CX3CL1 secreted in the tumor microenvironment serves as a chemoattractant playing a critical role in metastasis of CX3CR1 expressing cancer cells. CX3CR1 can be expressed in both cancer and immune-inhibitory myeloid cells to facilitate their migration. We generated a novel monoclonal antibody against mouse CX3CR1 that binds to CX3CR1 and blocks the CX3CL1-CX3CR1 interaction. We next explored the immune evasion strategies implemented by the CX3CL1-CX3CR1 axis and find that it initiates a resistance program in cancer cells that results in 1) facilitation of tumor cell migration, 2) secretion of soluble mediators to generate a pro-metastatic niche, 3) secretion of soluble mediators to attract myeloid populations, and 4) generation of tumor-inflammasome. The CX3CR1 monoclonal antibody reduces migration of tumor cells and decreases secretion of immune suppressive soluble mediators by tumor cells. In combination with anti-PD-1 immunotherapy, this CX3CR1 monoclonal antibody enhances survival in an immunocompetent mouse colon carcinoma model through a decrease in tumor-promoting myeloid populations. Thus, this axis is involved in the mechanisms of resistance to anti-PD-1 immunotherapy and the combination therapy can overcome a portion of the resistance mechanisms to anti-PD-1.

Autoantibodies against thyroid proteins are present in several thyroid diseases. Thyroid-stimulating hormone receptor (TSHR) is a G-protein-coupled receptor (GPCR) that binds to thyroid-stimulating hormone (TSH) and stimulates production of thyroxine (T4) and triiodothyronine (T3). When agonized by anti-TSHR autoantibodies, aberrant production of thyroid hormone can lead to Graves' Disease (GD). In Hashimoto's thyroiditis (HT), anti-TSHR autoantibodies target the thyroid for immune attack. To better understand the role of anti-TSHR antibodies in thyroid disease, we generated a set of rat antimouse (m)TSHR monoclonal antibodies with a range of affinities, blocking of TSH, and agonist activity. These antibodies could be used to investigate the etiology and therapy of thyroid disease in mouse models and as building blocks in protein therapeutics that target the thyroid for treatment in either HT or GD.

As severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) evolves to escape natural antibodies, it also loses sensitivity to therapeutic antibody drugs. By contrast, evolution selects for binding to ACE2, the cell-surface receptor required for SARS-CoV-2 infection. Consistent with this, we find that an ACE2 decoy neutralizes antibody-resistant variants, including Omicron, with no loss in potency. To identify design features necessary for in vivo activity, we compare several enzymatically inactive, Fc effector–silenced ACE2-Fc decoys. Inclusion of the ACE2 collectrin-like domain not only improves affinity for the S protein but also unexpectedly extends serum half-life and is necessary to reduce disease severity and viral titer in Syrian hamsters. Fc effector function is not required. The activity of ACE2 decoy receptors is due, in part, to their ability to trigger an irreversible structural change in the viral S protein. Our studies provide a new understanding of how ACE2 decoys function and support their development as therapeutics to treat ACE2-dependent coronaviruses.

The PD-1 pathway is a cornerstone in immune regulation. While the PD-1 pathway has received considerable attention for its role in contributing to the maintenance of T cell exhaustion in chronic infection and cancer, the PD-1 pathway plays diverse roles in regulating host immunity beyond T cell exhaustion. Here, we discuss emerging concepts in the PD-1 pathway, including (1) the impact of PD-1 inhibitors on diverse T cell differentiation states including effector and memory T cell development during acute infection, as well as T cell exhaustion during chronic infection and cancer, (2) the role of PD-1 in regulating Treg cells, NK cells, and ILCs, and (3) the functions of PD-L1/B7-1 and PD-L2/RGMb/neogenin interactions. We then discuss the emerging use of neoadjuvant PD-1 blockade in the treatment of early-stage cancers and how the timing of PD-1 blockade may improve clinical outcomes. The diverse binding partners of PD-1 and its associated ligands, broad expression patterns of the receptors and ligands, differential impact of PD-1 modulation on cells depending on location and state of differentiation, and timing of PD-1 blockade add additional layers of complexity to the PD-1 pathway, and are important considerations for improving the efficacy and safety of PD-1 pathway therapeutics.

829 Background: Microsatellite instability-high (MSI-H) metastatic colorectal cancer (mCRC) patients who have failed chemotherapy have shown response to checkpoint blockade. We investigate optimal third-line treatment in MSI-H mCRC with regard to overall survival, quality of life years gained (QALYs), and cost-effectiveness. Methods: A Markov Model was created for a base case of a 57 year old man with MSI-H mCRC refractory to two lines of chemotherapy. Treatments compared were nivolumab, nivolumab with ipilimumab, and trifluridine/tipiracil. Patients could remain stable, progress to fourth-line chemotherapy or palliative care, experience drug toxicity, die from age/sex mortality, or die from cancer over their simulated lifetimes. Transitions between health states were based on the CheckMate 142 and RECOURSE trials. Outcomes were survival or unadjusted life years, QALYs, and incremental cost-effectiveness ratios (ICERs). The willingness to pay threshold was $100,000/QALY. Results: Nivolumab with ipilimumab was the most effective strategy as it yielded more unadjusted life-years (4.24) and QALYs (2.53) compared to nivolumab (3.95 LY, 2.33 QALYs) and trifluridine/tipiracil (0.74 LY, 0.07 QALYs). However, nivolumab with ipilimumab was not cost-effective compared to nivolumab and neither treatment strategy was cost-effective compared to trifluridine/tipiracil. Sensitivity analysis found nivolumab monotherapy could be cost-effective with decrease in drug cost to $2000/dose. Conclusions: Our modeling analysis finds that both single and dual checkpoint blockade yield significantly increased overall survival and QALYs for MSI-H mCRC compared to third-line chemotherapy, but were not cost-effective because of nivolumab cost. Decreases in drug pricing and/or duration of maintenance nivolumab could make nivolumab monotherapy cost-effective. [Table: see text]

Background Patients with microsatellite instability-high (MSI-H)/mismatch repair-deficient (dMMR) metastatic colorectal cancer (mCRC) show a significant response to checkpoint inhibitor therapies, but the economic impact of these therapies is unknown. A decision analytic model was used to explore the effectiveness and cost burden of MSI-H/dMMR mCRC treatment. Methods The treatment of hypothetical patients with MSI-H/dMMR mCRC was simulated in 2 treatment scenarios: a third-line treatment and an exploratory first-line treatment. The treatments compared were nivolumab, ipilimumab and nivolumab, trifluridine and tipiracil (third-line treatment), and mFOLFOX6 and cetuximab (first-line treatment). Disease progression, drug toxicity, and survival rates were based on the CheckMate 142, study of TAS-102 in patients with metastatic colorectal cancer refractory to standard chemotherapies (RECOURSE), and Cancer and Leukemia Group B/Southwest Oncology Group 80405 trials. The analyzed outcomes included survival (life-years), quality-adjusted life-years (QALYs), and incremental cost-effectiveness ratios (ICERs). Results Ipilimumab with nivolumab was the most effective strategy (10.69 life-years and 9.25 QALYs for the third line; 10.69 life-years and 9.44 QALYs for the first line) in comparison with nivolumab (8.21 life-years and 6.76 QALYs for the third line; 8.21 life-years and 7.00 QALYs for the first line), trifluridine and tipiracil (0.74 life-years and 0.07 QALYs), and mFOLFOX6 and cetuximab (2.72 life-years and 1.63 QALYs). However, neither checkpoint inhibitor therapy was cost-effective in comparison with trifluridine and tipiracil (nivolumab ICER, $153,000; ipilimumab and nivolumab ICER, $162,700) or mFOLFOX6 and cetuximab (nivolumab ICER, $150,700; ipilimumab and nivolumab ICER, $158,700). Conclusions This modeling analysis found that both single and dual checkpoint blockade could be significantly more effective for MSI-H/dMMR mCRC than chemotherapy, but they were not cost-effective, largely because of drug costs. Decreases in drug pricing and/or the duration of maintenance nivolumab could make ipilimumab and nivolumab cost-effective. Prospective clinical trials should be performed to explore the optimal duration of maintenance nivolumab.

Abstract 3713 Background: The complementarity determining region, or idiotype, of the surface immunoglobulin receptor is a tumor-specific marker on B-cell lymphomas that is unique to each patient. Antibodies against idiotype can induce complete regression of lymphoma in patients, but since this therapeutic approach requires the generation of a custom monoclonal antibody for each patient, it has not been practical. Objective: Here we describe a method for targeting the idiotype on the surface of a B-cell lymphoma by using synthetic idiotype-ligands covalently linked to a recombinant IgG Fc domain (Figure 1A). These peptide idiotype-ligands can be identified through oligopeptide library screens and produced inexpensively by automated solid-phase synthesis. Linkage of idiotype-ligands to the Fc domain serves two purposes: to enhance their pharmacokinetics and to augment their anti-tumor effect by activating immune effector functions. Since each patient-specific peptide can be chemically linked to a common IgG Fc domain, this modular construct design yields a patient-specific therapeutic that does not require the production of a custom biologic macromolecule for each patient. Results: Idiotype peptide-ligands were produced by solid-phase synthesis and covalently linked to the amino-terminus of a recombinant mouse IgG2a Fc domain by native chemical ligation, a method for site-selective polypeptide ligation. The resulting peptibody demonstrated targeted killing of a human lymphoma cell line in vitro by crosslinking surface immunoglobulin and triggering activation-induced death. Additionally, the peptibody triggered complement-mediated cytotoxicity of opsonized lymphoma cells in vitro and activated natural killer cells co-cultured with opsonized lymphoma cells. The peptibody exhibited a favorable pharmacokinetic profile and peptibody treatment was sufficient to clear tumor in SCID mice challenged intravenously with a luciferase-labeled human lymphoma cell line ( p = 0.0018; Figure 1B-D). Conclusions: Idiotype-specific peptibodies demonstrate multimodal activity against lymphoma cells in vitro and clear human lymphoma in a disseminated xenograft model. The modular design of this therapeutic may enable a personalized and targeted therapy that is feasible to produce for patients with B-cell lymphoma. Disclosures: No relevant conflicts of interest to declare.

Purpose Combining tumor antigens with an immunostimulant can induce the immune system to specifically eliminate cancer cells. Generally, this combination is accomplished in an ex vivo, customized manner. In a preclinical lymphoma model, intratumoral injection of a Toll-like receptor 9 (TLR9) agonist induced systemic antitumor immunity and cured large, disseminated tumors. Patients and Methods We treated 15 patients with low-grade B-cell lymphoma using low-dose radiotherapy to a single tumor site and—at that same site—injected the C-G enriched, synthetic oligodeoxynucleotide (also referred to as CpG) TLR9 agonist PF-3512676. Clinical responses were assessed at distant, untreated tumor sites. Immune responses were evaluated by measuring T-cell activation after in vitro restimulation with autologous tumor cells. Results This in situ vaccination maneuver was well-tolerated with only grade 1 to 2 local or systemic reactions and no treatment-limiting adverse events. One patient had a complete clinical response, three others had partial responses, and two patients had stable but continually regressing disease for periods significantly longer than that achieved with prior therapies. Vaccination induced tumor-reactive memory CD8 T cells. Some patients' tumors were able to induce a suppressive, regulatory phenotype in autologous T cells in vitro; these patients tended to have a shorter time to disease progression. One clinically responding patient received a second course of vaccination after relapse resulting in a second, more rapid clinical response. Conclusion In situ tumor vaccination with a TLR9 agonist induces systemic antilymphoma clinical responses. This maneuver is clinically feasible and does not require the production of a customized vaccine product.

Abstract Abstract 722 Immunomodulating monoclonal antibodies (mAb) directed against immune cell targets can be used to enhance antitumor immune responses. CD137 (4-1BB) is a costimulatory molecule expressed on a variety of activated immune cells, including T and NK cells. Anti-CD137 agonistic mAb has demonstrated antitumor activity in various tumor models and has now entered clinical trials for the treatment of solid tumors (BMS-663513). Here, we investigate the therapeutic potential of anti-CD137 mAb in lymphoma. Using microarray gene expression data, we compared CD137 mRNA expression across 17 different types of cancer. We found that lymphoma tumor specimens significantly over-expressed CD137 mRNA compared to other tumors (p<0.0001). Single cell analysis of primary lymphoma samples of various histologies (follicular lymphoma [n=29], mantle cell lymphoma [n=14], and diffuse large B cell lymphoma [n=11]) revealed that CD137 was expressed by a significant proportion of tumor-infiltrating T cells, particularly in the CD8 subset (mean[range]=6.1%[2.5-11.5], 15.4%[2.7-40.9] and 18.7%[6.7-51.7], respectively). In contrast, CD137 was not expressed by the tumor cells. These results suggest that the target of anti-CD137 mAb is present and selectively expressed on cells with potential antitumor activity in patients with lymphoma. Using a murine model, we then investigated the anti-lymphoma activity of anti-CD137 agonistic mAb in vivo. BALB/c mice, bearing large and established A20 lymphoma tumors, were treated with anti-CD137 mAb or other immunomodulating mAbs (anti-OX40, anti-CTLA4, anti-GITR) for comparison. The mAbs were given i.p. on days 5 and 10 post tumor inoculation. Anti-CD137 mAb demonstrated potent anti-lymphoma activity in vivo, and appeared to be significantly superior to the other immunomodulating mAbs tested (Figure 1). The antitumor effect of anti-CD137 mAb was not due to direct targeting of the malignant cells as A20 tumor cells do not express CD137. Depletion experiments revealed that anti-CD137 therapy required both NK and CD8 T cells. Analysis of tumor-bearing mice showed that anti-CD137 therapy increased the number of CD8 T cells and reduced the number of Tregs at the tumor site. Anti-CD137 therapy also induced long-lasting antitumor immunity as cured mice harbored antitumor IFN-g producing memory CD8 T cells and were protected from tumor re-challenge more than 100 days after initial therapy. In conclusion, this study demonstrates for the first time that anti-CD137 agonistic mAb has potent antitumor activity in lymphoma. These results support the evaluation of anti-CD137 mAb in clinical trials for patients with lymphoma. Disclosures: No relevant conflicts of interest to declare.

Despite the success of passive immunotherapy with monoclonal antibodies (mAbs), many lymphoma patients eventually relapse. Induction of an adaptive immune response may elicit active and long-lasting antitumor immunity, thereby preventing or delaying recurrence. Immunomodulating mAbs directed against immune cell targets can be used to enhance the immune response to achieve efficient antitumor immunity. Anti-CD137 agonistic mAb has demonstrated antitumor efficacy in various tumor models and has now entered clinical trials for the treatment of solid tumors. Here, we investigate the therapeutic potential of anti-CD137 mAb in lymphoma. We found that human primary lymphoma tumors are infiltrated with CD137+ T cells. We therefore hypothesized that lymphoma would be susceptible to treatment with anti-CD137 agonistic mAb. Using a mouse model, we demonstrate that anti-CD137 therapy has potent antilymphoma activity in vivo. The antitumor effect of anti-CD137 therapy was mediated by both natural killer (NK) and CD8 T cells and induced long-lasting immunity. Moreover, the antitumor activity of anti-CD137 mAb could be further enhanced by depletion of regulatory T cell (T(regs)). These results support the evaluation of anti-CD137 therapy in clinical trials for patients with lymphoma.