In our studies of life-supporting GalT-KO pig-to-baboon kidneys, we have found that some recipients developed increased serum creatinine with growth of the grafts, without histologic or immunologic evidence of rejection. We hypothesized that the rapid growth of orthotopic pig grafts in smaller baboon recipients may have led to deterioration of organ function. To test this hypothesis for both kidneys and lungs, we have assessed whether the growth of outbred (Yorkshire) organ transplants in miniature swine is regulated by intrinsic (graft) or extrinsic (host environment) factors. Yorkshire kidneys exhibited persistent growth in miniature swine, reaching 3.7x their initial volume over 3 months vs. 1.2x for miniature swine kidneys over the same time period. Similar rapid early growth of lung allografts was observed and, in this case, led to organ dysfunction. For xenograft kidneys, a review of our results suggests that there is a threshold of 25cm3/kg for kidney graft volume/recipient body weight that induces cortical ischemia in transplanted GalT-KO kidneys in baboons. These results suggest that intrinsic factors are responsible, at least in part, for growth of donor organs and that this property should be taken into consideration for growth-curve mismatched transplants, especially for life-supporting organs transplanted into a limited recipient space.

To our knowledge, this is the first evidence of histologically viable porcine lung grafts beyond 7 days in baboons. Our results indicate that GalT-KO pig lungs are highly susceptible to acute humoral rejection and that this may be mitigated by transgenic expression of hCD47.

BackgroundProgrammed cell death protein 1/programmed death-ligand 1 (PD-1/PD-L1) blocking antibodies including cemiplimab have generated profound clinical activity across diverse cancer types. Tumorous PD-L1 expression, as assessed by immunohistochemistry (IHC), is an accepted predictive marker of response to therapy in some cancers. However, expression is often dynamic and heterogeneous, and therefore not reliably captured by IHC from tumor biopsies or archival samples. Thus, there is significant need for accurate whole-body quantification of PD-L1 levels.MethodsWe radiolabeled the novel human anti-PD-L1 antibody REGN3504 with zirconium-89 (89Zr) using the chelator p-SCN-Bn-Deferoxamine to enable non-invasive immuno-positron emission tomography (immuno-PET) of PD-L1 expression. PET imaging assessed the localization of 89Zr-REGN3504 to multiple human tumor xenografts. Mice genetically humanized for PD-1 and PD-L1 were used to assess the biodistribution of 89Zr-REGN3504 to normal tissues and the estimated human radiation dosimetry of 89Zr-REGN3504 was also determined. Pharmacokinetics of REGN3504 was assessed in monkeys.ResultsClear localization of 89Zr-REGN3504 to human tumor xenografts was observed via PET imaging and ex vivo biodistribution studies demonstrated high (fourfold to sixfold) tumor:blood ratios. 89Zr-REGN3504 specifically localized to spleen and lymph nodes in the PD-1/PD-L1 humanized mice. 89Zr-REGN3504 immuno-PET accurately detected a significant reduction in splenic PD-L1 positive cells following systemic treatment with clodronate liposomes. Radiation dosimetry suggested absorbed doses would be within guidelines for other 89Zr radiolabeled, clinically used antibodies. Pharmacokinetics of REGN3504 was linear.ConclusionThis work supports the clinical translation of 89Zr-REGN3504 immuno-PET for the assessment of PD-L1 expression. Future clinical studies will aim to investigate the utility of 89Zr-REGN3504 immuno-PET for predicting and monitoring response to anti-PD-1 therapy.

Conflict of interest:All authors are employees of Regeneron Pharmaceuticals and as such are shareholders and may be named on patent applications and patents. They otherwise have no conflicts of interest to declare.

Persistent antigen exposure and inflammatory signals in tumors induce expression of various co-inhibitory or immune checkpoint receptors on T cells, including programmed death protein 1 (PD-1) and Lymphocyte-Activation Gene 3 (LAG-3). Therapeutic antibodies blocking such co-inhibitory receptors have produced durable antitumor responses as single agents and in combinations. In order to monitor LAG-3 expression and potential changes in expression due to therapeutic intervention, we have developed a radionuclide-conjugated antibody to LAG-3 for immuno-PET. The fully human anti-LAG3 antibody REGN3767 was radiolabeled with the positron-emitting radionuclide Zirconium-89 (89Zr) using the bifunctional chelator p-SCN-Bn-Deferoxamine (DFO). 89Zr-REGN3767 demonstrated high radiochemical purity and immunoreactivity in cell binding assays. The ability of 89Zr-REGN3767 to successfully identify LAG-3 expression in vivo was initially assessed using MC38 mouse tumors expressing human LAG-3 (MC38/hLAG-3) implanted into immune-deficient mice. 89Zr-REGN3767 demonstrated higher uptake in MC38/hLAG-3 tumors compared to an 89Zr-isotype control antibody using immuno-PET, and specificity was confirmed by ex vivo biodistribution at day 6 post radiotracer injection (~35 and ~5 %ID/g for 89Zr-REGN3767 and 89Zr-isotype, respectively). Furthermore, a dose titration study of 89Zr-REGN3767 in immune deficient mice co-implanted subcutaneously with Raji lymphoma cells and human peripheral blood mononuclear cells (hPBMCs) demonstrated the ability of 89Zr-REGN3767 to target LAG-3-expressing intratumoral T-cells. 89Zr-REGN3767 immuno-PET and ex vivo biodistribution demonstrated specific localization to Raji/hPBMC co-implanted tumors; this uptake was significantly higher at antibody doses of 0.03 - 0.3 mg/kg than at 5 mg/kg. Doses of 0.03-0.3 mg/kg 89Zr-REGN3767 were also able to detect LAG-3 positive T cells in the spleen. This study shows the ability of 89Zr-REGN3767 to successfully image LAG-3 expressed on intratumoral and splenic T lymphocytes. This work supports the clinical translation of anti-LAG-3 immuno-PET for the assessment of LAG-3 expression, with the goal to investigate its utility for predicting and monitoring response to checkpoint blockade therapy. Citation Format: Marcus P. Kelly, Richard Tavare, Jason T. Giurleo, Sosina Makonnen, Carlos Hickey, Makenzie A. Danton, T Cody Arnold, Dangshe Ma, Jie Dai, Jerry Pei, Jessica R. Kirshner, William C. Olson, Gavin. Thurston. Immuno-PET detection of LAG-3 expressing intratumoral lymphocytes using the zirconium-89 radiolabeled fully human anti-LAG-3 antibody REGN3767 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3033.