Positive Tumor Cell Lines Research Articles

Generation and Characterization of SORT1-Targeted Antibody-Drug Conjugate for the Treatment of SORT1-Positive Breast Tumor.

Antibody-drug conjugates (ADCs) have greatly improved the outcomes of advanced breast tumors. However, the treatment of breast tumors with existing ADCs is still hindered by many issues, such as tumor antigen heterogeneity and drug resistance. Therefore, ADCs against new targets would provide options for the treatment of these challenges. Sortilin-1 (SORT1) may be a promising target for ADC as it is upregulated in breast cancer. To evaluate the possibility of SORT1 as an ADC target, a humanized antibody_8D302 with high affinity against SORT1 was generated. Additionally, 8D302 was conjugated with MMAE and DXd to generate two ADCs_8D302-MMAE and 8D302-DXd, respectively. Both 8D302-MMAE and 8D302-DXd showed effective cytotoxicity against SORT1 positive breast tumor cell lines and induced bystander killing. Consequently, 8D302-MMAE showed relatively better anti-tumor activity than 8D302-DXd both in vitro and in vivo, but 8D302-DXd had superior safety profile and pharmacokinetics profile over 8D302-MMAE. Furthermore, SORT1 induced faster internalization and lysosomal trafficking of antibodies and had a higher turnover compared with HER2. Also, 8D302-DXd exhibited superior cell cytotoxicity and tumor suppression over trastuzumab-DXd, a HER2-targeted ADC. We hypothesize that the high turnover of SORT1 enables SORT1-targeted ADC to be a powerful agent for the treatment of SORT1-positive breast tumor.

Abstract 2961: HLA-G-TCB (RG6353), a T cell bispecific antibody for the treatment of solid tumors in monotherapy and combination with immunomodulator agents

Abstract T-cell bispecific antibodies (TCB) represent a class of therapeutic agents that specifically activate T cells to attack and kill tumor cells. TCBs have demonstrated efficacy in the clinic and are also approved. TCB mediated efficacy in solid tumors, however, has been more challenging to demonstrate. HLA-G-TCB specifically targets human leukocyte antigen G (HLA-G) on tumor cells and cluster of differentiation 3 epsilon chain (CD3ε) expressed on T cells. HLA-G is expressed in a broad range of solid tumor indications and its role in mediating immune suppression makes it an attractive cancer immunotherapy target. Anti-tumor activity of HLA-G-TCB was demonstrated in vitro using different HLA G positive tumor cell lines. T-cell activation, IFN𝛾; secretion, and cytotoxicity induced by HLA G-TCB were shown to be dose dependent and correlating to the HLA G density on cell surface and the percentage of HLA G positive cells. Administration of HLA-G TCB as monotherapy (5mg/kg) to hematopoietic stem cell-humanized NSG mice (Humice) bearing SKOV3 HLA-G tumors resulted in strong tumor growth inhibition resulting in tumor-free animals (7 out of 8). Additionally, in a patient derived xenograft (PDX) tumor model with physiological (low/patchy) HLA-G expression, a significant dose-dependent efficacy ranging from 10-90% was achieved in monotherapy (0.05-5 mg/kg). Treatment of Humice bearing PDX tumors with a combination of HLA-G-TCB and a co-stimulatory immunomodulator resulted in remarkable anti-tumor activity resulting in tumor eradication in 100% of the mice treated with the combination (8 out of 8). A strong synergistic effect was also observed in checkpoint inhibitor resistant PDX tumor model, also resulting in tumor-free mice (5 out of 8) treated with a combination of HLA-G TCB and an immunomodulatory bispecific antibody. In line with the mode of action of TCBs, tumor growth inhibition was accompanied by increased cytokine secretion (including IFN𝛾;), tumoral T cell infiltration and activation as reflected by increased expression of T cell activation markers including CD69, CD25 and Granzyme B. Evaluation of HLA-G expression on tumor cell lines upon IFN𝛾; stimulation in vitro and in PDX tumors treated with HLA-G TCB revealed that HLA-G expression can be upregulated upon IFN𝛾; stimulation and TCB treatment respectively and may positively impact the anti-tumor activity of HLA-G-TCB in vivo. These promising preclinical findings reflect the potential and support the clinical evaluation of HLA-G TCB in treating patients with solid tumors in monotherapy and in combination. HLA-G TCB (RG6353) is being advanced to the clinic in BP44068, an open-label, multicenter, phase 1 study to evaluate safety, pharmacokinetics, and preliminary anti-tumor activity in patients with unresectable and/or metastatic HLA-G expressing solid tumors. Citation Format: Meher Majety, Carina Hage, Alexander Bujotzek, Anneliese Schneider, Martin Lechmann, Nitya Nair, Stefan Zimmerman, Anthony Morel, Christian Klein, Pablo Umana. HLA-G-TCB (RG6353), a T cell bispecific antibody for the treatment of solid tumors in monotherapy and combination with immunomodulator agents [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2961.

Abstract 1521: Unlocking the therapeutic index of highly potent site-specific ADCs in solid tumors

Abstract CD276 (B7-H3) is a transmembrane protein of the B7 family, which includes many prominent immune checkpoint targets, and is expected to play a role in T-cell stimulation, tumor-infiltration, as well as other tumorigenic functions with poor prognosis when upregulated. CD276 is highly expressed in solid tumors, cancer initiating stem cells, and tumor associated vasculature with minimal to no expression in healthy tissue. This promising expression profile has piqued interest for various targeted therapeutic modalities, such as antibody-drug conjugates (ADCs). Developing effective ADCs for solid tumors remains a challenge due to inefficient conjugation technologies which are either non-homogeneous, unstable, or do not position payloads at optimal sites. BrickADCs minimize hydrophobicity, optimize payload release kinetics, and tune biophysical properties to improve efficacy, safety, and bioavailability, resulting in improved therapeutic indexes. Potent payloads such as pyrrolobenzodiazepines have been underutilized due to toxicity, preventing their widespread use. BrickBio’s unique bioconjugation methodology enables precise (site-specific), flexible (unfettered site accessibility and chemistry), efficient, and scalable generation of ADCs which overcome these limitations. In this work, UAAs (unnatural amino acids) were incorporated into full-length anti-CD276 antibodies and conjugated with optimized pyrrolobenzodiazepine (PBD) payloads. BrickADCs leverage a proprietary Site-Select Panel to find optimal sites that shield the toxicity of highly potent, hydrophobic PBD payloads while maintaining their high activity to generate anti-CD276-UAA-PBD ADCs. The highest performing BrickADCs, developed by a rapid screen of PBD payloads conjugated to different sites in the antibody backbone, exhibit high therapeutic efficacy in a variety of CD276 positive solid tumor cell lines (eg. colon, ovarian, lung, sarcoma, etc) and in mouse xenograft models with a 3x differential in complete response. The tuned hydrophobicity and release kinetics have resulted in ADCs that have an increased therapeutic index, particularly due to their improved safety profile compared to cysteine conjugation.The BrickADC platform enables an increased therapeutic window for potent payloads across solid tumors, opening the floodgates for further development of ADCs which leverage payloads that have been plagued by poor therapeutic index. Continued work is underway for the CD276 pipeline candidates as they progress towards IND, as well as investigation into other targets and conjugate modalities. Citation Format: James S. Italia, Colby A. Souders, Sreedhar Reddy Suthe, Myer Hussain, Zhi Li, John Boyce, Audrey Warner, Abhishek Chatterjee. Unlocking the therapeutic index of highly potent site-specific ADCs in solid tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1521.

Construction of CAR-T cells targeting TM4SF1 and its anti-tumor capacity in ovarian cancer

Ovarian cancer (OC) is the most lethal gynecological malignancy with a 5-year survival rate of 49.1% on average. In clinical practice, cytoreduction and chemotherapy remain the conventional treatment for advanced OC. However, the overall prognosis remains poor, which urges oncologists to develop new treatments. Chimeric antigen receptor (CAR)-T therapy as a branch of immunotherapy had gained a success in treating hematological malignancies. TM4SF1, a potential biomarker in many tumors, was validated highly expressed in ovarian cancer. Here we constructed a 3rd generation CAR-T agent targeting TM4SF1 to treat ovarian cancer. CAR-T cells showed a specific cytotoxicity against TM4SF1 positive tumor cell lines in vitro and repressed SKOV3-derived tumor growth in vivo. This is the first time reporting a CAR-T therapy targeting TM4SF1 in ovarian cancer. Our results suggested that TM4SF1 could be a very promising target in curing OC and showed the possibility of TM4SF1-based immunotherapy.

Abstract 3434: APVO442 is a distinct PSMA x CD3 targeted bispecific candidate designed to optimize T cell fitness and distribution to solid tumors

Abstract APVO442 is a bispecific therapeutic candidate targeting PSMA and CD3 for redirection of T cell responses against PSMA-expressing solid tumors. The candidate bispecific was designed to have unique pharmacokinetic and safety properties to potentially maximize robust anti-tumor responses. APVO442 is designed with Aptevo’s ADAPTIR-FLEX࣪ technology to generate a molecule with low-affinity monovalent CD3 engagement, paired with high-affinity bivalent PSMA binding intended to be tumor - dependent while inducting optimal T-cell stimulation within the tumor microenvironment. The unique engineering of APVO442 reduces the potential peripheral CD3 T cell binding, minimizing the potential for on-target toxicity, such as peripheral cytokine release, while simultaneously increasing the likelihood to deliver an effective concentration of APVO442 locally within solid tumors. Preclinical data demonstrate that APVO442 is able to activate T cells and mediate directed cytotoxicity against PSMA-expressing tumor targets with comparable potency to α-PSMA x α-CD3 constructs with varying CD3 affinity or avidity both in vitro and in vivo. The CD3 activity is dependent on PSMA crosslinking as activity is not observed in the absence of tumor target, in line with the silenced Fc engineered into APVO442. Importantly, cytokine release was not observed in peripheral immune subsets in the absence of tumor targets, despite a beneficial cytokine profile observed in the presence of PSMA positive tumor cell lines. The unique biology elicited by APVO442 induces distinct kinetic activation of downstream transcriptional regulators following monovalent TCR engagement of APVO442 associated with reduced early signaling, but overall, a more sustained activation than higher affinity variants. In addition, APVO442 induces a distinct phenotypic profile on human T cells associated with optimal function and memory responses only in the presence of tumor targets. Further, combination of APVO442 with additional costimulatory agonists supports improved T cell activation and anti-tumor cytotoxicity in vitro. These data support the potential for unique clinical combination of APVO442 with immuno-oncology modalities that may support improved benefit in PSMA positive indications such as metastatic castration resistant prostate cancer. APVO442 is undergoing further preclinical in vitro and in vivo characterization in support of the IND-enabling package. Citation Format: Rebecca Gottschalk, Robert Miller, Brian Woodruff, Ashly Lucas, Elizabeth Haglin, Peter Pavlik, Michelle H. Nelson, Hilario J. Ramos. APVO442 is a distinct PSMA x CD3 targeted bispecific candidate designed to optimize T cell fitness and distribution to solid tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3434.

Abstract 609: Anti-HER3 radioimmunotherapy enhances the anti-tumor effects of CD47 blockade in solid tumors

Abstract Background: Cancer immunotherapy strategies targeting blockade of the CD47-SIRPα immunosuppressive signal have made significant progress in recent years. However, monotherapies have not shown meaningful clinical responses in solid tumors. Therefore, therapeutic combinations are being explored to improve patient outcomes. CD47 is a macrophage checkpoint inhibitor that acts as a “don’t eat me” signal on cancer cells to evade innate immune detection and destruction. Targeted radiation to cancer cells will upregulate calreticulin (CRT), a pro-phagocytic “eat me” signal. We therefore hypothesize that we can enhance the efficacy of anti-CD47 antibodies by combining them with appropriate targeted antibody radioconjugates (ARC). In this experiment we chose to study an anti-HER3 radioconjugate, as HER3 is overexpressed in a variety of cancers including breast, ovarian, lung, gastric and prostate and is associated with poor clinical prognosis. Additionally, upregulation of HER3 is implicated in the acquired resistance against HER1 or HER2 targeted therapies. Here, we demonstrate enhanced therapeutic efficacy of a novel Actinium-225 (225Ac) armed HER3 specific targeting ARC (225Ac-HER3-ARC) and a CD47 blocking antibody (anti-CD47) combination in preclinical solid tumor models. Methods: The anti-HER3 antibody (AT-02) was radiolabeled with 225Ac. 225Ac-HER3-ARC biological activity was evaluated using human recombinant HER3 and receptor positive tumor cell lines. 225Ac-HER3-ARC mediated CRT upregulation and cytotoxicity was evaluated using flow cytometry and MTS assay, respectively. The benefits of the 225Ac-HER3-ARC and anti-CD47 combination to enhance macrophage phagocytosis was evaluated by flow cytometry. We further evaluated the therapeutic benefits of the 225Ac-HER3-ARC and CD47 combination in human HER3+ tumor xenograft mouse model. Results: The 225Ac-HER3-ARC retains similar binding properties to native antibody and demonstrates specific cytotoxicity on tumor cells. CRT was upregulated by 225Ac-HER3-ARC in HER3+ cells. Furthermore, the combination of 225Ac-HER3-ARC and anti-CD47 enhances in vitro macrophage mediated tumor cell phagocytosis compared to each agent alone. Importantly, the in vivo 225Ac-HER3-ARC and CD47 antibody combination shows enhanced antitumor effect with reduced toxicity and improved survival benefit in a human preclinical solid tumor model compared to anti-CD47 agent alone. Conclusions: We demonstrate enhanced efficacy of the 225Ac-HER3-ARC and CD47 blocking antibody combination in vitro and in a preclinical solid tumor animal model. This approach is an encouraging strategy to potentially improve antitumor responses in patients with HER3+ tumors. Consequently, the findings obtained in this study along with the need to develop better therapies for patients with HER3+ tumors support the further preclinical development of HER3-ARC. Citation Format: Denis Beckford-Vera, Jason Li, Caroline Jennings, Megan McCloskey, Amanda Chin, Qing Liang, Jesse Hwang, Monideepa Roy, Mary Chen, Helen Kotanides. Anti-HER3 radioimmunotherapy enhances the anti-tumor effects of CD47 blockade in solid tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 609.

A Phase I Study of CD19 Chimeric Antigen Receptor-T Cells Generated By the PiggyBac Transposon Vector for Acute Lymphoblastic Leukemia

Introduction: Chimeric antigen receptor-modified T cells targeting CD19 (CD19.CAR-T cells) have shown clinical success in patients with hematological malignancies. Despite the encouraging results obtained with this novel therapy, a major concern to its global spread, particularly in developing countries, is its high cost. We developed a method of non-viral gene transfer using piggyBac transposon to reduce the cost of CAR-T therapy. In preclinical study, the median number and transduction efficiency of CAR-T cells obtained from 2x10 7 PBMC in 9 donors were 1.0x10 8 (range, 0.58-1.8x10 8) and 51% (range, 29-73%), respectively. The major subset of CAR-T cells was phenotypically CD8+CD45RA+CCR7+, closely related T-memory stem cells. Ex vivo, CD19.CAR-T cells showed cytotoxic effect on CD19 positive tumor cell lines. In NSG mice model, CD19.CAR-T cells successfully inhibit tumor growth. CAR gene integration sites were determined by inverse polymerase chain reaction and subsequent next-generation sequencing using MiSeq and equally distributed throughout the genome without preference for specific sites. The pre-clinical testing in mouse demonstrated safe toxicity profile at the 50 times dose of CD19.CAR-T cells. We started a human clinical trial to define feasibility, toxicity, maximum tolerated dose and clinical response of CD19.CAR-T cells (jRCTa040190099).Methods: We report the results of cohort 1 of the study in which the safety and efficacy of autologous CD19.CAR-T in patients with relapsed or refractory B-precursor acute lymphoblastic leukemia were evaluated. We engineered autologous T cells via the piggyBac transposon system with CD19.CAR-expression transposon vector and piggyBac transposase-expression vector to express CD19.CAR incorporating CD28 costimulatory domain. We designed this phase I trial using a modified 3 + 3 design to enroll 3-12 patients with relapsed or refractory acute lymphoblastic leukemia in both children and adults. In this study, patients in cohorts 1 (16-60 years old) and 2 (1-15 years old) receive 1 × 10 5 CAR-transduced T cells per kg. Patients in cohorts 3 and 4 (both 1-60 years old) receive 3 × 10 5 and 1 × 10 6 CAR-transduced T cells per kg, respectively. All patients receive 25mg/m 2/d of fludarabine and 250mg/m 2/d of cyclophosphamide for 3 days followed by a single infusion of CAR-T cells.Results: Three patients were enrolled in cohort 1 and infused with 1 × 10 5 CAR-transduced T cells per kg. All patients had previously undergone allogeneic hematopoietic stem cell transplantation. All patients had achieved a hematological complete response with salvage treatment before CAR-T therapy. None of the patients had dose-limiting toxicities (DLT) defined as nonhematological toxicities above grade 4 or cytokine release syndrome (CRS) above grade 4 or graft versus host disease (GVHD) above 4, or grade 3 nonhematological toxicities and GVHD not improved to grade 2 within 4 weeks after CAR-T infusion. There was no occurrence of non-hematological adverse events above grade 3. CRS was observed in one patient (grade 1) who also developed headache due to infiltration of CAR-T cells into the spinal fluid. In two patients, B cell aplasia lasted 2 and 9 months, respectively. Elevation of serum cytokine levels was observed in all patients and the peak time point was 7-21 days after CAR-T cell infusion.Conclusions: CD19.CAR-T cell infusion produced by the piggyBac transposon gene engineering system was safe in cohort 1 of our study. As no patients had DLT in cohort 1, we are enrolling the patients in further cohorts. DisclosuresMurata: MSD: Honoraria; Kyowa Kirin: Honoraria; Sumitomo Dainippon Pharma: Honoraria; FUJIFILM: Honoraria; Toyama Chemical: Honoraria; Novartis: Honoraria; JCR Pharmaceutical: Honoraria; Astellas: Honoraria; Miyarisan Pharmaceutical: Honoraria; Asahi Kasei: Honoraria; GlaxoSmithKline: Honoraria; Celgene: Honoraria; Otsuka Pharmaceutical: Honoraria.

Genotyping and Characterization of HPV Status, Hypoxia, and Radiosensitivity in 22 Head and Neck Cancer Cell Lines.

Simple SummaryCell lines are widely used for research, but even commonly used cell lines may not be well characterized, making validation of published data and generalizing the results to the clinic problematic. Here, we have established a head and neck squamous cell carcinomas (HNSCC) cell line database. We believe that these cell lines are suitable models to study HNSCC disease in vitro and in vivo and may provide a valuable tool for investigators wishing to study the relation between e.g., hypoxia and radiosensitivity in HNSCC.To study head and neck squamous cell carcinomas (HNSCC) in vitro, a large variety of HNSCC cell lines have been developed. Here, we characterize a panel of 22 HNSCC cell lines, thereby providing a tool for research into tumor-specific treatment options in HNSCC. Both human papillomavirus (HPV) positive and HPV negative tumor cell lines were collected from commercial and collaborative sources. Short tandem repeat profiling was used to confirm or characterize the identity of the cell lines. Targeted sequencing was performed using a standard pathology single molecule Molecular Inversion Probe panel to detect mutations for 23 tumor suppressors and oncogenes. HPV status, p16 status, radiosensitivity data, and hypoxia data are summarized from all cell lines. We detected HPV transcripts in five cell lines, all of which overexpressed p16. One HPV negative cell line was also p16 positive. We detected mutations in KIT (SCCNij185), PIK3CA (SCCNij185), and CDKN2A (UT-SCC-5 and UT-SCC-38). TP53 mutations were the most frequent, occurring in 16/22 cell lines. HPV infection and TP53 mutations were almost mutually exclusive, with the exception of 93-VU-147T. The cell lines exhibited a wide range of sensitivities towards hypoxia and irradiation. Here, we provide a description of a set of frequently used HNSCC cell lines with diverse characteristics as found in HNSCC patients.

Preparation of a novel tri-specific T cell engager targeting CD19 antigen and its anti-leukemia effect exploration

目的制备一种新的靶向CD19的三特异性T细胞衔接器融合蛋白（19TriTE），研究其对CD19阳性血液肿瘤的免疫治疗作用。方法通过分子克隆技术构建19TriTE表达质粒，并通过真核蛋白表达系统成功表达该融合蛋白。体外实验验证19TriTE对T细胞的活化与增殖作用及介导T细胞发挥对CD19阳性肿瘤细胞的特异性细胞毒作用。结果①成功构建19TriTE融合蛋白表达质粒，并通过真核蛋白表达系统成功表达。②19TriTE可以特异性结合T细胞和Nalm6细胞，与T细胞的平衡解离常数为19.21 nmol/L，与Nalm6细胞的平衡解离常数为11.67 nmol/L。③2 nmol/L浓度的19TriTE与T细胞及Nalm6细胞共培养时，T细胞中CD69阳性表达率为35.4％，CD25阳性表达率为49.8％，较对照组显著升高。④19TriTE在浓度为1 nmol/L时即可显著促进T细胞增殖，第12天时，T细胞绝对计数由初始1×106扩增至7.4×107，增殖74倍。⑤19TriTE融合蛋白能明显介导T细胞杀伤CD19阳性靶细胞且与剂量呈正相关，浓度为10 nmol/L时，靶细胞裂解率达50％。⑥脱颗粒实验验证，CD19阳性靶细胞存在时，19TriTE可以显著激活T细胞且与剂量呈正相关。⑦将19TriTE融合蛋白分别与过表达RFP及Luciferase基因的靶细胞及T细胞共培养，19TriTE能够介导T细胞杀伤CD19阳性的靶细胞。结论本研究成功构建及表达19TriTE融合蛋白，体外实验验证其可以有效活化T细胞，并促进T细胞增殖。同时，能够结合CD19阳性靶细胞和T细胞，促进T细胞发挥体外抗白血病作用，为进一步临床研究奠定基础。

Preclinical Development of MGC018, a Duocarmycin-based Antibody–drug Conjugate Targeting B7-H3 for Solid Cancer

B7-H3, also referred to as CD276, is a member of the B7 family of immune regulatory proteins. B7-H3 is overexpressed on many solid cancers, including prostate cancer, renal cell carcinoma, melanoma, squamous cell carcinoma of the head and neck, non-small cell lung cancer, and breast cancer. Overexpression of B7-H3 is associated with disease severity, risk of recurrence and reduced survival. In this article, we report the preclinical development of MGC018, an antibody-drug conjugate targeted against B7-H3. MGC018 is comprised of the cleavable linker-duocarmycin payload, valine-citrulline-seco duocarmycin hydroxybenzamide azaindole (vc-seco-DUBA), conjugated to an anti-B7-H3 humanized IgG1/kappa mAb through reduced interchain disulfides, with an average drug-to-antibody ratio of approximately 2.7. MGC018 exhibited cytotoxicity toward B7-H3-positive human tumor cell lines, and exhibited bystander killing of target-negative tumor cells when cocultured with B7-H3-positive tumor cells. MGC018 displayed potent antitumor activity in preclinical tumor models of breast, ovarian, and lung cancer, as well as melanoma. In addition, antitumor activity was observed toward patient-derived xenograft models of breast, prostate, and head and neck cancer displaying heterogeneous expression of B7-H3. Importantly, MGC018 exhibited a favorable pharmacokinetic and safety profile in cynomolgus monkeys following repeat-dose administration. The antitumor activity observed preclinically with MGC018, together with the positive safety profile, provides evidence of a potentially favorable therapeutic index and supports the continued development of MGC018 for the treatment of solid cancers. GRAPHICAL ABSTRACT: http://mct.aacrjournals.org/content/molcanther/19/11/2235/F1.large.jpg.

Abstract 1070: AUTO7: Anti-PSMA humanized CAR T-cell with improved persistence and resistance to tumor microenvironment for metastatic castration resistant prostate cancer (mCRPC)

Abstract Background. Prostate cancer (PCa) is the most common cancer in men and metastatic disease has no curative options. Here we describe a combinatorial/multi-modular CAR T therapeutic for prostate cancer termed AUTO7. The therapeutic consists of 6 functional modules. The encoded modules are: 1) an anti-PSMA CAR based on a novel humanized binder; 2) the safety switch; 3) dominant negative TGFβRII to induce TGFβ1 resistance (dnTGFβRII); 4) truncated SHP2 (dSHP2) for PD1/PD-L1 pathway inhibition; 5) a constitutively active IL-7 receptor (IL7R_CCR) to induce proliferation and finally 6) an IL-12 module (IL12) for lymphocyte recruitment/activation. Methods. PSMA-binder was generated by CDR grafting of an anti-PSMA antibody derived from genetically vaccinated rats. CAR T-cells were generated by transduction of T-cells with vectors containing the respective modules. CAR T-cells were evaluated in vitro in co-culture assays with PSMA positive cell lines for redirected lysis, cytokine secretion, T cell proliferation, resistance to immunosuppression and IL-12 expression. Antitumor activity of AUTO7 containing the CAR and all 6 modules was assessed in vivo in an established human xenograft in NSG mice. Results. AUTO7 CAR T-cells were highly potent in cytotoxicity assays against PSMA positive tumor cell lines showing no difference to a CAR alone control. AUTO7 CAR T cells encoding the dnTGFβRII and the dSHP2 showed resistance to both TGFβ1 and PD1/PDL1 mediated immunosuppression in vitro whilst the IL7R_CCR module conferred cytokine independent viability and proliferation. Finally, a module designed to enable the CAR T-cells to secrete IL-12 showed potent efficacy by its ability to activate fresh PBMC to release IFNγ. We demonstrated AUTO7 efficacy in an in-vivo model in which human PC3-PSMA PCa cells were engrafted into NSG mice for 3 weeks, prior to i.v. infusion of CAR T-cells. Although T cells transduced with CAR alone was unable to control tumor growth, AUTO7 CAR T-cells completely eradicated tumor with no signs of toxicity. Furthermore, examination of the IL12 module in immunocompetent models also showed no signs of cytokine mediated toxicity. Conclusions. Our results demonstrate the feasibility and efficacy of the multi-modular AUTO7 product. Addition of IL7R_CCR, IL-12, dnTGFβRII and dSHP2 modules to our anti-PSMA CAR product enhanced T cell functions by extending persistence, proliferation, activation and resistance to TGFβ1 and PD1/PDL1 driven immune inhibition. Citation Format: Marco Della Peruta, Victoria Koullourou, Mohamed El-Kholy, Mathew Robson, Philip Wu, Matteo Righi, Mathieu Ferrari, Giulia Agliardi, James Sillibourne, Simon Thomas, Shimobi Onuoha, Shaun Cordoba, Martin Pule. AUTO7: Anti-PSMA humanized CAR T-cell with improved persistence and resistance to tumor microenvironment for metastatic castration resistant prostate cancer (mCRPC) [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1070.

Sequence-Defined Heteromultivalent Precision Glycomacromolecules Bearing Sulfonated/Sulfated Nonglycosidic Moieties Preferentially Bind Galectin-3 and Delay Wound Healing of a Galectin-3 Positive Tumor Cell Line in an In Vitro Wound Scratch Assay.

Within this work, a new class of sequence-defined heteromultivalent glycomacromolecules bearing lactose residues and nonglycosidic motifs for probing glycoconjugate recognition in carbohydrate recognition domain (CRD) of galectin-3 is presented. Galectins, a family of β-galactoside-binding proteins, are known to play crucial roles in different signaling pathways involved in tumor biology. Thus, research has focused on the design and synthesis of galectin-targeting ligands for use as diagnostic markers or potential therapeutics. Heteromultivalent precision glycomacromolecules have the potential to serve as ligands for galectins. In this work, multivalency and the introduction of nonglycosidic motifs bearing either neutral, amine, or sulfonated/sulfated groups are used to better understand binding in the galectin-3 CRD. Enzyme-linked immunosorbent assays and surface plasmon resonance studies are performed, revealing a positive impact of the sulfonated/sulfated nonglycosidic motifs on galectin-3 binding but not on galectin-1 binding. Selected compounds are then tested with galectin-3 positive MCF 7 breast cancer cells using an in vitro would scratch assay. Preliminary results demonstrate a differential biological effect on MCF 7 cells with high galectin-3 expression in comparison to an HEK 293 control with low galectin-3 expression, indicating the potential for sulfonated/sulfated heteromultivalent glycomacromolecules to serve as preferential ligands for galectin-3 targeting.

Promotion of cellular senescence by THG-1/TSC22D4 knockout through activation of JUNB

Induction of cellular senescence in cancerous cells is an important strategy which is used in the treatment of cancer. However, cancer cells are capable of exhibiting resistance to cellular senescence through inactivation of tumor suppressors. Because of this, establishment of a route to cellular senescence induction in cancer cells is a crucial direction for developing future cancer therapies. In this study, we demonstrate the involvement of TSC-22 homologous gene-1 (THG-1, also called TSC22D4) in the suppression of cellular senescence. CRISPR/Cas9 gene editing was used to establish THG-1 knockout (KO) cells in a THG-1 positive esophageal tumor cell line. It was found that THG-1 KO cells exhibited delayed cell proliferation as well as cellular senescence. The elevated expression of the CDK inhibitor P21(CDKN1A) was also identified in senescent cells. Through the investigation of the upstream pathway for induction of P21(CDKN1A), the JUNB pathway was identified to play a critical role in P21(CDKN1A) transcription; in fact, the siRNA-mediated knockdown of JUNB reduced the abundance of P21(CDKN1A) mRNA and cellular senescence in THG-1 KO cells. These findings provide a novel insight into the induction of cellular senescence in THG-1 positive cancer cells.

Abstract C033: HPN328: An anti-DLL3 T cell engager for treatment of small cell lung cancer

Abstract HPN328: An Anti-DLL3 T Cell Engager for Treatment of Small Cell Lung Cancer Delta-like protein 3 (DLL3) is a Notch inhibitory ligand is expressed in more than 70% of small cell lung cancers (SCLCs), while there is little to no surface expression in normal adult tissues outside of the CNS *1. SCLC is an aggressive neuroendocrine tumor that represents about 15 percent of all lung cancers. Although often responsive to standard of care treatment, relapse is common, with a median progression-free survival of 2–3 months and median overall survival (OS) of 8–13 months and 5-year OS rate of <5%*2. DLL3 thus represents an attractive potential target for T cell–redirecting immunotherapy in SCLC. HPN328 is a tri-specific T cell activating construct (TriTAC) consisting of three binding domains: a N-terminal single chain Fv (scFv) that binds to CD3ϵ of the T cell receptor (TCR), a middle single domain antibody (sdAb) that binds to human serum albumin (HSA) to extend the half-life, and a C-terminal sdAb that binds to human DLL3. HPN328 is designed to simultaneously engage DLL3 on a target SCLC cell and CD3 on a T cell resulting in T cell activation, proliferation, and the eventual lysis of the target lung cancer cell. HPN328 is a highly stable single polypeptide of ~ 53 kDa expressed in CHO cells. It binds to human and cynomolgus monkey DLL3, albumin, and CD3ϵ with similar affinities. Flow cytometry analysis of T cells from various normal donors and a panel of DLL3 positive and DLL3 negative tumor cell lines confirmed binding of HPN328 to its native targets expressed on the cell surface. HPN328 induces potent killing of DLL3 expressing SCLC cell lines in vitro. In co-cultures of T cells from normal human donors, target tumor cells, and HSA, HPN328 mediated dose-dependent and DLL3-dependent cytotoxicity. Concomitant with target tumor cell killing, HPN328 also mediated dose-dependent upregulation of CD25 and CD69 on T cells in the TDCC co-cultures when DLL3 positive tumor cells were present. Consistent with the mechanism of action of CD3-based T cell engaging molecules, T cell derived cytokines, including TNFα, IL-2 and IFNγ, were detected. Nonclinical in vivo properties of HPN328 were evaluated in an NCI-H82 SCLC established tumor model. When administered to mice bearing human SCLC xenografts and human T cells, HPN328 eradicated subcutaneous tumors. In a single dose pilot toxicity study in cynomolgus monkeys, HPN328 was well tolerated at 1 and 10 mg/kg. Pharmacodynamic changes such as transient cytokine elevation mainly at the high dose were observed, consistent with the expected mechanism of action of T cell engagers. There were no clinically significant or adverse test article-related changes in hematology or clinical chemistry, and no apparent adverse findings at terminal and recovery necropsy. HPN328 exhibited linear PK properties in the given dose range with a serum half-life of 64 to 85 hours. Pharmacokinetic analysis supports weekly administration of HPN328 in humans. Preclinical and nonclinical characterization suggests that HPN328 is a highly efficacious, safe, and novel therapeutic candidate. A first-in-human phase 1 clinical trial is planned to evaluate HPN328 in SCLC. *1. Saunders, L et al. (2015) Sci Transl Med. 7(302): 302ra136. *2. Navarro, A et al. (2017) Transl Lung Cancer Res. 6(1): S78–S83. Citation Format: Wade H Aaron, Richard Austin, Manasi Barath, Evan Callihan, Michael Cremin, Thomas Evans, Maria Gamez, Vaishnavi Ganti, Golzar Hemmati, Kathryn Kwant, Che-Leung Law, Bryan Lemon, Llewelyn Lao, Mary Ellen Molloy, Jessica O’Rear, Laura Sun, Holger Wesche, Stephen Yu, Timothy Yu. HPN328: An anti-DLL3 T cell engager for treatment of small cell lung cancer [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; 2019 Oct 26-30; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2019;18(12 Suppl):Abstract nr C033. doi:10.1158/1535-7163.TARG-19-C033

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

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.

Pharmacological effects of site specific conjugated anti-human epidermal growth factor receptor 2-antibody drug conjugate using unnatural amino acid technology

To investigate inhibitory activities of a homogenous anti-human epidermal growth factor receptor 2 (HER2)-antibody drug conjugate (ADC) on the proliferation of nine tumor cell lines with different levels of HER2 expressions, and its activities on the tumor growth of five xenograft mouse models. The HER2 expression levels of BT-474, Calu-3, MCF-7, MDA-MB-231, MDA-MB-468, SK-BR-3, SK-OV-3, HCC1954, NCI-N87 tumor cell lines were measured using QIFI KIT. For the in vitro anti-proliferation assay, serial diluted anti-HER2-ADC, ado-trastuzumab emtansine, AS269, pAF-AS269 and paclitaxel were added to the seeded cells, and after 72 or 96 hours of incubation, the cell proliferation was analyzed. For the in vivo activity, 5-6 weeks old mice were inoculated with four HER2 positive tumor cell lines HCC1954, BT-474, SK-OV-3, NCI-N87 or one HER2 negative tumor cell line MDA-MB-468. Different amounts of anti-HER2-ADC, ado-trastuzumab emtansine, trastuzumab, paclitaxel and phosphate buffered saline control were injected after the tumor volume reached a certain size, then the tumor growth inhibition was analyzed. The expression levels of the six high HER2-expression cell lines SK-OV-3, NCI-N87, SK-BR-3, Calu-3, HCC1954, BT-474 were between 430 000 to 800 000 receptors per cell, which were 50 times higher than those of the other three low HER2 expression tumor cell lines MDA-MB-231, MCF-7, MDA-MB-468. Anti-HER2-ADC had inhibition effects on cell lines with high level of HER2 expression in the in vitro anti-proliferation assay. The half maximal inhibitory concentrations of anti-HER2-ADC on SK-OV-3, NCI-N87, SK-BR-3, Calu-3, HCC1954, BT-474 tumor cell lines were 46 pmol/L, 17 pmol/L, 17 pmol/L, 161 pmol/L, 125 pmol/L, 50 pmol/L, respectively. Anti-HER2-ADC had a dose dependent antitumor activity in vivo in all the HER2 positive xenograft mouse models. In NCI-N87 xenograft tumor model, the same dose of anti-HER2-ADC showed better anti-tumor activity compared with trastuzumab and ado-trastuzumab emtansine, and its relative tumor proliferation rates were about 1/30 to 1/20 of the two. In HCC1954 xenograft tumor model, the complete regression of the tumor was observed. As expected, anti-HER2-ADC had no tumor inhibitory effects on MDA-MB-468 xenograft models with low HER2 expression. The antitumor activities of anti-HER2-ADC in HER2 positive xenograft tumor models were the same as or better than the activities of ado-trastuzumab emtansine. The homogenous site-specific anti-HER2-ADC obtained using unnatural amino acid technology can inhibit the growth of high HER2-expression tumor cells with high potency both in vivo and in vitro.

The Epstein\u2013Barr virus nuclear antigen-1 upregulates the cellular antioxidant defense to enable B-cell growth transformation and immortalization

Epstein–Barr virus (EBV) immortalizes human B-lymphocytes and is implicated in the pathogenesis of lymphoid and epithelial cell malignancies. The EBV nuclear antigen (EBNA)-1 induces the accumulation of reactive oxygen species (ROS), which enables B-cell immortalization but causes oxidative DNA damage and triggers antiproliferative DNA damage responses. By comparing pairs of EBV-negative and -positive tumor cell lines we found that, while associated with the accumulation of oxidized nucleotides, EBV carriage promotes the concomitant activation of oxo-dNTP sanitization and purging pathways, including upregulation of the nucleoside triphosphatase mut-T homolog 1 (MTH1) and the DNA glycosylases 8-oxoguanine-glycosylase-1 (OGG1) and mut-Y homolog (MUTYH). Expression of EBNA1 was reversibly associated with transcriptional activation of this cellular response. DNA damage and apoptosis were preferentially induced in EBNA1-positive cell lines by treatment with MTH1 inhibitors, suggesting that virus carriage is linked to enhanced vulnerability to oxidative stress. MTH1, OGG1, and MUTYH were upregulated upon EBV infection in primary B-cells and treatment with MTH1 inhibitors prevented B-cell immortalization. These findings highlight an important role of the cellular antioxidant response in sustaining EBV infection, and suggests that targeting this cellular defense may offer a novel approach to antiviral therapy and could reduce the burden of EBV associated cancer.

Abstract 1922: MYB mimic peptides targeting human and murine MYB-NFIB positive tumor cells

Abstract Adenoid Cystic Cancer (ACC) is the most aggressive salivary gland tumor with no effective systemic therapy. MYB/MYBL1 fusion event underlies the etiology of ACC with few recurrent variants focused on NOTCH and chromatin remodeling pathways and low overall mutational tumor burden. No chemotherapy or targeted drugs have been approved for ACC. Therefore, there is an urgent need to develop novel therapeutic strategies. To study MYB biology and test new therapeutic agents we have developed and characterized the phenotype of a new murine conditional MYB-NFIB transgene model and have also generated a mammary tumor cell line expressing the human MYB fusion transgene with ACC-like features. We have also generated new human MYB-NFIB positive ACC tumor cell lines and xenografts from patient samples and have tested these tumor cells with newly developed MYB mimic peptides. We analyzed the phenotype of mice carrying conditional MMTV-CRE MYB-NFIB transgene alone or after crossing to heterozygosity with p16/ARF null alleles. We observed shortened survival in two different mouse transgene strains due to development of B-cell leukemia manifested by elevated WBC, low platelets, and marked splenomegaly. We also noted sporadic solid tumors including the development of a murine mammary tumor which generated a MYB-NFIB positive tumor cell line for testing new therapies. In addition, we have generated patient derived xenografts (PDX) and the successful generation of a human ACC tumor cell line from fresh ACC patient biopsies. Using shRNA reagents we observed reduced tumor cell growth and viability assayed by MTS assay following Notch1 and MYB knockdown. We also tested four candidate MYB inhibitors, celastrol, all-trans retinoic acid, and the peptidomimetic inhibitors, MYBMIM and CRYBMIM, against murine and human ACC tumor samples with immortalized normal cells as controls and will discuss these results. Citation Format: Yue Jiang, Lauren Forbes, Jianping Li, Shelby Freeberg, Chunxia Cao, Maria Zajac-Kaye, Jonathan Licht, Alex Kentsis, Frederic Kaye. MYB mimic peptides targeting human and murine MYB-NFIB positive tumor cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1922.

Abstract 2370: Synergistic effect of carboplatin on farletuzumab (FAR) tumor cell killing via downregulation of the humoral immunosuppressive CA125 protein and enhanced antibody dependent cellular cytotoxicity (ADCC)

Abstract Farletuzumab (FAR) is a humanized monoclonal antibody (mAb) that targets folate receptor alpha (FRA), a cell surface protein highly overexpressed on a large number of cancers. Clinical evidence suggests that FAR enhances the anti-tumor effects of carboplatin and taxane in ovarian cancer patients whose tumors express low CA125 levels. In light of the recent evidence that CA125 has a direct effect on suppressing the humoral immune mechanisms [antibody dependent cellular cytotoxicity (ADCC) and complement dependent cytotoxicity (CDC)] of FAR (1, 2) and the findings that chemotherapeutic agents can enhance the ADCC activity of certain mAbs, we tested the potential synergistic effects that carboplatin may have on FAR immune effector function. Here we show the immunosuppressive effects that CA125 has on FAR-mediated tumor cell killing in vivo and demonstrate that carboplatin is able to suppress cell surface CA125 expression leading to enhanced FAR ADCC on FRA-expressing ovarian tumor cells. CA125 has been previously found to suppress FAR-mediated tumor cell killing in vitro. To determine its effect in vivo, we tested single agent FAR against isogenic tumor cells in mouse xenografts. OVCAR-3 is a FRA positive ovarian tumor cell line that expresses high levels of CA125 on its cell surface. A CA125 shRNA knockdown line (OV-KD) has been previously generated and shown to be more susceptible to FAR ADCC than its parental counterpart. Xenograft studies using these lines found that FAR had significant single agent tumor killing in the OV-KD xenografts in contrast to OVCAR-3 xenografts. We next tested FAR in vivo killing when used in combination with carboplatin. These data showed that carboplatin had a synergistic effect when combined with FAR treatment as compared to single agent treatment alone. To determine if this mechanism involved enhanced ADCC, we conducted similar studies in vitro whereby OVCAR-3 cells were treated with a sublethal concentration of carboplatin for 72 hrs, washed and then tested for FAR ADCC killing. These data found that FAR had enhanced ADCC activity on carboplatin treated cells as compared to untreated cells. Finally, to explore the potential mechanism by which this effect may occur, we examined the steady-state levels of both the mannose-6-phosphate receptor (M6PR), reported to be upregulated by chemotherapeutic drugs leading to enhanced ADCC via higher granzyme B uptake and the CA125 immunosuppressive factor. Our data found that both M6PR and CA125 expression were significantly suppressed following carboplatin exposure. These data support a mechanism by which carboplatin may enhance the humoral immune response for FAR and potentially other anti-tumor mAbs via downregulation of the immunosuppressive CA125 protein. 1. OncoTarget 8:66747-57, 20172. 2. EJI 201847707, 2018 Citation Format: J. Bradford Kline, Keiji Furuuchi, James Fulmer, Jennifer McDonough, Luigi Grasso, Nicholas C. Nicolaides. Synergistic effect of carboplatin on farletuzumab (FAR) tumor cell killing via downregulation of the humoral immunosuppressive CA125 protein and enhanced antibody dependent cellular cytotoxicity (ADCC) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2370.

PF444 IDENTIFICATION OF T‐CELL RECEPTORS FROM THE ALLOGENEIC‐HLA REPERTOIRE SPECIFIC FOR MELANOMA‐ASSOCIATED ANTIGENS

Background:Melanoma‐associated antigens (MAGE) are highly expressed in multiple myelomas as well as solid tumors, but silent in normal cells except for male germ line cells. This tumor‐restricted expression makes it an excellent target for adoptive T cell therapy. However, due to thymic selection high‐affinity T cells against self‐antigens presented in the context of self‐HLA are deleted from the T‐cell repertoire of a patient. To overcome this problem, we identify T‐cell receptors (TCRs) from the allogeneic‐HLA repertoire instead of the autologous‐HLA repertoire.Aims:To identify high‐affinity T‐cell receptors from the allogeneic‐HLA repertoire specific for melanoma‐associated antigens.Methods:In this study we selected MAGE peptides by peptide elution experiments from HLA class I and subsequent mass spectrometric analysis of tumor cells. Only peptides binding in the most frequently occurring HLA‐alleles were selected. From the identified peptides, HLA‐tetramers were formed to isolate peptide specific CD8+ T cells from healthy allogeneic donors. Avidity of the selected T‐cell clones was determined by stimulation assays including a broad range of MAGE expressing tumor cell lines. To examine off‐target recognition, T‐cell clones were stimulated with MAGE negative cell lines and EBV‐transformed B lymphoblastic cells expressing all prevalent HLA molecules.Results:MAGE peptides were eluted from multiple myelomas, EBV‐transformed lymphoblastic cells, acute myeloid leukemia and ovarium carcinomas. For MAGE‐A1, T‐cell clones specific for four different epitopes presented in the context of HLA‐A∗01:01, HLA‐A∗03:01, HLA‐B∗07:02 and HLA‐C∗07:02 were selected. For MAGE‐A3, T‐cell clones specific for one epitope presented in the context of HLA‐A∗01:01 and HLA‐B∗35:01 were identified. The selected T‐cell clones demonstrated efficient recognition of MAGE‐A1 or MAGE‐A3 positive multiple myeloma and solid tumor cell lines without detectable cross‐reactivity.Summary/Conclusion:MAGE‐A1 and MAGE‐A3 specific T‐cell clones were isolated from the allogeneic‐HLA repertoire of healthy individuals. The identified clones were activated upon exposure to MAGE‐A1 or MAGE‐A3 positive tumor cell lines and showed limited off‐target recognition. These newly identified TCRs could potentially be used for TCR‐gene therapy strategies targeting MAGE‐A1 or MAGE‐A3 positive tumors.