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In recent years, immune checkpoint inhibitors (ICIs) have improved treatment for several types of cancer. However, the overall response rates remain low, and drug resistance can occur in patients after initial responses. This resistance is thought to be due in part to tumor-associated macrophages (TAMs), which can deploy a number of mechanisms to suppress anti-tumor immunity, resulting in tumor immune escape. Triggering receptor expressed on myeloid cells-2 (TREM2), a transmembrane receptor that is part of the immunoglobulin superfamily, is highly expressed on the surface of TAMs. Moreover, the expression level of TREM2 is reported to be higher in cancerous tissues vs normal tissues, with higher expression associated with poorer patient outcomes across multiple solid tumor types. Conversely, mice that lack Trem2 experience slower tumor cell expansion and are more responsive to anti-PD-1 immunotherapy, suggesting that TREM2 may be a promising therapeutic target for improving responses to ICI immunotherapies. To better assist the efficacy evaluation of anti TREM2 antibodies, Biocytogen has generated a humanized TREM2 mouse model. In these mice, exons 1-5 and the 3’UTR of mouse Trem2 gene, which encodes the full-length protein, were replaced by its human counterpart. In homozygous mice, human TREM2 protein expression is detected. The distribution of basal leukocyte subpopulations of blood, spleen, and lymph nodes in humanized B-hTREM2 mice were similar and comparable to those in wild-type C57BL/6 mice. Using the MC38 tumor model, we showed modest efficacy of an anti-human TREM2 antibody in inhibiting tumor growth in vivo. In summary, TREM2 humanized mice provide a powerful preclinical model for in vivo evaluation of anti-human TREM2 antibodies for cancer treatment. Citation Format: Ruili Lv, Jia Yu, Zhiyuan Shen, James Jin. Generation of humanized TREM2 mice for preclinical evaluation of therapeutics targeting tumor-associated macrophages. [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 5209.

In recent years, an increasing number of therapeutic antibodies have shown to be effective for the treatment of cancer and other diseases. However, limitations in the traditional discovery process, including immune tolerance of highly homologous genes, challenges with antibody sequence humanization, clone selection, and model selection for drug efficacy and safety evaluation, often hinder the process of identifying new therapeutic antibodies. The RenMice™ HiTS (Hyperimmune Target Specific) Platform is a library of chromosome engineered mice with fully human immunoglobulin variable domains replacing the mouse loci, each with a specific drug target gene knocked out. These mice are designed to establish robust immune responses and generate antibodies that bind to more epitopes of the target protein, including conserved domains. The platform is ideal for challenging targets, such as proteins with high homology across species, or multi-pass transmembrane proteins (e.g. GPCRs/ion channels). Here, we show that the platform can be used to generate antibodies that cross-react with multiple species, like human, monkey, dog, and mouse targets, by immunizing with both human and mouse or dog antigen. We provide examples for newer campaigns, including species cross-reactivity and internalization of novel antibodies targeting NECTIN-4, and high-throughput in vivo efficacy screening of novel anti-PD-1 antibodies in wild-type mice. In the future, we will evaluate the preliminary toxicity of these cross-reactive antibodies in preclinical animal models. Thus, selection of the best antibody candidate based on in vivo efficacy and safety allows for a streamlined and successful preclinical phase. In conclusion, the RenMice™ HiTS platform facilitates the generation of developable antibodies that recognize novel epitopes and challenging targets. Citation Format: Xiaoqian Zhang, Shufang Fu, Shujin Zhang, Xin Ji, Li Hui, James Jin, Jing Zhang. The RenMice™ HiTS (Hyperimmune target specific) Platform facilitates identification of novel therapeutic antibodies for challenging targets. [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 5320.

Regulatory T cells (Tregs) play an immunosuppressive role in the tumor microenvironment (TME), such that altering or depleting Treg function has emerged as an important anti-tumor strategy. Targeting CCR8, which is highly expressed in tumor-infiltrating Tregs, has shown its potency in reducing tumor burden and increasing responsiveness to immune-checkpoint blockade therapy. Collectively, this suggests a need for preclinical models that support Treg-targeted therapies. To address this, Biocytogen developed a triple humanized (B-hPD-1/hPD-L1/hCCR8) mouse model to evaluate in vivo efficacy of combination therapies. In this model, the full coding sequence of murine Ccr8 was replaced by the full coding sequence of human CCR8, while murine Pd-1 and Pd-l1 IgV domains were replaced by their human counterpart gene sequences. Human PD-1 and PD-L1 protein expression was confirmed in B-hPD-1/hPD-L1/hCCR8 mice by flow cytometry, and human CCR8 protein expression was observed in MC38 tumors harvested from B-hPD-1/hPD-L1/hCCR8 mice. Finally, combination therapy using anti-human PD-1 and anti-human CCR8 antibodies were effective in controlling B-hPD-L1 MC38 tumor growth in B-hPD-1/hPD-L1/hCCR8 mice. Altogether, this line provides a powerful preclinical model for in vivo evaluation of therapeutic antibodies. Citation Format: Linlin Wang, Xiao Liu, James Jin, Yuxiang Yu. A novel triple humanized B-hPD-1/hPD-L1/hCCR8 mouse model to evaluate in vivo efficacy of combination therapies [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 3233.

The use of immune-competent mouse models that express target human genes provide a promising preclinical platform aimed at developing novel immunotherapies. Emerging immunotherapies include targeting OX40 and OX40 ligand (OX40L), which can lead to enhanced T cell activation, proliferation, and effector function. Blocking the OX40/OX40L pathway improves autoantigen-specific T cell responses and decreases immunocompetence across several autoimmune diseases. To explore the potential of OX40 and OX40L antibody efficacy studies, we developed double humanized B-hOX40/hOX40L mice by replacing the extracellular domain sequences of murine Ox40 and Ox40l with the corresponding human sequences. We validated human OX40L gene expression by RT-PCR and OX40/OX40L protein expression by flow cytometry in B-hOX40/hOX40L mice. Additionally, percentages of splenic, blood, and lymph node immune cells were similar between B-hOX40/hOX40L and wild-type C57BL/6 mice. Altogether, this data demonstrates that B-hOX40/hOX40L mice are suitable for in vivo efficacy studies using anti-human OX40 and OX40L antibodies. Citation Format: Chonghui Liu, Jiawei Yao, Xiaofei Zhou, James Jin, Zan Zhang. Humanized OX40/OX40L mice as a tool for evaluating novel therapeutics. [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 5178.

Signal regulatory protein α (SIRPα) is a transmembrane protein with an extracellular region comprising three Ig-like domains and a cytoplasmic region containing immunoreceptor tyrosine-based inhibition motifs which mediate binding of the protein tyrosine phosphatases SHP1 and SHP2. SIRPα is especially abundant in myeloid cells such as macrophages and DCs, whereas it is expressed at very low levels in T, B, NK, and NK T cells. SIRPα inhibits phagocytosis in macrophages upon interacting with its ligand CD47, which is commonly upregulated on the surface of malignant cells. Thus, antibodies that block the CD47-SIRPα interaction should enhance macrophage phagocytosis in the tumor microenvironment and inhibit tumor growth, making anti-SIRPα antibodies promising as potential new tools for cancer immunotherapy. We are interested in subverting the negative feedback of the CD47-SIRPa axis, in order to treat cancer patients. Using Biocytogen’s antibody research platform, we obtained several high-affinity anti-human SIRPα mAb by high-throughput flow cytometry screening. Next, these murine antibodies were screened directly in single or double humanized mice models (B-hSIRPα or B-hSIRPα/hCD47) bearing a syngeneic humanized cancer cell line (MC38-hCD47), where we discovered candidates that have potent anti-tumor activity in vivo. Chimeric antibodies and humanized antibodies based on these candidates were generated and further examined in vivo. In addition, we uncovered synergistic anti-tumor activity of anti-human SIRPα mAbs in combination with anti-mouse PD-1 mAb in B-hSIRPα/hCD47 mice. In brief, our existing studies demonstrate that SIRPα is an ideal target for preclinical cancer models. Clinical evaluation of the translational merits for these novel anti-SIRPα antibodies is pending. Citation Format: James Jin, Benny(Yi) Yang, Chaoshe Guo, Yuelei Shen, Jian Ni. Anti-SIRPα antibodies as a potential weapon for cancer immunotherapy via accelerated screening in humanized mouse models [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 1212.

CD40, Cluster of Differentiation 40, is a member of the TNF-receptor superfamily. CD40 is a co-stimulatory protein found primarily on antigen presenting cells, including B cells, macrophages and dendritic cells. Its ligand, CD40L (CD154) is predominantly expressed on T helper cells, whose engagement activates antigen presenting cells and induces a variety of downstream effects. Whereas most immune checkpoint receptors are expressed on the surface of T cells, CD40 stands out as it is expressed by antigen presenting cells and controls the proper activation of these cells. In turn, CD40 activation exerts a significant impact on T cell-mediated activity against cancer cells. Several pharmaceutical companies are pursuinganti-CD40 therapiesfor treating hematological and solid cancers. Having an access to Biocytogen's antibody discovery and in vivo efficacy screening platform for immune checkpoints, we asked whether we could find suitable anti-CD40 reagents which can successfullycombat developed tumors in preclinical settings. We designed a powerful screening strategy using Biocytogen's humanized CD40 mouse (B-hCD40). Candidate antibodies were generated by immunizing wild-type mice with recombinant hCD40 protein. Then the hybridomas were screened by high-throughput flow cytometry. Instead of examining the immune modulating activity in cell culture, we applied these antibodies to B-hCD40 mice bearing MC38 tumors. Through this straightforwardscreening, several clones stood out by their unparallelingbenefits. These clones were further selected for humanization. Again, the cohort of recombinant humanized CD40 antibodies was screened in B-hCD40 mice. In the end, we discovered the leads with the most potent anti-tumor activity. In conclusion, our work successfully identified several CD40 antibodies that are highly effective for cancer treatment in the preclinical setting. Citation Format: James Jin, Yanan Guo, Yuelei Shen, Benny Yang. Fast screening of CD40 agonistic antibodies for cancer immunotherapy [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 5762.

Over the past years, monoclonal antibodies have been successfully utilized in clinical trials to block or activate key mediators of immune checkpoint pathways, including CTLA-4, PD-1, PD-L1 and OX40 et.al,. Although significant benefits including complete regression and long-term survival were reported among the majority of participants, not all patients respond. In order to optimize the efficacy and minimize toxicities of anticancer immunotherapies, immune checkpoint antibodies are being tested in combination with other conventional therapeutics (radiation, chemotherapy and targeted therapies) and newer immunotherapies (cancer vaccines, oncolytic viruses). However, along the IO drug development process, in vivo efficacy models have always been a rate-limiting step, especially for the combination therapy of two IO antibodies. Therefore, we generated humanized double knock-in mouse to evaluate the in vivo efficacy of combination therapy. For example, humanized double Knock-in B-hPD-1/hOX40 mice pairing with mouse cancer cells, demonstrated a synergistic effect of using hPD-1 and hOX40 antibodies. Additionally, more double knock-in mice targeting immune checkpoint molecules were developed, such as B-hPD-1/hPD-L1, B-hPD-1/hCTLA-4, B-hPD-1/hLAG3, B-hPD-1/hTIM3, B-hPD-1/hTIGIT, B-hPD-1/hBTLA, et al,. Humanized double knock-in mice, represent useful in vivo efficacy models for evaluation of the combination effects of various IO drugs. Citation Format: Tian Gan, James Jin, Chaoshe Guo, Yuelei Shen, Yanan Guo. Humanized double knock-in mouse models for evaluating in vivo efficacy of the combination therapy of immune-oncology drugs [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 5688.