. An aglycosylated variant of CTX-8573 lacking CD16a binding maintains cell killing activity, demonstrating that engagement of NKp30 alone is sufficient to promote innate cell activation and cytotoxicity, although activity is enhanced by CD16A engagement. Furthermore, CTX-8573 maintains its cytotoxic activity in presence of soluble BCMA or BCMA ligands APRIL and BAFF. CTX-8573 does not induce innate cell activation, cytokine production, or killing in the absence of BCMA expressing target cells, supporting a wide therapeutic window. Additionally, unlike daratumumab, CTX-8573 does not induce NK-cell fratricide. In-vivo, CTX-8573 demonstrates anti-tumor efficacy in multiple humanized mouse models including killing of low BCMA expressing cell lines. In Cynomolgus monkeys, CTX-8573 displays standard biphasic pharmacokinetics with a 16 day β-phase half-life and has no evidence of systemic immune activation as measured by C-reactive protein levels. Lastly, NKp30 expression is maintained on bone marrow NK cells from MM patients including the presence of a significant NKp30+CD16a- subpopulation. Conclusion: CTX-8573 represents a novel class of bispecific antibodies that promote potent tumor cell killing by NK and γδ T-cells through engagement of the activating receptors NKp30 and CD16a. CTX-8573 demonstrates strong anti-tumor efficacy in vitro and in vivo, a wide therapeutic window with no evidence of systemic toxicity, and monoclonal-like pharmacokinetics and manufacturability. Together, these data highlight the potential of CTX-8573 as a novel treatment for MM either alone or as a complement to existing therapies. Disclosures Watkins-Yoon: Compass therapeutics LLC: Employment, Equity Ownership. Guzman:Compass therapeutics LLC: Employment, Equity Ownership. Oliphant:Compass therapeutics LLC: Employment, Equity Ownership. Haserlat:Compass therapeutics LLC: Employment, Equity Ownership. Leung:Compass therapeutics LLC: Employment, Equity Ownership. Chottin:University of Louisiana at Lafayette: Employment. Ophir:Compass therapeutics LLC: Employment, Equity Ownership. Vekeria:Compass therapeutics LLC: Employment, Equity Ownership. Nanjappa:Compass therapeutics LLC: Employment, Equity Ownership. Markrush:Compass therapeutics LLC: Employment, Equity Ownership. McConaughy:Compass therapeutics LLC: Employment, Equity Ownership. Wang:Compass therapeutics LLC: Employment, Equity Ownership. Schilling:Compass therapeutics LLC: Employment, Equity Ownership. Kim:Compass therapeutics LLC: Employment, Equity Ownership. Wu:Compass Therapeutics LLC: Employment, Equity Ownership. Liu:Compass therapeutics LLC: Employment, Equity Ownership. Rogers:University of Louisiana at Lafayette: Employment. Villinger:University of Louisiana at Lafayette: Employment. Gong:Compass therapeutics LLC: Employment, Equity Ownership. Hamilton:Compass therapeutics LLC: Employment, Equity Ownership. Bobrowicz:Compass therapeutics LLC: Employment, Equity Ownership. Schuetz:Compass therapeutics LLC: Employment, Equity Ownership. Schmidt:Compass therapeutics LLC: Employment, Equity Ownership. Draghi:Compass therapeutics LLC: Employment, Equity Ownership.

Download Full-text

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

Blood ◽

10.1182/blood-2019-131540 ◽

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.

Download Full-text

Inducible MyD88/CD40 Allows AP1903-Dependent Costimulation to Control Proliferation and Survival of Chimeric Antigen Receptor-Modified T Cells

Blood ◽

10.1182/blood.v124.21.1121.1121 ◽

2014 ◽

Vol 124 (21) ◽

pp. 1121-1121 ◽

Cited By ~ 1

Author(s):

Aaron Foster ◽

Aruna Mahendravada ◽

Peter Chang ◽

Nicholas Shinners ◽

Kevin Slawin ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Cell Lines ◽

T Cell Activation ◽

Cell Activation ◽

First Generation ◽

Employment Equity ◽

Co Activation ◽

Equity Ownership

Abstract Introduction: Adoptive transfer of T cells genetically engineered to express chimeric antigen receptors (CARs) has begun to show impressive clinical results. The efficacy of T cell therapy is dependent not only on tumor recognition, but also on the survival and expansion of T cells following infusion. T cells modified with CAR constructs encoding costimulatory domains such as CD28 or 4-1BB have the capacity to rapidly proliferate in vivo, but severe toxicities have been observed due to unchecked T cell activation. Thus, strategies to regulate T cell activation in vivowould allow physicians to prevent toxicities and maximize anti-tumor efficacy. Here, we describe a novel T cell costimulation switch, inducible MyD88/CD40 (iMC), that can be activated by a small molecule chemical inducer of dimerization, AP1903, to enhance survival and drive T cell proliferation. Methods: T cells were activated with anti-CD3/28 antibodies and subsequently transduced with a biscistronic retrovirus encoding myristolated tandem AP1903 binding domains (FKBPv36), cloned in-frame with MyD88 and CD40 cytoplasmic signaling molecules, and truncated CD19 to identify transduced T cells (SFG-iMC.2A.ΔCD19). Control vectors without signaling elements, or with only MyD88 or cytoplasmic CD40 were also used to generate gene-modified T cell lines. iMC activation was measured by treating T cells with and without AP1903 and measuring cytokine production by ELISA, and assessing cell surface activation markers by flow cytometry. Co-activation of T cells through CD3ζ in combination with iMC was accomplished using anti-CD3 antibodies, or by co-transducing T cells with first generation CAR constructs recognizing prostate stem cell antigen or CD19 (PSCA.ζ or CD19.ζ, respectively), and coculturing T cells with PSCA+ (Capan-1) or CD19+ tumor cell lines (Raji, Daudi and Nalm-1) with and without AP1903. Efficacy of iMC-modified CAR T cells were assessed using NOD scid gamma (NSG) immune deficient mice engrafted with tumor cell lines followed by intravenous injection of T cells. The iMC costimulatory molecule was subsequently activated in vivo by intraperitoneal injection of AP1903 (5 mg/kg). Tumor burden was assessed and T cell expansion in vivowas measured by bioluminescent imaging using an IVIS instrument. Results: T cells transduced with iMC produce cytokines (e.g. IFN-γ, TNF-α, IL-6) in response to AP1903. iMC activation permits T cell survival in the absence of growth cytokines, such as IL-2, but by itself is not sufficient to induce IL-2 production or autonomous growth. Interestingly, AP1903-induction of MyD88 or cytoplasmic CD40 alone showed minimal T cell activation, suggesting potential synergy of the two signaling molecules. However, co-activation of the T cell receptor (TCR) with soluble anti-CD3 and iMC with AP1903 upregulated CD25 expression, induced IL-2 production and promoted T cell expansion. Importantly, endogenous TCR signaling could be substituted by a PSCA-specific CAR linked to the CD3 ζ endodomain (PSCA.ζ CAR), where co-activation of iMC by AP1903, and CAR by tumor cells expressing PSCA (Capan-1) induced high levels of IL-2 secretion, CD25 upregulation and rapid T cell proliferation. Similar results were achieved using T cells transduced with iMC-enabled CD19 CAR (SFG-iMC.2A.CD19.ζ) when cocultured with CD19+lymphoma cell lines. Treatment of tumor bearing immunodeficient mice with T cells modified with iMC and PSCA.ζ CAR showed enhanced antitumor efficacy when mice were administered with AP1903 dimerizer. Bioluminescence imaging also demonstrated marked proliferation and persistence of iMC-transduced T cells in response to AP1903 administration. Following AP1903 withdrawal, T cell levels declined, consistent with the requirement for costimulation in combination with CAR activation. Summary: Inducible MyD88/CD40 represents a novel activation switch that can be used to provide a controllable costimulatory signal to T cells transduced with a first generation CAR. The separation of the cytolytic signal 1 (CD3 ζ) domain from signal 2 costimulation (iMC) provides a unique mechanism by which T cells can be expanded only in response to both AP1903 and tumor antigen, or reduced in number by withdrawal of AP1903-induced iMC costimulation. Disclosures Foster: Bellicum Pharmaceuticals: Employment, Patents & Royalties. Mahendravada:Bellicum Pharmaceuticals: Employment. Chang:Bellicum Pharmaceuticals: Employment. Shinners:Bellicum Pharmaceuticals: Employment. Slawin:Bellicum Pharmaceuticals: Employment, Equity Ownership, Patents & Royalties. Spencer:Bellicum Pharmaceuticals: Employment, Equity Ownership, Patents & Royalties.

Download Full-text

Pre-Clinical Development of Adct-402, a Novel Pyrrolobenzodiazepine (PBD)-Based Antibody Drug Conjugate (ADC) Targeting CD19-Expressing B-Cell Malignancies

Blood ◽

10.1182/blood.v126.23.1564.1564 ◽

2015 ◽

Vol 126 (23) ◽

pp. 1564-1564 ◽

Cited By ~ 7

Author(s):

Francesca Zammarchi ◽

David G. Williams ◽

Lauren Adams ◽

Karin Havenith ◽

Simon Chivers ◽

...

Keyword(s):

Single Dose ◽

B Cell ◽

Cell Lines ◽

Xenograft Model ◽

B Cell Malignancies ◽

Employment Equity ◽

Equity Ownership ◽

Anti Tumor Activity

Abstract Human CD19 antigen is a 95 kilodalton type I transmembrane glycoprotein belonging to the immunoglobulin superfamily (Wang, Wei, & Liu, 2012). The role of CD19, both in health and disease, is well studied, and the therapeutic efficacy and safety of CD19 modulation have been well defined over several decades (Scheuermann & Racila, 1995). In normal human tissue, expression of CD19 is limited to the various stages of B-cell development and differentiation (except plasma cells) and its expression is maintained on the majority of B-cell malignancies, including B-cell leukemia and non-Hodgkin lymphomas of B-cell origin. CD19 has rapid internalization kinetics and it is not shed into the circulation (Blanc et al., 2011; Gerber et al., 2009). All these features make CD19 an attractive target for the development of an ADC to treat B-cell malignancies. ADCT-402 is an ADC composed of a humanized antibody directed against human CD19, stochastically conjugated via a valine-alanine cleavable, maleimide linker to a PBD dimer cytotoxin. PBD dimers are highly efficient anticancer drugs that covalently bind in the minor groove of DNA and form cytotoxic DNA interstrand cross-links. The average drug to antibody ratio of ADCT-402 is 2.3 ± 0.3, as shown by hydrophobic interaction chromatography and reverse-phase HPLC. In vitro, ADCT-402 demonstrated potent cytotoxicity in a panel of human-derived cell lines of differing levels of CD19, while its potency was strongly reduced in CD19-negative cell lines. In vivo, ADCT-402 demonstrated dose-dependent anti-tumor activity in a subcutaneously implanted human Burkitt's lymphoma-derived Ramos xenograft model, where a single dose at 0.33 mg/kg induced significantly delayed tumor growth compared to the vehicle-treated mice and at 0.66 mg/kg and 1 mg/kg gave 4/10 and 10/10 tumor-free survivors, respectively. In the same model, ADCT-402 showed remarkably superior anti-tumor activity compared to both maytansinoid- and auristatin-based CD19-targeting ADCs, when they were tested at the same dose and schedule (1 mg/kg, single dose). Moreover, ADCT-402 mediated an impressive increase in survival compared to both vehicle-treated and isotype control ADC-treated mice in the disseminated Ramos xenograft model when tested as a single dose at 0.33 mg/kg or 1 mg/kg. For example, a single dose of ADCT-402 at 1 mg/kg resulted in 10/10 survivors at day 91, while there were 0/10 survivors at day 19 in the group of animals treated with either the vehicle control or with a single dose of the non-binding, control ADC at 1 mg/kg. In rat, a single dose of ADCT-402 at 2 mg/kg was well tolerated with no adverse signs or hematologic effects. Altogether, these data show the potent and specific anti-tumor activity of ADCT-402 against CD19-expressing B-cell malignancies, both in vitro and in vivo, and warrant further development of this ADC into the clinic. Disclosures Zammarchi: ADC Therapeutics: Employment. Williams:Spirogen/Medimmune: Employment. Adams:Spirogen/Medimmune: Employment, Equity Ownership. Havenith:ADC Therapeutics: Employment. Chivers:ADC Therapeutics: Employment. D'Hooge:Spirogen/Medimmune: Employment, Equity Ownership. Howard:ADCT Spirogen/Medimmune: Employment, Equity Ownership, Patents & Royalties. Hartley:ADCT Spirogen/Medimmune: Employment, Equity Ownership, Membership on an entity's Board of Directors or advisory committees. van Berkel:ADC Therapeutics: Employment, Equity Ownership, Patents & Royalties.

Download Full-text

Targeting dendritic cells with a PD-L1 based bispecific antibody rejuvenates specific anti-tumor T cells

10.21203/rs.3.rs-39214/v1 ◽

2020 ◽

Author(s):

Longchao Liu ◽

Jiahui Chen ◽

Joonbeom Bae ◽

Zhida Liu ◽

Eric Hsu ◽

...

Keyword(s):

T Cells ◽

Dendritic Cells ◽

T Cell ◽

Bispecific Antibody ◽

Lymphoblastic Leukemia ◽

Severe Toxicity ◽

Dependent Manner ◽

Therapeutic Doses ◽

Anti Tumor Activity

Abstract Bispecific T-cell engagers (BiTEs) that preferentially target tumor-associated antigens (TAA) to reengage CD3 signaling have been approved to treat acute B-cell lymphoblastic leukemia. However, their applications in solid tumors have been hampered due to short half-life, weak anti-tumor activity, and severe toxicity at therapeutic doses. To explore new targets, we designed a bispecific antibody (BsAb) which simultaneously targets CD3 and immune checkpoint PD-L1. Compared with conventional TAA based targeting, PDL1xCD3 generates far superior anti-tumor immune responses in vivo. Mechanistically, blockade of PD-L1 on dendritic cells instead of tumor cells can potently rejuvenate preexisting tumor reactive CD8 T cells in a B7-1/2 dependent manner for a durable anti-tumor responses. This study argues that targeting DC-T cell instead of current tumor-T cell can achieve much better T cell rejuvenation in BsAb therapy.

Download Full-text

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

Blood ◽

10.1182/blood-2019-131586 ◽

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.

Download Full-text

CC-92480 Is a Novel Cereblon E3 Ligase Modulator with Enhanced Tumoricidal and Immunomodulatory Activity Against Sensitive and Resistant Multiple Myeloma Cells

Blood ◽

10.1182/blood-2019-124338 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 1812-1812 ◽

Cited By ~ 3

Author(s):

Antonia Lopez-Girona ◽

Courtney G. Havens ◽

Gang Lu ◽

Emily Rychak ◽

Derek Mendy ◽

...

Keyword(s):

Cell Lines ◽

Acquired Resistance ◽

E3 Ligase ◽

Immunomodulatory Activity ◽

Employment Equity ◽

Ic50 Value ◽

Ic50 Values ◽

Equity Ownership ◽

Induction Of Apoptosis ◽

Low Levels

Lenalidomide- and pomalidomide-based therapies are effective drugs in the treatment of patients with multiple myeloma (MM), however most patients with MM eventually relapse or become resistant. CC-92480, a novel cereblon (CRBN) E3 ligase modulator (CELMoD) with multiple activities including potent immunomodulation and single-agent antiproliferative effects, is being investigated in a phase 1 clinical trial (CC-92480-MM-001; NCT03374085) for patients with relapsed/refractory MM (RRMM). The present study investigates the preclinical data and mechanism of action of CC-92480 in MM models. CELMoD agents bound to CRBN confer differentiated substrate-degradation specificity on the CRL4CRBN E3 ubiquitin ligase. CRBN-modulator agents mediate destruction of Ikaros and Aiolos, transcription factors that contribute to myeloma cell survival. CC-92480 was found to produce rapid, deep, and sustained degradation of Ikaros and Aiolos, with superior antimyeloma activity. Accordingly, in a CRBN protein competitive binding assay, CC-92480 displaced a Cy-5-labeled CELMoD analog from CRBN with a 50% inhibitory concentration (IC50) value of 0.03 μM, whereas lenalidomide competed with an IC50 value of 1.27 μM in the same assay, demonstrating a higher binding affinity of CC-92480 for CRBN. Additionally, CC-92480 promoted the recruitment of Ikaros to the CRBN E3 ligase complex more effectively than pomalidomide in 2 orthogonal CRBN/Ikaros binding assays; it also triggered a more extensive cellular ubiquitination of Ikaros, and a faster, more efficient depletion of cellular Ikaros and Aiolos than pomalidomide. In various MM cell lines, including those with acquired resistance to lenalidomide or pomalidomide and low levels of CRBN, CC-92480 produced robust degradation of Ikaros and Aiolos followed by strong reduction of 2 additional and highly critical transcription factors, c-Myc and interferon regulatory factor 4, which are linked to the induction of apoptosis as measured by cleaved caspase-3. The tumoricidal activity of CC-92480 was shown to be CRBN dependent, since the effect was prevented by complete loss of CRBN or by the stabilization of Ikaros and Aiolos. CC-92480 displayed broad and potent antiproliferative activity across a panel of 20 MM cell lines that are either sensitive, have acquired resistance, or are refractory to lenalidomide or pomalidomide; the cell lines also contained diverse chromosomal translocations and oncogenic drivers typically found in MM patients. Approximately half of the MM cell lines evaluated were highly sensitive to CC-92480, with IC50 values for antiproliferative activity ranging from 0.04 to 5 nM; only 2 cell lines had IC50 values > 100 nM. CC-92480 inhibits cell proliferation and induces apoptosis in MM cell lines that are not sensitive to lenalidomide or pomalidomide. This panel of cell lines includes both refractory cell lines and resistant cell lines generated through continuous exposure to lenalidomide and pomalidomide that acquired low levels of CRBN protein or mutations in the CRBN gene. CC-92480 also induced deep destruction of Ikaros and Aiolos in cultures of peripheral blood mononuclear cells (PBMCs), which led to the activation of T cells and increased production of the cytokines interleukin-2 and interferon gamma. These responses occurred at the range of CC-92480 concentrations that show potent tumoricidal effect against MM cells. The T cell activation and enhanced cytokine production by CC-92480 led to the potent and effective immune-mediated killing of MM cells in co-cultures with PBMCs. CC-92480 is a potent antiproliferative and proapoptotic novel CELMoD with enhanced autonomous cell-killing activity in MM cells that are either sensitive, resistant, or have acquired resistance to lenalidomide and pomalidomide. CC-92480 has a unique and rapid degradation profile stemming from the enhanced efficiency to drive the formation of a protein-protein interaction between Ikaros and Aiolos and CRBN, inducing cytotoxic effects in a CRL4CRBN-dependent fashion that leads ultimately to the induction of apoptosis, even in the context of low or mutated CRBN protein. Additionally, similar to lenalidomide, CC-92480 conserves immunomodulatory activity against MM cells. These data support the clinical investigation of CC-92480 in patients with RRMM. Disclosures Lopez-Girona: Celgene Corporation: Employment. Havens:Pfizer: Employment, Equity Ownership; Celgene: Equity Ownership. Lu:Celgene Corporation: Employment, Equity Ownership. Rychak:Celgene Corporation: Employment, Equity Ownership. Mendy:Celgene Corporation: Employment. Gaffney:Celgene: Employment. Surka:Celgene: Employment, Equity Ownership. Lu:Celgene Corporation: Employment, Equity Ownership. Matyskiela:Celgene corporation: Employment. Khambatta:Celgene: Employment. Wong:Celgene Corporation: Employment, Equity Ownership. Hansen:Celgene Corporation: Employment. Pierce:Celgene Corporation: Employment, Equity Ownership. Cathers:Global Blood Therapeutics (GBT): Employment; Celgene Corporation: Equity Ownership. Carmichael:Celgene plc: Employment, Equity Ownership.

Download Full-text

PD-L1 Checkpoint Blockade Augments Anti-Tumor Immune Response of GD2 or HER2-Bsab Ex Vivo Armed T-Cells (EVAT) Therapy in Osteosarcoma

Blood ◽

10.1182/blood-2019-131325 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 1959-1959

Author(s):

Jeong A Park ◽

Hong fen Guo ◽

Hong Xu ◽

Nai-Kong V. Cheung

Keyword(s):

Immune Response ◽

T Cells ◽

T Cell ◽

Immune Checkpoint ◽

Bispecific Antibody ◽

Ex Vivo ◽

Activated T Cells ◽

The Impact ◽

Anti Tumor Activity

Background Ex Vivo Armed T-cells (EVAT) carrying zeptomoles (10-21M) of T-cell engaging GD2-bispecific antibody (GD2-EVAT) or HER2-bispecific antibodies (HER2-EVAT) have potent anti-tumor activity against GD2(+) and/or HER2(+) solid tumors. Strategies to further optimize this approach are highly relevant. PD-1 is a key immune checkpoint receptor expressed mainly by activated T-cells and mediates immune suppression by binding to its ligands PD-L1 or PD-L2. Upregulation of PD-L1 has been found in many cancers including osteosarcoma and associated with aggressive disease and poor outcome. While the use of immune checkpoint inhibitors (ICIs) seems logical, the ideal timing when combined with T-cell engaging bispecific antibody (T-BsAb) or EVAT has yet to be defined. Here, we described the effects of anti-PD-1 or anti-PD-L1 antibodies on GD2-EVAT or HER2-EVAT therapy and explored the impact of its timing in the treatment of osteosarcoma which is GD2(+), HER2(+) and PD-L1(+). Methods GD2-BsAb and HER-BsAb were built using the IgG(L)-scFv format (Can Immunol Res, 3:266, 2015, Oncoimmunology, PMID:28405494). T-cells from healthy volunteer donors were isolated, and cultured ex vivo in the presence of CD3/CD28 beads plus 30 IU/mL of interleukin 2 (IL-2). Between day 7 and day 14, activated T-cells (ATCs) were harvested and armed for 20 minutes at room temperature with GD2-BsAb or HER2-BsAb. In vivo anti-tumor activity against GD2(+), HER2(+), and PD-L1(+) osteosarcoma cell line xenografts was tested in BALB-Rag2-/-IL-2R-γc-KO mice. Anti-human PD-1 antibody (pembrolizumab, anti-PD-1) or anti-human PD-L1 antibody (atezolizumab, anti-PD-L1) were tested for synergy with GD2-EVAT or HER2-EVAT therapy. Results The PD-1 expression increased among T-cells that circulated in the blood, that infiltrated the spleen or the tumor after EVAT therapy. While anti-PD-L1 combination therapy with GD2-EVAT or HER2-EVAT improved anti-tumor response against osteosarcoma (P=0.0123 and P=0.0004), anti-PD-1 did not (all P>0.05). The addition of anti-PD-L1 significantly increased T-cell survival in blood and T-cell infiltration of tumor when compared to GD2-EVAT or HER2-EVAT alone (all P<0.0001). Treatment of GD2-EVAT or anti-PD-L1 plus GD2-EVAT downregulated GD2 expression on tumors, but anti-PD-1 plus GD2-EVAT did not. For the next step we tested the impact of different combination schedules of ICIs on GD2-EVAT therapy. Concurrent anti-PD-1 (6 doses along with GD2-EVAT therapy) interfered with GD2-EVAT, while sequential anti-PD-1 (6 doses after GD2-EVAT) did not make a significant effect (P>0.05). On the other hand, while the concurrent use of anti-PD-L1 did not show benefit on GD2-EVAT, sequentially administered anti-PD-L1 produced a significant improvement in tumor control when compared to anti-PD-L1 or GD2-EVAT alone (P=0.002 and P=0.018). When anti-PD-L1 treatment was extended (12 doses after GD2-EVAT), the anti-tumor effect was most pronounced compared to GD2-EVAT alone (P <0.0001), which translated into improved survival (P=0.0057). These in vivo anti-tumor responses were associated with increased CD8(+) tumor infiltrating lymphocytes (TILs) of tumor. Conclusion In the arming platform, large numbers of target-specific T-cells can be generated, and this EVAT therapy is a highly effective cellular treatment with high potency in preclinical models. In addition, the advantage of ex vivo cytokine release following T-cell arming and activation could reduce or avoid life threatening cytokine storm if such activation was to proceed in vivo. Adoptive T-cell therapy induced immune response upregulates the inhibitory immune checkpoint PD-1/PD-L1 pathway, and combination treatment with anti-PD-L1 antibody, especially when combined as sequential therapy and continuously treated, significantly improved anti-tumor effect of EVAT, partly through increase in CD8(+) TILs infiltration. Disclosures Xu: MSK: Other: co-inventors in patents on GD2 bispecific antibody and HER2 bispecific antibody. Cheung:Ymabs: Patents & Royalties, Research Funding.

Download Full-text