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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.

Background Interleukin-12 (IL12) is a proinflammatory cytokine produced by activated antigen-presenting cells that induces differentiation of Th1 cells and increased proliferation and cytotoxicity of T and NK cells. Stimulation of these cells by IL12 leads to production of high levels of IFNγ. These immune-stimulating aspects of IL12 may help to establish an inflammatory tumor microenvironment critical for anti-tumor responses. Preclinical studies in mice revealed that native IL12 can dramatically shrink syngeneic tumors, however clinical studies in humans resulted in severe toxicity and a small therapeutic window, limiting response rates. Prior work at Xencor demonstrated that reduced-potency IL15/IL15Rα-Fc fusion proteins exhibited superior pharmacokinetics, pharmacodynamics, and safety in non-human primates through reduction of receptor-mediated clearance. Applying similar principles to IL12, we created IL12 heterodimeric Fc-fusions (IL12-Fc) with reduced potency to improve tolerability, slow receptor-mediated clearance, and extend half-life. Methods IL12 is a heterodimeric protein consisting of two subunits, so we engineered IL12-Fc fusions by fusing the IL12p35 subunit to one side of a heterodimeric (and inactive) Fc domain, and the IL12p40 subunit to the other side. These Fc-fusions were tuned for optimal activity by introducing amino acid substitutions at putative receptor-interface positions and screening for reductions of in vitro potency. In vitro activity was assessed on human PBMCs by measuring signaling in a STAT4 phosphorylation assay and IFNγ production in a mixed-lymphocyte reaction (MLR). In vivo anti-tumor activity was assessed by engrafting MCF-7 cells into PBMC engrafted NSG MHC class I and II double-knockout mice and by measuring tumor volume, lymphocyte activation/proliferation, and IFNγ production over time. Results IL12-Fc were produced with good yield and purity. An IL12-Fc potency series was created, and variants had up to a 10,000-fold reduction in STAT4 signaling potency and IFNγ production in an MLR assay compared to native IL12-Fc. Anti-tumor activity in the huPBMC-MCF7 model was achieved with potency-reduced IL12-Fc as a single-agent and in combination with anti-PD1, with weaker variants maintaining anti-tumor activity at higher dose levels. Analysis of peripheral lymphocytes indicated increased numbers of T and NK cells as well as activation of CD8+ T cells, as evidenced by upregulation of CD25. Increased expression of immune checkpoints including PD1 was also observed. Analysis of serum indicated up to 200-fold increases in IFNγ levels. Conclusions Combined, these data indicate that potency-reduced IL12-Fc retain strong anti-tumor activity, while potentially overcoming safety and tolerability issues related to small therapeutic index associated with recombinant native IL12 or IL12-Fc agents.

BackgroundInterleukin-12 (IL12) is a proinflammatory cytokine that induces differentiation of Th1 cells and increased cytotoxicity of T and NK cells. Stimulation by IL12 leads to production of IFNγ and an inflammatory tumor microenvironment critical for anti-tumor responses. Studies in mice revealed IL12 can dramatically shrink syngeneic tumors, however human clinical studies resulted in severe toxicity and a small therapeutic window, limiting response rates. Prior work at Xencor demonstrated that reduced-potency IL15/IL15Rα-Fc fusion proteins exhibited superior therapeutic index (TI) in non-human primates (NHP) by reducing receptor-mediated clearance. Applying similar principles to IL12, we created IL12 heterodimeric Fc-fusions (IL12-Fc) with reduced potency to improve TI.MethodsIL12 is a heterodimer of two subunits, so we engineered IL12-Fc fusions by fusing the IL12p35 subunit to one side of a heterodimeric (and inactive) Fc domain, and IL12p40 to the other side. These Fc-fusions were tuned for optimal activity by introducing amino acid substitutions at putative receptor-interface positions and screening for reductions of in vitro potency. In vitro activity was assessed on human PBMCs by measuring signaling in a STAT4 phosphorylation assay and IFNγ production in a mixed-lymphocyte reaction (MLR). In vivo anti-tumor activity of human IL12-Fc was assessed in huPBMC-NSG-DKO and huCD34+ MCF7 xenograft models. Surrogate mouse potency-reduced IL12-Fc were evaluated in syngeneic tumor models. Tolerability and pharmacodynamic activity were assessed in NHP.ResultsAn IL12-Fc potency series was created, and variants had up to a 10,000-fold reduction in STAT4 signaling and IFNγ production in an MLR assay compared to wild-type IL12-Fc. Anti-tumor activity was achieved with potency-reduced IL12-Fc as single-agents and in combination with anti-PD1, with weaker variants maintaining anti-tumor activity at higher dose levels. Analysis of peripheral lymphocytes indicated increased numbers of T and NK cells as well as activation of CD8+ T cells. Increased expression of immune checkpoints including PD1 was also observed. Analysis of serum indicated up to 200-fold increases in IFNγ levels. Surrogate potency-reduced IL12-Fc had improved tolerability and greater selectivity of IFNγ production in tumors compared to spleen and less production of IL10 compared to wild-type IL12-Fc. In NHP, potency-reduced IL12-Fc had superior exposure with slower, more sustained accumulation of IFNγ and IP10, and a more gradual dose-dependent peak response, as well as more sustained margination of T and NK cells compared to wild-type IL12-Fc.ConclusionsPotency-reduced IL12-Fc retain strong anti-tumor activity, while potentially overcoming safety and tolerability issues related to narrow TI associated with wild-type IL12 or IL12-Fc agents.

Interleukin-12 (IL12) is a proinflammatory cytokine produced by activated antigen-presenting cells that induces differentiation of Th1 cells and increased proliferation and cytotoxicity of T and NK cells. Stimulation of these cells by IL12 leads to production of high levels of IFNγ. These immune-stimulating aspects of IL12 may help to establish an inflammatory tumor microenvironment critical for anti-tumor responses. Preclinical studies in mice revealed that native IL12 can dramatically shrink syngeneic tumors, however clinical studies in humans resulted in severe toxicity and a small therapeutic window, limiting response rates. Prior work at Xencor demonstrated that reduced-potency IL15/IL15Rα-Fc fusion proteins exhibited superior pharmacokinetics, pharmacodynamics, and safety in non-human primates through reduction of receptor-mediated clearance. Applying similar principles to IL12, we created IL12 heterodimeric Fc-fusions (IL12-Fc) with reduced potency to improve tolerability, slow receptor-mediated clearance, and extend half-life. IL12 is a heterodimeric protein consisting of two subunits, so we engineered IL12-Fc fusions by fusing the IL12p35 subunit to one side of a heterodimeric (and inactive) Fc domain, and the IL12p40 subunit to the other side. These Fc-fusions were tuned for optimal activity by introducing amino acid substitutions at putative receptor-interface positions and screening for reductions of in vitro potency. In vitro activity was assessed on human PBMCs by measuring signaling in a STAT4 phosphorylation assay and IFNγ production in a mixed-lymphocyte reaction (MLR). In vivo anti-tumor activity of human IL12-Fc were assessed in a human PBMC engrafted mouse MCF7 tumor model. Surrogate mouse IL12-Fc were evaluated in additional murine tumor models. Tolerability and pharmacodynamic activity were assessed in non-human primates. IL12-Fc were produced with good yield and purity. An IL12-Fc potency series was created, and variants had up to a 10,000-fold reduction in STAT4 signaling potency and IFNγ production in an MLR assay compared to native IL12-Fc. Anti-tumor activity was achieved with potency-reduced IL12-Fc as a single-agent and in combination with anti-PD1, with weaker variants maintaining anti-tumor activity at higher dose levels. Analysis of peripheral lymphocytes indicated increased numbers of T and NK cells as well as activation of CD8+ T cells. Increased expression of immune checkpoints including PD1 was also observed. Analysis of serum indicated up to 200-fold increases in IFNγ levels. Combined, these data indicate that potency-reduced IL12-Fc retain strong anti-tumor activity, while potentially overcoming safety and tolerability issues related to narrow therapeutic index associated with recombinant native IL12 or IL12-Fc agents. Citation Format: Matthew J. Bernett, Ke Liu, Christine Bonzon, Rumana Rashid, Nicole Rodriguez, Nargess Hassanzadeh-Kiabi, Connie Ardila, Katrina Bykova, Michael Hackett, Norm Barlow, Irene Leung, Duc-Hanh Nguyen, Araz Eivazi, Seung Y. Chu, Rajat Varma, Umesh S. Muchhal, John R. Desjarlais. IL12 heterodimeric Fc-fusions engineered for reduced potency exhibit strong anti-tumor activity and improved therapeutic index compared to native IL12 agents [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 1743.

There is a large unmet need for targeted therapies to treat ovarian cancer and other solid tumors. A promising strategy is the use of T cell engaging bispecific antibodies that recruit T cells to kill cancer cells by simultaneously binding to tumor-associated antigens (TAA) on cancer cells and CD3 on T cells. Effective and safe use of these therapeutics depends on selective targeting of cancer cells over normal tissue, feasible when a TAA is more highly expressed on cancer cells versus normal tissues. Selectivity can also be improved by using a mixed valency “2 + 1” bispecific format, coupled with target affinity tuning, to achieve avid binding to the TAA. Claudin-6 (CLDN6) is a tetraspan membrane protein, a member of the claudin family of tight junction proteins, and has been identified as a promising TAA for ovarian cancer due to its high expression on ovarian and other cancer tissues and low expression on normal tissue. However, a complicating factor is that many proteins in the claudin family have high sequence identity. Most similar to CLDN6 is CLDN9, and the two proteins differ at only 3/76 residues in their extracellular loops, creating a challenge for the development of highly selective CLDN6 antibodies. We analyzed CLDN6 and CLDN9 expression using a combination of normal tissue gene expression data (GTEx), cancer cell genomics data (TCGA), and immunohistochemistry (IHC) on a panel of cancer and normal tissues. Data confirmed the tumor-specific expression pattern of CLDN6 but indicated high CLDN9 expression in normal tissues, suggesting that strong antibody selectivity for CLDN6 versus CLDN9 is critical. We created CLDN6-selective T cell engaging bispecific antibodies by starting with a highly selective monoclonal antibody humanized from a mouse hybridoma. Selectivity was further improved by engineering the CDR regions. Variant antibodies were screened for binding to transiently transfected HEK293E cells that highly expressed either CLDN6, CLDN9, or other homologous claudin family members. Selective variants were converted into the XmAb® 2 + 1 bispecific antibody format and tested in T cell-dependent cellular cytotoxicity (TDCC) assays using cancer cell lines expressing CLDN6, or HEK293E cells transfected with claudin homologs. A set of lead CLDN6 x CD3 bispecifics displaying a range of potencies on CLDN6+ cancer cell lines and high selectivity for CLDN6 over CLDN9 and other homologous claudins was identified. Tolerability and pharmacokinetics of these molecules are currently being assessed in non-human primates, and activity will be evaluated in humanized mouse models of ovarian cancer. Citation Format: Matthew S. Faber, Sung-Hyung Lee, Yoon Kyung Kim, Jing Qi, Kendra N. Avery, Duc-Hanh T. Nguyen, Rumana Rashid, Araz Eivazi, Seung Y. Chu, Juan E. Diaz, Connie Ardila, Ruschelle Love, Alex Nisthal, Norman J. Barlow, Christine Bonzon, Umesh S. Muchhal, Matthew J. Bernett, John R. Desjarlais. Bispecific claudin-6 x CD3 antibodies in a 2 + 1 format demonstrate selectivity and activity on human ovarian cancer cells [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 1860.

Interleukin-12 (IL12) is a proinflammatory cytokine produced by activated antigen-presenting cells that induces differentiation of Th1 cells and increased proliferation and cytotoxicity of T and NK cells. Stimulation of these cells by IL12 leads to production of high levels of IFNγ. These immune-stimulating aspects of IL12 are promising for cancer treatment and may help to convert immunologically suppressed “cold” tumors into inflamed “hot” tumors. Preclinical studies in mice revealed that IL12 can have a dramatic effect on shrinking syngeneic tumors, however clinical studies in humans have resulted in severe toxicity and a small therapeutic window, limiting response rates. Prior work at Xencor demonstrated that reduced-potency IL15/IL15Rα-Fc fusion proteins exhibited superior pharmacokinetics, pharmacodynamics, and safety in non-human primates through reduction of receptor-mediated clearance. Applying similar principles to IL12, we created various IL12 heterodimeric Fc-fusions (IL12-Fc) with reduced potency in order to improve tolerability, slow receptor-mediated clearance, and prolong half-life. IL12 is a heterodimeric protein consisting of two subunits, and thus we engineered IL12-Fc fusions by fusing the IL12p35 subunit to one side of a heterodimeric (and inactive) Fc domain, and the IL12p40 subunit to the other side. These Fc-fusions were tuned for optimal activity by introducing amino acid substitutions at putative receptor-interface positions and screening for reductions of in vitro potency. In vitro activity was assessed on normal human PBMCs by measuring signaling in a STAT4 phosphorylation assay and IFNγ production in a mixed-lymphocyte reaction (MLR). In vivo anti-tumor activity was assessed by engrafting pp65-MCF-7 cells into human PBMC engrafted NSG MHC class I and II double-knockout mice and by measuring tumor volume, lymphocyte activation/proliferation, and IFNγ production over time. IL12-Fc were produced with good yield and purity. An IL12-Fc potency series was created, and weaker variants had up to a 10,000-fold reduction in STAT4 signaling potency and IFNγ production in an MLR assay compared to a wild-type IL12-Fc. In vivo anti-tumor activity in the huPBMC-pp65-MCF7 model was achieved with reduced-potency IL12-Fc as a single-agent and in combination with anti-PD1, with weaker variants maintaining anti-tumor activity at higher dose levels. Analysis of peripheral lymphocytes indicated increased numbers of T and NK cells as well as activation of CD8+ T cells, as evidenced by upregulation of CD25. Increased expression of immune checkpoints including PD1 was also observed. Analysis of serum cytokine indicated up to 200-fold increases in IFNγ levels. Combined, these data indicate that reduced-potency IL12-Fc retain strong anti-tumor activity, with potential for improvement of therapeutic index. Citation Format: Rajat Varma, Ke Liu, Christine Bonzon, Rumana Rashid, Nicole Rodriguez, Nargess Hassanzadeh-Kiabi, Connie Ardila, Seung Y. Chu, Umesh S. Muchhal, John R. Desjarlais, Matthew J. Bernett. Potency-reduced IL12 heterodimeric Fc-fusions exhibit strong anti-tumor activity [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 5549.

Bispecific antibodies enable therapeutic modalities inaccessible to traditional mAbs, such as T cell engagers that bind both CD3 on T cells and a target antigen on tumor cells. These agents provide synthetic immunity by expanding, activating, and redirecting T cells against a target of interest. 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. Prostate cancer (PC) is one of the most prevalent cancers in men, and end stage (castration-resistant prostate cancer) has no curative treatment option. Prostate specific membrane antigen (PSMA), a type II transmembrane protein with a large extracellular domain, has long generated interest as a therapeutic target. It is highly overexpressed in PC compared to normal tissue, and its expression has been shown to correlate with malignancy. Previous attempts to target PSMA include antibody-based radiotherapy and antibody drug conjugates, which have shown some success but can be hampered by the inherent toxicity of the modality. Building upon the XmAb® heterodimeric Fc platform, we generated bispecific antibodies in an XmAb 2+1 Fab2-scFv-Fc format that are bivalent for PSMA and monovalent for CD3. Reducing the affinity of both specificities encouraged avid binding and strong activity on high PSMA expressing cell lines while minimizing reactivity on low expressing cell lines, matching PSMA's differential expression pattern. To ensure biologically valid antigen densities were being considered, IHC was conducted on paraffin embedded arrays of tumor tissue, normal tissue, and cell lines. Upon matching the staining intensity between the three sample types, we identified cell lines with corresponding PSMA antigen density that could serve as proxies of tumor and normal tissue. In vitro T cell-dependent cellular cytotoxicity (TDCC) assays on the relevant cell lines confirmed selective potency on high versus low expressing cells. Citation Format: Alex Nisthal, Matthew Dragovich, Erik W. Pong, Veronica Zeng, Michael Hedvat, Christine Bonzon, Kendra Avery, Rumana Rashid, Connie Ardila, Umesh S. Muchhal, Gregory L. Moore, Seung Y. Chu, John R. Desjarlais. Affinity tuned XmAb®2+1 PSMA x CD3 bispecific antibodies demonstrate selective activity in prostate cancer models [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 5663.

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.

Somatostatin receptor 2 (SSTR2) is highly expressed in neuroendocrine tumors (NETs) and small cell lung cancer (SCLC). Treatment options for NETs include somatostatin analogs and radionuclides; however, such therapies suffer from short half-life, modest efficacy, and toxicities due to inhibition of other SSTRs. Reasoning that a targeted immunotherapy against SSTR2 would provide a new therapeutic modality for NETs, we designed XmAb18087, a humanized and affinity-optimized bispecific antibody that engages T cells to stimulate redirected T cell-mediated cytotoxicity (RTCC) of SSTR2+ tumor cells. XmAb18087 possesses an Fc domain that maintains long half-life, yet lacks binding to Fcγ receptors to reduce Fc-mediated effector functions. XmAb18087 stimulated robust RTCC of SSTR2+ cell lines including medullary thyroid carcinoma (TT), lung carcinoma (A549), and CHO cells overexpressing SSTR2, with EC50s of ~1 to 100 ng/ml. XmAb18087 also upregulated CD69 and CD25 activation markers on CD4 and CD8 T cells. T cell responses were target-specific, because SSTR2‾ cell lines were not depleted, and because a control bispecific (anti-RSV x CD3) was ineffective. In addition, XmAb18087 (3 mg/kg weekly) reduced tumor burden of an established A549 xenograft in NSG mice engrafted with 107 human PBMC. We next assessed XmAb18087 activity in cynomolgus monkeys. As SSTR2 is not expressed in peripheral blood, target cell depletion cannot be monitored in vivo. However, CD3 bispecifics induce effects such as lymphocyte extravasation, cytokine induction, and T cell activation, which can serve as pharmacologic markers for activity in target organs. XmAb18087 dosed once at 30 or 60 μg/kg rapidly activated peripheral T cells, as quantified by CD69 and CD25 induction (peaking at ~8-12 hr). T cells rapidly extravasated from blood, with a nadir by 8 hr. Cytokines IL6 and TNF were rapidly induced, peaking at ~1-8 hr and returning to baseline by 48 hr. To explore repeat dosing, XmAb18087 was dosed at 1 or 10 μg/kg on Days 0 and 7 in a second study. The first dose of both 1 and 10 μg/kg again stimulated peripheral T cell activation, extravasation, and cytokine induction. These responses decreased markedly after the second dose, suggesting that SSTR2+ target cells remained depleted for at least 7 days. In summary, these results on human cells, in mice, and in monkeys support clinical assessment of XmAb18087 in SSTR2+ cancers including NETs and SCLC. In monkeys, T cell activation, extravasation, and cytokine induction were readily measured in peripheral blood and are indicative of T cell-mediated depletion of SSTR2+ target cells. Importantly, these responses may also serve as useful surrogate markers of NET depletion in clinical trials of XmAb18087. Citation Format: Sung-Hyung Lee, Seung Y. Chu, Rumana Rashid, Sheryl Phung, Irene W. Leung, Umesh S. Muchhal, Gregory L. Moore, Matthew J. Bernett, Suzanne Schubbert, Connie Ardila, Christine Bonzon, Paul Foster, David E. Szymkowski, John R. Desjarlais. Anti-SSTR2 × anti-CD3 bispecific antibody induces potent killing of human tumor cells in vitro and in mice, and stimulates target-dependent T cell activation in monkeys: A potential immunotherapy for neuroendocrine tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3633. doi:10.1158/1538-7445.AM2017-3633