Abstract Immune checkpoint inhibitors (ICIs) has revolutionized the treatment landscape of various cancers by reinvigorating the exhausted T cells in patients. However, the therapeutic efficacy is largely confined due to the primary or acquired resistance to anti-PD-1 (L1) therapy in many patients. Recently, the mechanisms of resistance to ICIs has been extensively elucidated from different aspects and emerging sequencing data from clinical samples has pointed to IFN-γ signaling defects and antigen presentation loss in patients who are resistant to PD-1(L1) blockade. The loss-of-function mutations in JAK1 and JAK2 results in lack of response to IFN-γ signaling and incapacity to upregulate PD-L1 and MHC-I, which subsequently leads to noninflammatory TMEs and resistance to anti-PD-1/PD-L1. The loss-of-function of B2M is likely a common resistance mechanism that the intratumoral cytotoxic CD8+ T cells are failed to be boosted owing to antigen presentation loss. In order to obtain higher clinical benefits in various cancers, it’s appealing and urgent to develop mechanism-based strategies to overcome resistance to ICI-based immunotherapy. To this end, we developed a genetically acquired resistant tumor model with B2M mutations in syngenic MC38 tumor cells using CRISPR/Cas9 technology, which is ‘hot’ tumor to PD-1/PD-L1 blockade. The expression of B2M was detected by western blotting and flow cytometry. Mouse B2M-knockout MC38 tumors became resistant to PD-1/PD-L1 blockade in vivo. Notably, we found a remarkable increase of CD8+ T cell infiltration in B2m-KO MC38 tumors in comparison to parental MC38 tumors upon anti-PD-1 treatment, which further explains that antigen presentation loss results in inactivation of cytotoxic CD8+ T cells within the tumor microenvironments (TMEs), subsequently leading to resistance and poor survival to ICI therapy.In summary, our data consistently indicated the importance of MHC-I expression in T cell activation in the TMEs and the possibility to dig out the pathway involved in MHC-I expression independent of IFN-γ signaling by CRISPR-based screening to overcome MHC-I deficiency-induced resistance in the future. In addition, it’s possible to leverage the cytotoxic NK cells and CD4+ T cells in antitumor immunity against MHC-II+ tumors even with the loss of MHC-I molecules. For example, high dose of IL-2RB-biased IL-2 agonist preferentially binding to the dimeric IL-2R can stimulate Teff and NK cells independent of immune checkpoint expression. Likewise, cytokine modified anti-PD-1 fusion formulation like anti-PD-1-IL-2 mutein and anti-PD-1-IL-15 mutein are promising to overcome the resistance. Furthermore, it has been suggested that the resistance due to genetic mutations of MHC-I could be overcome by combining NF-B targeted therapies. Citation Format: Yanlei Zhang, Hechun Ma, Boang Han, Yi Li, Ping Yang, Zhen Li, Dongxiao Feng, Lei Ci, Ruilin Sun, Daniel X. He. Genetic ablation of B2M leads to resistance to PD-1/PD-L1 blockade in vivo [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2666.

Background Depletion of tumor-resident CCR8+ Treg cells has been demonstrated remarkable antitumor effects. A few hCCR8-targeted therapeutic antibodies have been developed and are now undergone clinical testing in patients with advanced or metastatic solid tumors. However, the challenge remains in preclinical evaluation for human-specific CCR8 mAbs in immunocompetent system.1–3 Therefore, we developed the first-generation chimeric h/m CCR8 knockin mouse model, i.e., hCCR8 (v1) for in vivo test of anti-hCCR8 mAbs. Whereas, we found that MC38 syngeneic tumors displayed no responses to anti-hCCR8 blockade in hCCR8 (v1) mice, which has been a 'hot' tumor model to ICIs or Treg-depletion. We guess that the conformational change of the 7-transmembrane GPCR does matter, because hCCR8 (v1) mice express chimeric h/mCCR8 proteins that only aa 1–280 are humanized and aa 281–355 are still mouse sequence.4 Hence, we developed the second-generation hCCR8 knockin mouse model, i.e., hCCR8 (v2) for this, in which only full-length of human CCR8 proteins are expressed for homozygous hCCR8 (v2). Here we demonstrated that hCCR8 (v2) mice have higher level of hCCR8 protein expression on the activated CD4+ and CD8+ T cells as well as Treg cells. Intriguingly, we observed distinct antitumor effects in hCCR8 (v2) mice engrafted with MC38 tumors treated with anti-hCCR8 at both low and high dose. Methods The hCCR8 (v2) knockin mice was established via CRISPR/Cas9 engineering that the entire coding sequence of mouse CCR8 is replaced by full-length human CCR8 in C57BL/6 mice. The expression of full-length hCCR8 protein in hCCR8 (v2) knockin mice was validated by FACS. Then, the binding and EC50 of BMS-986340 and anti-hCCR8 (BD, clone 433H) was determined by FACS using the activated splenocytes from hCCR8 (v2) knockin mice by anti-mCD3/anti-mCD28.2/mIL-2. Finally, hCCR8 (v2) knockin mice were engrafted with MC38 syngeneic tumors and treated with anti-hCCR8 (BMS-986340) via i.p. injection to evaluate tumor growth inhibition (TGI) of anti-hCCR8 mAbs in vivo. Results Anti-hCCR8 mAb led to ~38% TGI at 2 mg/kg and ~50% TGI at 10 mg/kg in MC38 tumor model, respectively. We observed the tumor-resident CCR8+ Treg populations decreased significantly and CD4+/CD8+ T cells proliferation as well as PD-1 upregulation in CD4+/CD8+ T cells 24h post 6th dose. Conclusions The hCCR8(v2) mouse model is a more suitable preclinical humanized model distinguished with the previous hCCR8(v1) mouse model, enabling the in vivo evaluation of various human-specific CCR8 mAbs alone or in combination with immune checkpoint inhibitors like PD-1 and CTLA-4. References Plitas G, Konopacki C, et al. Regulatory T Cells Exhibit Distinct Features in Human Breast Cancer. I 2016;45(5):1122–1134. Damme H, Dombrecht B, et al. Therapeutic depletion of CCR8+ tumor-infiltrating regulatory T cells elicits antitumor immunity and synergizes with anti-PD-1 therapy. J Immunother Cancer. 2021;9(2):e001749. Kidani Y, Nogami W, et al. CCR8-targeted specific depletion of clonally expanded Treg cells in tumor tissues evokes potent tumor immunity with long-lasting memory. Proc Natl Acad Sci U S A. 2022;119(7):e2114282119. He X, Li Q. In vitro and in vivo characterization of CCR8 humanized mouse model (HuGEMM™). Mol Cancer Ther. 2021;20:Abstract P003. Ethics Approval All studies were conducted following an approved IACUC protocol (SMOC-IACUC NO. 2022–0049). Although this study was not conducted in accordance with the FDA Good Laboratory Practice regulations, 21 CFR Part 58, all experimental data management and reporting procedures were in strict accordance with applicable Shanghai Model Organisms Center, Inc. Guidelines. Guidelines and Standard Operating Procedures. The methods and results in this study accurately reflect the raw data generated during the execution of the study.

Background The development of antibody-based therapeutics for the neurological diseases and glioma is largely hampered due to the blood-brain barrier (BBB). It is appealing to identify more promising targets that are highly selectively expressed on brain endothelial cells (BECs) which could be leveraged to transport antibodies across the BBB into the brain.1 2 TFR1 (transferrin receptor 1), also known as CD71, is such an example which has been most extensively studied for receptor-mediated transcytosis (RMT) for drug delivery to the brain.3 4 However, actively proliferating cells, reticulocytes as well as tumor cells can also express TFR1. The increased expression of TFR1 is associated with the poor prognosis in various cancer types, for example, NSCLC and esophageal squamous cell carcinoma. Thus, TFR1 can also be a therapeutic target against tumor by developing either TFR1-targeted antibody drug conjugations (ADC) via RMT or TFR1 mAb-mediated ADCC.5 6 Taken together, we developed a humanized TFR1 knockin mouse model, which could be a powerful preclinical model to enable assessment of the efficacy, safety and PK/PD of therapeutic antibodies specifically against human TFR1/CD71 in vivo. Methods We developed the humanized TFR1 knockin mouse model by insertion of whole human TFR1 coding sequence plus WPRE/polyA stop cassette into mouse Cd71 gene locus in C57BL/6 background via ES cell-based gene targeting. To characterize the hTFR1 knockin mice, firstly, we verified hTFR1 expression on the brain endothelial cells by immunohistochemistry (IHC) and then we performed flow cytometry analysis to characterize hTFR1 expression on activated T cells after in vitro stimulation by PHA-L and anti-mCD3/mCD28 for various exposure time. Additionally, the hTFR1 expression on bone marrow-derived erythroid cells was confirmed by FACS. Finally, we treated the hTFR1 knockin mice with an anti-human TFR1 antibody and control antibody via i.v. injection to evaluate the in vivo PK and brain uptake capability across the BBB. Results We observed increased expression of hTFR1 on activated T lymphocytes upon stimulation. Brain tissues and serums were taken from the hTFR1 knockin mice i.v. injected with a human-specific TFR1 antibody (10 mg/kg) and a control antibody (10mg/kg) as a single dose 18 hours post treatment for PK analysis. The antibody concentrations in brain and serum were quantified by ELISA. Human-specific TFR1-binding antibody exhibited higher serum clearance and enhanced brain uptake against blood-brain barrier. Conclusions Our humanized TFR1 knockin mice provides a powerful tool to evaluate the in vivo efficacy, safety and PK/PD of the therapeutic antibodies targeting human TFR1/CD71 in the preclinical investigations. References Bell RD, Ehlers MD. Breaching the blood-brain barrier for drug delivery. Neuron. 2014;81(1):1–3. Terstappen GC, et al. Strategies for delivering therapeutics across the blood-brain barrier. Nat Rev Drug Discov. 2021;20(5):362–383. Niewoehner J, et al. Increased brain penetration and potency of a therapeutic antibody using a monovalent molecular shuttle. Neuron. 2014;81(1):49–60. Hultqvist G, et al. Bivalent brain shuttle increases antibody uptake by monovalent binding to the transferrin receptor. Theranostics. 2017;7(2):308–318. Johnson M, et al. Phase I, first-in-human study of the probody therapeutic CX-2029 in adults with advanced solid tumor malignancies. Clin Cancer Res. 2021;27(16):4521–4530. Candelaria PV, et al. Antibodies Targeting the Transferrin Receptor 1 (TfR1) as Direct Anti-cancer Agents. Front Immunol. 2021;12:607692. Ethics Approval All studies were conducted following an approved IACUC protocol (SMOC IACUC No. 2022–0008). Although this study was not conducted in accordance with the FDA Good Laboratory Practice regulations, 21 CFR Part 58, all experimental data management and reporting procedures were in strict accordance with applicable Shanghai Model Organisms Center, Inc. Guidelines. Guidelines and Standard Operating Procedures. The methods and results in this study accurately reflect the raw data generated during the execution of the study.