Tolerance In Nonhuman Primates Research Articles

(167) - Comparing Delayed versus Simultaneous Bone Marrow Transplantation to Achieve Lung Allograft Tolerance in Non-Human Primates

(167) - Comparing Delayed versus Simultaneous Bone Marrow Transplantation to Achieve Lung Allograft Tolerance in Non-Human Primates

Abstract 1872: A DLL3/CD3/CD137 trispecific T cell engager shows potent antitumor activity in small cell lung cancer models

Abstract Background: Despite the approval of immune checkpoint inhibitors (ICIs), prognosis of small cell lung cancer (SCLC) remains poor. DLL3 is upregulated in SCLC whereas expression in normal tissues is minimal, representing the favorable profile as a therapeutic target. T cell engager (TCE) is a potent immunotherapy that redirects T cells to tumors expressing a specific antigen. Unlike ICIs, TCEs do not require the recognition of tumor antigens by T cells and thus could be an alternative approach to target tumors where the benefit of ICIs is limited such as SCLC. CD137 (4-1BB) is a costimulatory molecule that promotes T cell activation, proliferation, and survival. Since CD137 agonists synergize with CD3-mediated T cell activation, inducing concurrent CD137 costimulation is a promising strategy to unleash the potential of TCEs. We therefore developed a DLL3/CD3/CD137 trispecific T cell engager composed of two CD3/CD137 dual specific Fabs and one extra DLL3 Fab (DLL3 trispecific, RG6524). Results: We initially investigated the CD3 and CD137 signal transduction in Jurkat cells harboring NFAT or NF-κB reporter cocultured with DLL3 positive cells. DLL3 trispecific showed dose-dependent increase in NFAT and NF-κB activity, indicating that target engagement successfully activated CD3 and CD137 signaling. In the in vitro assay using human PBMC, DLL3 trispecific induced cytotoxicity against SCLC cell lines with EC50s in the two-digit pM range. In vivo efficacy was evaluated in SCLC xenograft models established by engrafting SCLC cell lines into immune humanized NOG mice. DLL3 trispecific showed greater tumor growth inhibition compared to a traditional bispecific T cell engager. Furthermore, flow cytometry-based immunophenotyping revealed that DLL3 trispecific increased T cell number in tumors and improved IFN-γ production from CD8 T cells. We also explored the combination with platinum-based drugs, which are widely used for SCLC, and showed that DLL3 trispecific enhanced the efficacy of platinum-based drugs. These data demonstrated that DLL3 trispecific has the potent in vivo efficacy and the potential for clinical use. We next evaluated whether cytokine release syndrome (CRS) mitigating approaches affect antitumor efficacy. Although prophylactic use of a steroid significantly reduced cytokine production, tumor growth inhibition by DLL3 trispecific remained unchanged. Likewise, tocilizumab treatment did not reduce the efficacy, suggesting that CRS mitigation did not abrogate the therapeutic benefit. We finally assessed the tolerability in non-human primates. DLL3 trispecific did not reach the maximum tolerated dose and was well tolerated up to 16 mg/kg Q2D, the highest dose tested. Conclusion: Our data showed that DLL3 trispecific has potent activity and is well suited for clinical application in SCLC. These findings provide a rationale for the clinical testing of DLL3 trispecific. Citation Format: Hirofumi Mikami, Shu Feng, Sotaro Naoi, Yumiko Azuma, Yoko Kayukawa, Toshiaki Tsunenari, Kentaro Asanuma, Ryutaro Iwabuchi, Hiroaki Nagano, Junko Shinozuka, Masaki Yamazaki, Haruka Kuroi, Siok Wan Gan, Priyanka Chichili, Shun Shimizu, Yutaka Matsuda, Shinya Ishii, Shogo Kamikawaji, Yasuko Kinoshita, Yuichiro Shimizu, Akihisa Sakamoto, Masaru Muraoka, Noriyuki Takahashi, Tatsuya Kawa, Hirotake Shiraiwa, Kenji Kashima, Futa Mimoto, Mika Kamata-Sakurai, Takehisa Kitazawa, Tomoyuki Igawa. A DLL3/CD3/CD137 trispecific T cell engager shows potent antitumor activity in small cell lung cancer models [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 1872.

Abstract 2636: Amanitin-based ADCs targeting Guanylyl cyclase C (GCC) as novel therapeutic modality for treatment of colorectal cancer

Abstract Background: Amatoxin-based antibody drug conjugates (so called ATAC®s) constitute a new class of antibody-drug conjugates (ADCs) which utilize amanitin as toxic payload. Amanitin binds to the RNA pol II and thereby efficiently inhibits cellular transcription. In the current study, in vitro and in vivo data of an ATAC® targeting GCC (guanylyl cyclase C) are presented. GCC is a cell surface receptor expressed in >95% of colorectal cancer, and in approximately 65% of esophageal, gastric, and pancreatic tumors. In healthy conditions GCC expression is restricted to the gastrointestinal tract, and more specific to the apical brush border of the intestinal epithelium (luminal site). Thus, GCC in healthy tissue is not exposed to the circulation but upon tumor progression in GI malignancies it becomes accessible for i.v. injected targeted therapeutics. This tumor-specific accessibility for drugs in circulation makes GCC a highly attractive target for ATAC®s. Material and Methods: ATAC®s: Cysteine-reactive amanitin-linker constructs were synthesized at Heidelberg Pharma and conjugated site-specifically to engineered cysteine residues of a proprietary anti-GCC antibody yielding ATAC®s with a DAR of 2.0. Animal models: Subcutaneous (s.c.) mouse xenograft models with the GCC overexpressing cell line HEK293-GUCY2C mono2 or colon cancer PDX models (Charles River) were performed in single-dose and multiple-dosing experiments. Tolerability was assessed in NMRI nude mice and non-human primates (NHP). Results: All anti-GCC ATAC®s with amanitin-linker derivatives, optimized for use in solid tumors, showed favorable in vitro cytotoxicity with picomolar activity on GCC+ cell lines and no cytotoxic activity on target-negative cells. In mouse xenograft models, the optimized anti-GCC ATAC®s caused dose-dependent tumor regression in HEK293-GUCY2C mono2 s.c. xenografts. Even in colorectal cancer PDX models, anti- GCC ATAC®s led to a substantial anti-tumor effect. Multiple dosing improved this anti-tumor efficacy even further without negative impact on toxicity. The MTD of the ATAC®s tested in mice differed between 7.5 and 50 mg/kg depending on linker chemistry. Safety profiling in cynomolgus monkeys revealed good tolerability for all selected ATAC®s. Hematology and clinical chemistry parameters were unaffected apart from a moderate and transient increase in liver enzymes. Conclusions: Targeted cytotoxic drug delivery to GCC+ cell lines was achieved by using anti-GCC ATAC®s optimized for the use in solid tumors. The mode of action of the payload amanitin led to a pronounced anti-tumor effect in vitro and in vivo in PDX models with good tolerability in non-human primates. The use of anti-GCC ATAC®s with their novel mode of action in the therapy of GCC positive cancers, such as colorectal cancer represents a promising approach in cancer therapy. Citation Format: Alexandra Braun, Andreas Pelz, Franziska Ebeling, Kristin Decker, Anikó Pálfi, Michael Kulke, Andreas Pahl, Torsten Hechler. Amanitin-based ADCs targeting Guanylyl cyclase C (GCC) as novel therapeutic modality for treatment of colorectal cancer [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 2636.

Active immunization with a structurally aggregated PD-L1 antigen breaks T and B immune tolerance in non-human primates and exhibits in vivo anti-tumoral effects in immunocompetent mouse tumor models

Despite the clinical success of the programmed death ligand 1 (PD-L1) blocking therapy in cancer treatment, only a subset of patients exhibits durable responses, therefore further exploration of other immunotherapeutic alternatives are needed. This paper reported the development of the PKPD-L1Vac vaccine, a new protein vaccine candidate that uses aluminum phosphate as an adjuvant and as an antigen the extracellular domain of human PD-L1 fused to a 47 amino-terminal portion of the LpdA protein from N. meningitides (PKPD-L1). The PKPD-L1 antigen has different physical and biological characteristics than those found in the natural molecule and in others PD-L1 vaccine candidates. The quimeric protein has a reduced binding capacity to the PD-1 and CD80 receptors to decrease their pro-tumoral activity. Besides, the distinctive feature of the PKPD-L1 polypeptide to be structurally aggregated could be desirable for its immunogenic properties. PKPD-L1Vac elicited anti-PD-L1-specific IgG antibodies and T lymphocyte-mediated immunity in mice and non-human primates. The vaccine administration demonstrated antitumor activity on CT-26 and B16–F10 primary tumor models in mice. Moreover, the immunization with PKPD-L1Vac increased the tumor-infiltrating lymphocytes and decreased the proportion of CD3+CD8+PD1+high anergic T cells in CT-26 tumor tissues, suggesting that the vaccine may remodel the tumor microenvironment. In summary, the PKPD-L1Vac vaccine exhibits very promising preclinical results and deserves to move forward to a phase I clinical trial.

(360) Use of Anti-Il-6 or Anti-IL-6r Therapy in a Mixed Chimerism Protocol to Achieve Heart Allograft Tolerance in Non-Human Primates

(360) Use of Anti-Il-6 or Anti-IL-6r Therapy in a Mixed Chimerism Protocol to Achieve Heart Allograft Tolerance in Non-Human Primates

Targeting CD74 in B-cell non-Hodgkin lymphoma with the antibody-drug conjugate STRO-001.

Overexpression of CD74, a type II transmembrane glycoprotein involved in MHC class II antigen presentation, has been reported in many B-cell non-Hodgkin lymphomas (NHLs) and in multiple myeloma (MM). STRO-001 is a site-specific, predominantly single-species antibody-drug conjugate (ADC) that targets CD74 and has demonstrated efficacy in xenograft models of MM and tolerability in non-human primates. Here we report results of preclinical studies designed to elucidate the potential role of STRO-001 in B-cell NHL. STRO-001 displayed nanomolar and sub-nanomolar cytotoxicity in 88% (15/17) of cancer cell lines tested. STRO-001 showed potent cytotoxicity on proliferating B cells while limited cytotoxicity was observed on naïve human B cells. A linear dose-response relationship was demonstrated in vivo for DLBCL models SU-DHL-6 and U2932. Tumor regression was induced at doses less than 5 mg/kg, while maximal activity with complete cures were observed starting at 10 mg/kg. In MCL Mino and Jeko-1 xenografts, STRO-001 starting at 3 mg/kg significantly prolonged survival or induced tumor regression, respectively, leading to tumor eradication in both models. In summary, high CD74 expression levels in tumors, nanomolar cellular potency, and significant anti-tumor in DLBCL and MCL xenograft models support the ongoing clinical study of STRO-001 in patients with B-cell NHL.

Clinical trials for renal allograft tolerance induction through combined hematopoietic stem cell transplantation: A narrative review.

During the last four decades, development of effective immunosuppressants has significantly improved short-term results of organ transplantation. However, long-term results have not been satisfactory due to chronic rejection or complications caused by immunosuppressive drugs. Therefore, induction of immunological tolerance of the transplanted organ is considered essential to improve the long-term results. Despite numerous tolerance strategies that have been successful in murine models, inducing hematopoietic chimerism through donor hematopoietic stem cell transplantation is the only method that reproducibly induces kidney allograft tolerance in nonhuman primates or humans. Combining kidney and hematopoietic stem cell transplantation to achieve allograft tolerance has now been attempted with different chimerism strategies. This review summarizes the status of current clinical trials on the induction of allograft tolerance. We also summarize recent studies to extend the chimerism approach to deceased donor transplant recipients.

Abstract 2080: LAG3-targeted IL15/IL15Rα-Fc (LAG3 x IL15) fusion proteins for preferential TIL expansion

Abstract The IL2Rβ/γc binding human cytokines IL2 and IL15 aid in the activation, proliferation, and survival of T and NK cells, and their therapeutic potential has been well established in animal models and human trials. However, therapeutic approaches utilizing these cytokines have suffered from low tolerability, fast clearance, and limited therapeutic window due to extensive activity on peripheral lymphocytes. Conversely, higher drug concentration and prolonged exposure are desirable to allow lymphocyte activation and proliferation at the tumor site, but this can be difficult to achieve due to dose-limiting toxicities associated with this axis. IL15 functions as a stabilized heterodimeric complex with membrane-bound IL15Rα on the surface of monocytes and DCs, which is presented in trans to lymphocytes expressing IL2Rβ/γc. We hypothesized that we could selectively target tumor-reactive TILs by combining a reduced potency IL15/IL15Rα heterocomplex with an immune checkpoint(CP)-targeting arm to bias binding and activation to CP-positive TILs, potentially improving therapeutic index. Lymphocyte activation gene 3 (LAG3) was chosen as the CP targeting-arm due to its frequent co-expression with PD1, bias to CD8+ T cells, ability to easily combine with anti-PD1 agents, and recent promising results with anti-LAG3 agents in the clinic. First, potency-reduced IL15/IL15Rα were created by engineering amino acid substitutions in IL15 - at the IL2Rβ/γc interface - that reduced in vitro potency by at least 1000-fold. We then designed LAG3 x IL15 fusion proteins containing single-chain IL15/IL15Rα and LAG3-targeting arms attached to a heterodimeric-Fc region, relying on targeting avidity to recover potency on LAG3+ cells. In vitro proliferation of lymphocytes in human PBMCs, stimulated with sub-optimal concentrations of anti-CD3 to induce LAG3 expression, was monitored by CFSE dilution or by counting Ki67+ cells after incubation with LAG3 x IL15 for 4 days. In vivo activity was evaluated using humanized mouse models by measuring the extent of human leukocyte expansion. Lead LAG3 x IL15 were evaluated for pharmacodynamic activity, pharmacokinetics, and tolerability in non-human primates. LAG3 x IL15 showed >500-fold selectivity compared to a non-targeted IL15 in an in vitro proliferation assay of lymphocytes stimulated for induced LAG3 expression. In vitro potency was greatest on effector memory CD8 T cells. LAG3 x IL15 were 3-fold more potent on CD8 T cells compared to CD4 T cells and had very weak activity on NK cells, consistent with minimal LAG3 expression on this population. In mouse models, treatment with LAG3 x IL15 promoted significantly increased numbers of T cells. Moreover, LAG3 x IL15 combined productively with anti-PD1 to promote additional T cell expansion. These results demonstrate that LAG3 x IL15 show a promising profile of selective IL15 delivery to TIL with minimal peripheral activity. Citation Format: Matthew J. Bernett, Suzanne Schubbert, Michael Hackett, Lukasz J. Ochyl, Lizett E. Scott, Christine Bonzon, Rumana Rashid, Kendra N. Avery, Irene W. Leung, Nicole Rodriguez, Connie Ardila, Umesh S. Muchhal, Norman J. Barlow, Rena Bahjat, John R. Desjarlais. LAG3-targeted IL15/IL15Rα-Fc (LAG3 x IL15) fusion proteins for preferential TIL expansion [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 2080.

Successful Use of Anti-IL-6R Therapy to Achieve Cardiac Allograft Tolerance in Non-Human Primates

<h3>Purpose</h3> In contrast to kidney allografts, attempts to achieve tolerance of isolated cardiac allografts in non-human primates (NHPs) using a mixed chimerism strategy have been unsuccessful. IL-6 is a pro-inflammatory cytokine known to play an important role in allograft rejection. We investigated whether adding anti-IL-6R therapy to our mixed chimerism protocol would enable the induction of tolerance in NHP recipients of MHC mismatched heart allografts. <h3>Methods</h3> Ten cynomolgous NHPs underwent heart and bone marrow transplantation (BMTx) with non-myeloablative conditioning including total body and thymic irradiation, ATGAM, anti-CD154 mAb, and cyclosporine until post-operative day (POD) 28, with or without anti-IL-6R therapy (tocilizumab, 10 mg/kg IV on POD 0, 7, 14, 21, 28, 56, 84). Group A (n=2) received allogeneic BMTx without anti-IL-6R therapy, Group B (n=6) received allogeneic BMTx with anti-IL-6R therapy, and Group C (n=2) received autologous BMTx with anti-IL-6R therapy. <h3>Results</h3> In Group A, both recipients experienced allograft failure due to rejection on POD 104 and 132 [Table]. In Group B, 3 recipients achieved long-term cardiac allograft survival of >400 days off immunosuppression; one recipient is ongoing at POD 360. Two rejected on POD 169 and 383. For Groups A and B, peak lymphocyte chimerism was 10.1% and 18.1%, respectively and duration of lymphocyte chimerism was 57 and 178 days, respectively. In Group C, one recipient rejected on POD 131 and one is ongoing at POD 120. There was no significant difference in magnitude of peripheral T_reg expansion between groups (p=0.77). Compared to Group A, Groups B and C had lower CRP levels (mean 12.3 vs 54.3 μg/mL) and higher free serum IL-6 (mean 23.0 vs <7.7 pg/mL) while receiving anti-IL-6R therapy. <h3>Conclusion</h3> Tolerance of cardiac allografts has been achieved for the first time in NHPs using a combination of IL-6 signaling blockade and mixed chimerism. This approach represents significant progress towards clinical cardiac allograft tolerance.

Abstract 915: Preclinical evaluation of anti-CD37 antibody-targeted amanitin conjugates (ATAC) in B-cell malignancies

Abstract Background: ATACs (antibody-targeted amanitin conjugates) comprise a new class of antibody-drug conjugates (ADCs) using amanitin as a toxic payload. Amanitin binds to the RNA pol II and thereby efficiently inhibits the cellular transcription process. In the current study, in vitro and in vivo data of ATACs targeting CD37 (member of the tetraspanin family) are presented. CD37 is a transmembrane protein expressed exclusively on cells of the immune system and mainly on mature B-cells, as well as in many B-cell malignancies, including Richter's Syndrome (RS) or Richter´s transformation. RS is a transformation of B cell chronic lymphocytic leukemia, refractory to treatment and carries a poor prognosis, and hence is considered an ideal target for amanitin-based ADCs. Material and methods: Cell lines: CD37-positive: Mec-2 (B-chronic lymphocytic leukaemia); Raji-Luc, Ramos (Burkitt´s lymphoma); CD37-negative: HL-60 (acute lymphocytic leukemia) Antibody: anti-CD37 engineered monoclonal antibody produced at Heidelberg Pharma Toxic warhead: Cysteine reactive amanitin-linker constructs were synthesized at Heidelberg Pharma and conjugated site-specifically to the antibody. Cell proliferation assay: Quantitative determination of cell viability was performed by CellTiter Glo 2.0 assay (Promega). Animal models: Disseminating Mouse xenograft tumor models (Mec-2 and Raji-Luc) and CD37-positive patient derived xenograft (PDX; Richter's Syndrome) models were performed in single-dose experiments. Tolerability was assessed in non-human primates (NHP). Results: All tested anti-CD37 ATACs showed in vitro cytotoxicity in the picomolar range on CD37-positive cell lines. No cytotoxic activity was observed on CD37-negative cells. In mouse xenograft models, 80 - 100% overall survival was achieved with two anti-CD37 ATACs at doses of 1/16 MTD (Mec-2 model) and 1/64 MTD (Raji Luc Model). Single-dose treatment caused rapid and complete tumor remission already 7 days after treatment. The tested ATACs revealed good tolerability in mice. In NHP, the anti CD37 ATACs revealed good tolerability. Hematology and clinical chemistry parameters were unaffected except liver transaminases and LDH: A mild to moderate and transient increase was observed. Conclusions: Targeted cytotoxic drug delivery to CD37 positive cell lines was achieved by using anti-CD37 ATACs. The mode of action of the payload amanitin led to an efficient anti-tumor potential in vitro and in vivo with good tolerability in non-human primates. Using ADCs in the therapy of B-cell lymphomas, including malignancies that underwent Richter's transformation, is a promising approach, especially by using a payload whose mode of action differs from other commonly used toxins, like ATACs. Citation Format: Stephanie Voss, Aniko Palfi, Christoph Mueller, Andreas Pahl, Torsten Hechler, Michael Kulke. Preclinical evaluation of anti-CD37 antibody-targeted amanitin conjugates (ATAC) in B-cell malignancies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 915.

Abstract 910: Amanitin-based ADCs targeting PSMA as novel therapeutic modality for prostate cancer therapy

Abstract Background: Antibody-targeted amanitin conjugates (ATACs) comprise a new class of antibody-drug conjugates (ADCs) using amanitin as toxic payload. Amanitin binds to the RNA pol II and thereby efficiently inhibits the cellular transcription process. In the current study, in vitro and in vivo data of an ATAC targeting PSMA (prostate specific membrane antigen) are presented. PSMA is a type II integral membrane glycoprotein and used as tumor marker due to its predominant expression on malignant prostate cells in prostate carcinoma. As PSMA expression increases with tumor aggressiveness, metastatic and disease recurrence, it is considered an ideal target for amanitin-based ADCs. Material and methods: Cell lines: PSMA+ cell lines: LNCap, 22RV1; PSMA- cell line: PC3 Antibody: cysteine engineered monoclonal anti-PSMA antibody (Albert Ludwig University Freiburg, medical center; derivatization and production at Heidelberg Pharma) Toxic warhead: Cysteine reactive amanitin-linker constructs were synthesized at Heidelberg Pharma and conjugated site-specifically to engineered cysteine residues of the anti-PSMA antibody yielding ATACs with a DAR of 2. Cell proliferation assay: Quantitative determination of cell viability was performed by BrdU-based chemiluminescent cell proliferation ELISA (Roche). Animal models: Subcutaneous Mouse xenograft tumor models with PSMA-positive cell lines 22RV1 and LNCap were performed in single-dose experiments. Tolerability was assessed in mice and non-human primates (NHP). Results: All anti-PSMA ATACs with optimized amanitin-linker variants for use in solid tumors showed favorable in vitro cytotoxicity with nano- to picomolar activity on PSMA+ cell lines and no cytotoxic activity on PSMA- cells. In mouse xenograft models, the optimized anti-PSMA ATACs caused dose-dependent tumor regression in LNCap s.c. xenografts. Complete remission was achieved after a single i.v. dose of ¼ MTD with 100% overall survival for the full duration of the studies (&amp;gt;160 days). The tolerability of the tested ATACs in mice differed between 15 and &amp;gt; 80 mg/kg Safety profiling in Cynomolgus monkeys revealed a good tolerability and therapeutic index for all selected amanitin-linker variants. Hematology and clinical chemistry parameters were unaffected except liver enzymes and LDH: A moderate and transient increase was observed. Low off target toxicity was confirmed by a non-binding ATAC. Conclusions: Targeted cytotoxic drug delivery to PSMA positive cell lines was achieved by using anti-PSMA ATACs optimized for the use in solid tumors. The mode of action of the payload amanitin led to an efficient anti-tumor potential in vitro and in vivo with good tolerability in non-human primates. Using ATACs in the therapy of PSMA positive prostate cancer is a promising approach, especially by using a cytotoxic agent whose mode of action differs from other commonly used toxins. Citation Format: Alexandra Braun, Aniko Palfi, Christoph Mueller, Torsten Hechler, Andreas Pahl, Michael Kulke. Amanitin-based ADCs targeting PSMA as novel therapeutic modality for prostate cancer therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 910.

Trph-222, a Novel Anti-CD22 Antibody Drug Conjugate (ADC), Has Signficant Anti-Tumor Activity in NHL Xenografts and Is Well Tolerated in Non-Human Primates

CD22 is a B-cell specific transmembrane glycoprotein belonging to the SIGLEC family of lectins. Its expression is restricted to B lineage cells from the early immature/pre-B-cell stage through the mature naïve stage, and is downregulated on the surface of plasma cells. CD22 is highly expressed on a wide range of malignancies arising from mature B-cells, including NHL (non-Hodgkin's lymphoma), DLBCL (diffuse large B cell lymphoma), MCL (mantle cell lymphoma), MZL, B-cell acute lymphoblastic leukemia, chronic lymphocytic leukemia and small lymphocytic leukemia. The restricted lineage of CD22 and rapid internalization upon antibody binding make CD22 an appealing target for an antibody-drug conjugate (ADC) therapeutic for B cell malignancies. Prior attempts at targeting CD22 using ADC technology have been limited clinically in NHL due to an unacceptable or poor therapeutic index. Subsequently the ADC field has focused on more stable and site specific conjugation of payloads to increase the therapeutic index of ADCs.TRPH-222 is an ADC comprised of an anti-CD22 antibody site-specifically modified at one site in each heavy chain to express formylglycine (FG), allowing for site-specific conjugation of a maytansinoid payload, a protease-insensitive spacer, and a functional group for coupling to an aldehyde on the antibody FG residues. This SMARTAG™ site specific non-cleavable conjugation enforces a maximum drug to antibody ratio (DAR) of 2.0.The aim of this study was to characterize the anti-tumor activity of TRPH-222 in mouse xenografts and tolerability in non-human primates (NHP).In the WSU-DLCL2 xenograft model, once weekly intravenous (IV) dosing of 1, 3 or 10mg/kg or TRPH-222 reduced tumor volume by 51%, 100% and 100% after 28 days. A once every three-week dose of 10mg/kg reduced tumor volume by 100% from pre-dosing measurements. In contrast, rituximab (30mg/kg, weekly) resulted in 55% tumor growth inhibition. In both SU-DHL-2 (DLBCL) and Granta-519 (MCL) xenograft models, weekly dosing at 10mg/kg resulted in tumor stasis.The tolerability and PK of TRPH-222 were evaluated in cynomolgus monkeys. Animals were dosed IV once every 3 weeks (the anticipated clinical dosing schedule) for either 2 (5, 12.5, or 25 mg/kg) or 3 (50 mg/kg) doses. TRPH-222 was well tolerated at all doses evaluated and all animals survived to study termination, with no TRPH-222-related effects on clinical observations or body weight. Aspartate aminotransferase (AST) activity was mildly increased in animals at ≥12.5 mg/kg/dose, while minimal increases in alanine aminotransferase (ALT) activity were noted at 50 mg/kg/dose. Minor microscopic findings of Kupffer cell hypertrophy/hyperplasia were noted in the livers of monkeys treated at 50 mg/kg IV; this finding was not considered adverse based on minimal severity and lack of degeneration/necrosis. Preliminary PK data demonstrate the stability of TRPH-222, as the ratio of the area under the curve (AUC) from day 0-42 for the ADC was ~76-88% that of the antibody backbone suggesting that the ADC has a long half-life and that the linker is stable in vivo.In conclusion, the studies reported here demonstrate that TRPH-222 has significant anti-tumor activity in a range of B-cell lymphoma models and that very high doses of TRPH-222 are well tolerated in monkeys. IND-enabling safety studies are ongoing to support clinical evaluation of TRPH-222 in 2018. These data support the argument that TRPH-222 will have a large therapeutic window due to the stability of conjugation of the maytansinoid payload to the anti-CD22 antibody; doses tolerated in the monkey study described above correspond to human equivalent doses significantly higher than for most ADCs approved or currently under clinical evaluation. To our knowledge, this is the highest dose of a maytansine containing ADC that has been tolerated in monkeys and raises the potential for a best in class therapy for NHL in addition to a best in class ADC technology. DisclosuresMaclaren:Triphase Accelerator: Employment. Levin:Triphase Accelerator: Employment. Lowman:Triphase Accelerator: Employment. Trikha:Triphase Accelerator: Employment; Encycle Therapeutics: Membership on an entity's Board of Directors or advisory committees.

Using Selective Bcl2 Inhibition to Induce Cardiac Allograft Tolerance

<h3>Purpose</h3> Cardiac allograft tolerance in non-human primates (NHPs) has been achieved by our group using a mixed chimerism model with allogeneic bone marrow transplant after non-myeloablative conditioning but not without kidney co-transplantation. B cell lymphoma 2 (Bcl2) inhibition has allowed for durable mixed chimerism with reduced conditioning in the murine skin transplant model. We investigated whether the Bcl2 inhibitor ABT199 could enhance mixed chimerism and induce cardiac allograft tolerance in the absence of kidney cotransplantation. <h3>Methods</h3> Fifteen cynomolgus recipients underwent mixed chimerism conditioning (ATGAM, total body and thymic irradiation) then organ and bone marrow transplantation. Group 1 recipients received heart alone (n=5), Group 2 recipients received heart and kidney cotransplants (n=7), and in Group 3, heart recipients received ABT199 in lieu of kidney cotransplants (n=3).Group 1 and 2 received 3Gy total body irradiation (TBI) while Group 3 received 1.5Gy TBI. All recipients underwent a 28-day course of immunosuppression with a calcineurin inhibitor and anti-CD154 mAb after which all immunosuppression was stopped. <h3>Results</h3> All five Group 1 recipients demonstrated cellular rejection (ISHLT 3R) by 96 days post bone marrow transplant (pBMTx). In contrast, no Group 2 recipients showed early rejection and 6/7 survived >400 days pBMTx. The first two Group 3 recipients developed GvHD and succumbed by days 59 and 68 pBMTx without evidence of rejection. However, after reducing the size of the donor BMT, the third recipient is now 76 days pBMTx with excellent allograft contractility and no evidence of GVHD. Mean peak lymphoid chimerism was 10% on day 26 pBMTx for Group 1, 26% on day 33 pBMTx for Group 2, and 57% on day 54 pBMTx for Group 3. (Figure 1) <h3>Conclusion</h3> After further refinement of the conditioning regimen, ABT199 may allow for the induction of heart allograft tolerance without the need for kidney co-transplantation and with a reduction in TBI thereby improving safety.

Effects of IL-2 and/ or Anti-IL6R Therapy on Long-Term Cardiac Allograft Survival in Non-Human Primates

Purpose Cardiac allograft tolerance in non-human primates (NHPs) has been achieved by our group using a mixed chimerism model with allogeneic bone marrow transplant after non-myeloablative conditioning but not without kidney co-transplantation. Transplantation of the heart alone results in graft rejection within 150-180 days. Our results suggest that the ability of the kidney to confer tolerance on the co-transplanted heart relates to its ability to induce the expansion/activation of host regulatory T cells (Tregs). We investigated whether IL-2 and/ or anti-IL6R therapy could substitute for the kidney and induce cardiac allograft tolerance by augmenting Tregs. Methods Sixteen cynomolgus NHPs underwent heart and donor bone marrow transplant with mixed chimerism conditioning including total body and thymic irradiation, ATGAM, anti-CD154 mAb, and cyclosporine until post-operative day (POD) 28. Recipients in Group A (n=5) received low dose IL-2 therapy (1M IU/m2 SC, POD -6 to 5) and an anti-IL6R mAb (tocilizumab, 10 mg/kg IV, POD 0, 7, 14, 21, 28, 56, 84, 112). Recipients in Group B (n=11) received tocilizumab alone (10 mg/kg IV, POD 0, 7, 14, 21, 28, 56, 84). Results In Group A, 3/5 recipients rejected at POD 127, 198, and 254 with acute cellular rejection (ACR), antibody mediated rejection (AMR), and donor specific antibodies (DSA). 2/5 died due to sepsis (POD11) or technical complications (POD7). In Group B, 4/11 recipients are ongoing, of which 2 are >430 days post-transplant without ACR. 1/11 rejected at POD 621 but only after donor skin transplant on POD 526. 1/11 failed to develop chimerism and rejected at POD 169. Graft failure/ death occurred in 5/11 due to technical or sedation complications (POD3, POD5 [n=2], POD9) and pancytopenia (POD65). Group A recipients exhibited an average 15-fold expansion in percent CD25+FoxP3+ of peripheral CD3+CD4+ cells compared to an average 3-fold expansion in Group B. Conclusion Despite its ability to promote Tregs, combined IL-2/ aIL6R treatment failed to achieve long-term allograft survival. We surmise that this was due to IL-2 stimulation of alloreactive memory T cells. In contrast, aIL6R mAb treatment alone achieved long-term allograft survival (>1 year) off immunosuppression in 3 recipients and represents a promising strategy towards clinical cardiac allograft tolerance.

Cardiac Allograft Tolerance Induction with Ox40L Costimulatory Blockade

<h3>Purpose</h3> Cardiac allograft tolerance in non-human primates (NHPs) has been achieved by our group via hematopoietic mixed chimerism with allogeneic bone marrow and kidney co-transplantation. This model has failed without the regulatory mechanisms associated with the donor kidney. We hypothesized that rapamycin and anti-OX40L mAb (KY1005, Kymab Ltd) could substitute for kidney co-transplantation and induce cardiac allograft tolerance by providing a favorable immunologic milieu for regulatory T cells (Tregs). <h3>Methods</h3> Fourteen cynomolgus NHPs underwent mixed chimerism conditioning (ATGAM, total body and thymic irradiation) then organ and bone marrow transplant. Heart Alone recipients received a heart transplant (n=5), Heart/Kidney recipients received a heart and kidney co-transplant (n=7). Heart/OX40L recipients received a heart transplant plus 40 days of anti-OX40L mAb (n=2). All groups underwent a 12 day course of aCD154 mAb. The Heart Alone and Heart Kidney groups received 28 days of cyclosporine while the Heart aOX40L group received 100 days of rapamycin. <h3>Results</h3> All Heart Alone recipients had cellular rejection (ISHLT 3R) by 200 days post bone marrow transplant (pBMT). No Heart/Kidney recipients showed early rejection. Similarly, no Heart/OX40L recipients showed early rejection and one is currently 258 days pBMT without clinical or pathologic evidence of rejection (Figure 1). The other recipient died of viral complications. Mean peak lymphoid chimerism was 10% for the Heart Alone group, 26% for the Heart/Kidney group, and 29% for the Heart/OX40L group. The anti-OX40L protocol also showed an expansion of the Treg population with a peak of 35% Tregs in the CD4+ population. <h3>Conclusion</h3> The OX40L protocol achieved robust lymphoid chimerism with concomitant expansion of Tregs. The surviving Heart/OX40L recipient achieved longer freedom from allograft rejection when compared to the Heart Alone group. OX40L costimulatory blockade may allow for heart allograft tolerance without kidney co-transplantation.

Abstract 2286: XmAb30819, an XmAb®2+1 ENPP3 x CD3 bispecific antibody for RCC, demonstrates safety and efficacy in in vivo preclinical studies

Abstract Bispecific T cell engagers simultaneously bind CD3 on T cells and tumor-associated antigens to promote T cell-mediated killing of tumor cells. While targeting lineage-restricted antigens such as CD19 or CD20 have found clinical success in hematopoietic cancers, targeting solid tumors requires high expressing tumor associated antigens (TAAs) with minimal normal tissue expression. One underexplored TAA is ectonucleotide pyrophosphatase/phosphodiesterase 3 (ENPP3) for renal cell carcinoma (RCC). An activation marker for basophils, ENPP3 is a type II transmembrane protein that can promiscuously hydrolyze a large variety of nucleotide-containing molecules. Although its specific function in the tumor microenvironment is unknown, bulk RNAseq data from the TCGA describes broad overexpression of ENPP3 in clear cell and papillary RCC and less frequently in hepatocellular carcinoma (HCC). This pattern of expression has been confirmed by IHC and makes ENPP3 a potential target for a CD3 bispecific approach. Building upon the XmAb® heterodimeric Fc platform, we generated bispecific antibodies in an XmAb 2+1 Fab2-scFv-Fc format that are bivalent for ENPP3 and monovalent for CD3. Reducing the affinity of both specificities encouraged avid binding and strong potency on high ENPP3 expressing cell lines while minimizing reactivity on low expressing cell lines, matching ENPP3's differential expression pattern. In vitro T cell-dependent cellular cytotoxicity (TDCC) assays on the relevant cell lines confirmed selective potency on high versus low expressing cells. In vivo potency was evaluated by xenograft tumor models in human PBMC-engrafted NSG mice. In a KU812 tumor model, the lead candidate, XmAb30819, exhibited complete responses both as a single agent and in combination with checkpoint blockade. When tested against RXF393, an RCC cell line that overexpresses ENPP3 upon tumor engraftment, XmAb30819 demonstrated complete responses at all tested doses. Tolerability in non-human primates was evaluated in an escalating dose model and then confirmed in an expansion cohort. Dose-dependent T cell margination was observed, and doses that were active in the mouse tumor studies were well-tolerated. Preclinical studies have demonstrated the safety and efficacy of XmAb30819, an XmAb 2+1 ENPP3 x CD3 bispecific antibody for RCC. Citation Format: Alex Nisthal, Sung-Hyung Lee, Yoon Kyung Kim, Christine Bonzon, Rumana Rashid, Kendra N. Avery, Liz Bogaert, Connie Ardila, Irene W. Leung, Nicole Rodriguez, Umesh S. Muchhal, Gregory L. Moore, Seung Y. Chu, John R. Desjarlais. XmAb30819, an XmAb®2+1 ENPP3 x CD3 bispecific antibody for RCC, demonstrates safety and efficacy in in vivo preclinical studies [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 2286.

Social Structure Predicts Eye Contact Tolerance in Nonhuman Primates: Evidence from a Crowd-Sourcing Approach

In most primates, eye contact is an implicit signal of threat, and often connotes social status and imminent physical aggression. However, in humans and some of the gregarious nonhuman primates, eye contact is tolerated more and may be used to communicate other emotional and mental states. What accounts for the variation in this critical social cue across primate species? We crowd-sourced primatologists and found a strong linear relationship between eye contact tolerance and primate social structure such that eye contact tolerance increased as social structures become more egalitarian. In addition to constituting the first generalizable demonstration of this relationship, our findings serve to inform the related question of why eye contact is deferentially avoided in some human cultures, while eye contact is both frequent and even encouraged in others.

Transient-mixed Chimerism With Nonmyeloablative Conditioning Does Not Induce Liver Allograft Tolerance in Nonhuman Primates.

Although short-term outcomes for liver transplantation have improved, patient and graft survival are limited by infection, cancer, and other complications of immunosuppression. Rapid induction of tolerance after liver transplantation would decrease these complications, improving survival and quality of life. Tolerance to kidneys, but not thoracic organs or islets, has been achieved in nonhuman primates and humans through the induction of transient donor chimerism. Since the liver is considered to be tolerogenic, we tested the hypothesis that the renal transplant transient chimerism protocol would induce liver tolerance. Seven cynomolgus macaques received immune conditioning followed by simultaneous donor bone marrow and liver transplantation. The more extensive liver surgery required minor adaptations of the kidney protocol to decrease complications. All immunosuppression was discontinued on postoperative day (POD) 28. Peripheral blood chimerism, recipient immune reconstitution, liver function tests, and graft survival were determined. The level and duration of chimerism in liver recipients were comparable to those previously reported in renal transplant recipients. However, unlike in the kidney model, the liver was rejected soon after immunosuppression withdrawal. Rejection was associated with proliferation of recipient CD8 T effector cells in the periphery and liver, increased serum interleukin (IL)-6 and IL-2, but peripheral regulatory T cell (Treg) numbers did not increase. Antidonor antibody was also detected. These data show the transient chimerism protocol does not induce tolerance to livers, likely due to greater CD8 T cell responses than in the kidney model. Successful tolerance induction may depend on greater control or deletion of CD8 T cells in this model.

Inducing Donor MHC Chimerism with Bone Marrow Derived Exosomes in Non-Human Primates

Cardiac allograft tolerance in non-human primates (NHPs) has been achieved by our group via hematopoietic mixed chimerism consisting of an allogeneic bone marrow (BM) and kidney co-transplantation from the same donor after non-myeloablative conditioning. In this setting, T cell tolerance is believed to rely on expression of donor MHC molecules in the host's primary and secondary lymphoid organs. Here, we investigated whether donor MHC chimerism could be achieved using donor bone marrow derived extracellular vesicles (exosomes) instead of cells. Exosomes were isolated through serial ultracentrifugation from supernatants of donor BM cells cultured for 48 hours (expressing H38 MHC class I epitope). Two H38- cynomolgus macaque recipients received conditioning consisting of ATGAM and thymic irradiation, with or without total body irradiation (TBI) before a heart and kidney co-transplantation. Both recipients underwent a short course of immunosuppression with a calcineurin inhibitor and co-stimulation blockade with anti-CD154 mAb. A single dose of H38+ donor bone marrow derived exosomes was infused into H38- recipients on post-operative day 2. Image flow cytometry was used to detect the presence of recipient cells displaying donor MHC class I on their surface (cross-dressed) in the blood, thymus, spleen and lymph nodes. Infusion of donor bone marrow derived exosomes resulted in the presence of recipient peripheral blood leukocytes displaying donor MHC class I molecules in both animals. Flow cytometric analysis showed that MHC class I chimerism was restricted to the myeloid cell compartment. Tissues collected from the total body irradiated NHP showed that H38+ cross-dressed cells were also present in the lymph nodes, thymus, and spleen. In a separate study, a NHP recipient treated with an exosome protocol without TBI is currently 54 days post-transplantation with a well-functioning cardiac allograft. Donor MHC chimerism in the form of MHC cross-dressing can be achieved in a NHP model using donor bone marrow derived exosomes. These findings suggest that bone marrow derived exosomes could be used in place of whole bone marrow to induce allograft tolerance. The use of exosomes would allow for the reduction of recipient conditioning and would eliminate the risk of GVHD making this approach clinically safer than traditional BMT.

Transplantation tolerance in nonhuman primates and humans.

This review focuses on our recent studies involving nonmyeloablative bone marrow transplantation as an approach to inducing organ allograft tolerance across MHC barriers in nonhuman primates and in patients. The clinical studies are focused on mechanisms of tolerance involved in a protocol carried out at Massachusetts General Hospital in HLA-mismatched haploidentical combinations for the induction of renal allograft tolerance. These studies, in which chimerism was only transient and GVHD did not occur, suggest an early role for donor-specific regulatory T cells in tolerance induction, followed by partial and gradual deletion of donor-reactive T cells. We utilized high-throughput sequencing methodologies in a novel way to identify and track large numbers of alloreactive T cell receptors (TCRs). This method has been shown to identify biologically significant alloreactive TCRs in transplant patients and pointed to clonal deletion as a major mechanism of long-term tolerance in these patients. More recently, we adapted this sequencing method to optimally identify the donor-specific regulatory T cell (Treg) repertoire. Interrogation of the early posttransplant repertoire demonstrated expansion of donor-specific Tregs in association with tolerance. Our studies suggest a role for the kidney graft in tolerance by these mechanisms in patients who had only transient chimerism. Nonhuman primate studies indicate that other organs, including the heart, the lungs and the liver, are less readily tolerated following a period of transient mixed chimerism. Our efforts to extend the reach of mixed chimerism for tolerance induction beyond the kidney are therefore focused on the addition of recipient Tregs to the protocol. This approach has the potential to enhance chimerism while further reducing the risk of GVHD.