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Programmed cell death 1 (PD-1) inhibitors have limited effect in pancreatic ductal adenocarcinoma (PDAC), underscoring the need to co-target alternative pathways. CXC chemokine receptor 4 (CXCR4) blockade promotes T cell tumor infiltration and is synergistic with anti-PD-1 therapy in PDAC mouse models. We conducted a phase IIa, open-label, two-cohort study to assess the safety, efficacy and immunobiological effects of the CXCR4 antagonist BL-8040 (motixafortide) with pembrolizumab and chemotherapy in metastatic PDAC (NCT02826486). The primary outcome was objective response rate (ORR). Secondary outcomes were overall survival (OS), disease control rate (DCR) and safety. In cohort 1, 37 patients with chemotherapy-resistant disease received BL-8040 and pembrolizumab. The DCR was 34.5% in the evaluable population (modified intention to treat, mITT; N = 29), including nine patients (31%) with stable disease and one patient (3.4%) with partial response. Median OS (mOS) was 3.3 months in the ITT population. Notably, in patients receiving study drugs as second-line therapy, the mOS was 7.5 months. BL-8040 increased CD8+ effector T cell tumor infiltration, decreased myeloid-derived suppressor cells (MDSCs) and further decreased circulating regulatory T cells. In cohort 2, 22 patients received BL-8040 and pembrolizumab with chemotherapy, with an ORR, DCR and median duration of response of 32%, 77% and 7.8 months, respectively. These data suggest that combined CXCR4 and PD-1 blockade may expand the benefit of chemotherapy in PDAC and warrants confirmation in subsequent randomized trials. Results from the phase IIa COMBAT trial combining CXCR4 and PD-1 inhibition in patients with metastatic cancer show encouraging clinical responses in association with enhanced antitumor immune activation.

Purpose: Pancreatic ductal adenocarcinoma (PDAC) is largely unresponsive to checkpoint inhibitors. Blockade of the CXCR4/CXCL12 axis increases intratumoral trafficking of activated T cells while restraining immunosuppressive elements. This study evaluates dual blockade of CXCR4 and PD1 with chemotherapy in PDAC. Patients and Methods: Multicenter, single-arm, phase II study to evaluate the safety and efficacy of motixafortide and pembrolizumab combined with chemotherapy in patients with de novo metastatic PDAC and disease progression on front-line gemcitabine-based therapy (NCT02826486). Subjects received a priming phase of motixafortide daily on days 1–5, followed by repeated cycles of motixafortide twice a week; pembrolizumab every 3 weeks; and nanoliposomal irinotecan, fluorouracil, and leucovorin every 2 weeks (NAPOLI-1 regimen). The primary objective was objective response rate (ORR). Secondary objectives included overall survival (OS), progression-free survival (PFS), disease control rate (DCR), safety, and tolerability. Results: A total of 43 patients were enrolled. The ORR according to RECISTv1.1 was 21.1% with confirmed ORR of 13.2%. The DCR was 63.2% with median duration of clinical benefit of 5.7 months. In the intention-to-treat population, median PFS was 3.8 months and median OS was 6.6 months. The triple combination was safe and well tolerated, with toxicity comparable with the NAPOLI-1 regimen. Notably, the incidence of grade 3 or higher neutropenia and infection was 7%, lower than expected for this chemotherapy regimen. Conclusions: Triple combination of motixafortide, pembrolizumab, and chemotherapy was safe and well tolerated, and showed signs of efficacy in a population with poor prognosis and aggressive disease.

Background CXCR4 mediates the retention and survival of acute myelogenous leukemia blasts in bone marrow and contributes to their resistance to chemotherapy. The authors evaluated a combination of the high-affinity CXCR4 antagonist BL-8040 with high-dose cytarabine (HiDAC) chemotherapy in a phase 2a study of patients with relapsed and refractory AML. Methods Forty-two patients received treatment with BL-8040 monotherapy for 2 days followed by a combination of BL-8040 with HiDAC for 5 days. Six escalating BL-8040 dose levels were investigated (0.5, 0.75, 1.0, 1.25, 1.5, and 2.0 mg/kg), and 1.5 mg/kg was selected as the dose for the expansion phase (n = 23). Results BL-8040 in combination with HiDAC was safe and well tolerated at all dose levels. Clinical response was observed with BL-8040 doses ≥1.0 mg/kg. The composite response rate (complete remissions plus complete remissions with incomplete hematologic recovery of platelets or neutrophils) was 29% (12 of 42) in all patients and 39% (9 of 23) in the 1.5-mg/kg phase. The median overall survival was 8.4 months for all patients, 10.8 months in the 1.5-mg/kg phase, and 21.8 months for responding patients in the 1.5-mg/kg cohort. Two days of BL-8040 monotherapy triggered the mobilization of blasts into peripheral blood, with significantly higher mean fold-changes in responders versus nonresponders. This was accompanied by a decrease in bone marrow blasts. Conclusions The current results demonstrate the efficacy of CXCR4 targeting with BL-8040 and support continued clinical development in acute myelogenous leukemia.

Background: Improving outcomes of PDAC with checkpoint inhibitors (CPIs) have been ineffective, underscoring the need to co-target alternative pathways. Preclinical data showed that CXCR4-SDF1 axis modulates the tumor microenvironment (TME) in PDAC and that CXCR4 inhibition enhances T cell access to the TME, increasing tumor sensitivity to CPIs. This was confirmed in the COMBAT Cohort 1 (CC1) study showing that the dual combination M+P increases activated CD8+ T cells and decreases myeloid derived suppressor cells (MDSCs) within the TME. Moreover, our pre-clinical studies showed that adding C to M+P resulted in improved efficacy vs C alone. The COMBAT Cohort 2 (CC2) aims to test the safety and efficacy of the triple combination of M+P+C in 2L mPDAC. Methods: Single arm phase 2a study in mPDAC. In cohort 2, patients with stage IV PDAC at diagnose who had progressed to 1L gemcitabine-based C received 5 days M priming, followed by M BIW + P Q3W plus C [Irinotecan liposomal injection/5-FU/LV (OFL)] Q2W. The primary endpoint was RR. Results: A total of 43 patients with stage 4 PDAC, 98% of whom were diagnosed with stage 4 disease, were enrolled. Median age was 68 (40-85) and 74.4% had liver disease. The safety profile was consistent with the individual profiles of each treatment alone. Of note, the incidence of ≥G3 neutropenia (G3Neu) was 7% and ≥G3 infection was 7%, which is lower than expected for C (OFL) alone (20% and 17%, respectively). The levels of T-cells increased during M priming and returned to normal values, which remained stable across the study despite the OFL treatment. For the evaluable patients (N=38) the ORR was 21.1% with a 13.2% confirmed ORR (defined as two consecutive assessments showing PR) and a 63.2% DCR (PR+SD). Median duration of clinical benefit was 5.6 months. Median OS and PFS were 6.5 months and 4.0 months, respectively (6.6 months and 3.8 months, respectively, for the ITT population). Conclusions: The triple combination of M+P+C is tolerable and shows encouraging results with cORR 13.2%, mPFS 4.0 months and mOS 6.5 months (compared to 7.7%, ~3 months and 4.7 months, respectively, on a historical basis for OFL alone in the stage 4 diagnosis subpopulation). SD of 42.1% and DCR of 63.2% were also higher than historical data on SoC chemotherapy used in 2L patients. The incidence of severe neutropenia and infections is lower than the historical data on C. The results from the CC2 suggest that M+P may expand the efficacy and safety benefit of OFL in PDAC, and warrants further investigation in a randomized study. Citation Format: Manuel Hidalgo, Teresa Macarulla, Valerya Semenisty, Erkut Borazanci, Jaime Feliu, Mariano Ponz-Sarvise, David Gutierrez Abad, Paul Oberstein, Angela Alistar, Andres Muñoz, Ravit Geva, Carmen Guillén-Ponce, Mercedes Salgado Fernandez, Amnon Peled, Marya Chaney, Irit Glicko-Kabir, Liron Shemesh-Darvish, Debby Ickowicz, Ella Sorani, Shaul E. Kadosh, Abi Vainstein-Haras, Bruno Bockorny. A multi-center phase 2a trial of the CXCR4 inhibitor motixafortide (BL-8040) (M) in combination with pembrolizumab (P) and chemotherapy (C), in patients with metastatic pancreatic adenocarcinoma (mPDAC) [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 CT177.

Background: We recently conducted a clinical trial assessing Pembrolizumab (P) and BL-8040 (BL) in patients with pancreatic cancer. We prospectively collected serial biopsies to help understand the impact of this treatment on the tumor microenvironment (TME). Methods: Twenty patients were enrolled and were treated with BL8040 (1.25mg/kg) monotherapy for 2 weeks followed by dual therapy with BL8040 (d1, 4, 8, 11) plus Pembrolizumab (200 mg IV, d1) every 3 weeks. Biopsies were collected at baseline (T0) and during therapy. Some patients had biopsies after BL monotherapy (Tm) and some after combined therapy (Tc) due to a protocol change mid-study. Malignant cells expressing PD-L1 were assessed using immunohistochemistry (IHC) and TME was studied using 3 multiplex immunofluorescence panels including one each for T-cells subpopulations, macrophages and myeloid-derived suppressor cells (MDSCs). Median densities of the different phenotypes were analyzed in tumor and stromal compartments and correlated with serial biopsies. Results: Of 20 patients enrolled and 15 were evaluable for response of whom 2 had stability and 1 had a partial response. 17 patients had at least one viable biopsy; 4 had biopsies at T0 and Tm, 3 had biopsies at T0, Tm, and Tc, and 2 had biopsies at T0 and Tc. In general, we noted that cytotoxic T cells increased from baseline when measured at Tm or Tc. In contrast, MDSC counts and overall T cells decreased at Tm or Tc from baseline. Additionally, we saw an increase in CD 68+ and PD-L1 expressing macrophages (P=0.059 and P=0.046, respectively). Patients who had clinical benefit (PR/SD) had greater numbers of cytotoxic T-cells (CD3+CD8+, median, 226.08 cells/mm2) and regulatory T cells (CD3+FOXP3+, median, 48.20 cells/mm2) at baseline than PD patients (median, 26.15 cells/mm2, P=0.03; 13.31 cells/mm2, P=0.016, respectively). Conversely, at T0, the lowest numbers of tumor cytotoxic T-cells activated CD3+CD8+GB+ (median, 0.34 cells/mm2) and highest numbers of PMN-cells (CD66b+CD11b+, median, 16.66 cells/mm2) were observed in patients with PD when compared with PR or SD (median, 4.54 cells/mm2, P=0.097; 3.38 cells/mm2, P=0.023, respectively), suggesting that highest densities of PMN-cells may contribute to downregulation of T-cells in those cases. Conclusions: Higher cytotoxic T-cells or PMN-cell counts at T0 may predict changes in the TME and radiologic response to treatment with BL8040+Pembrolizumab therapy in metastatic pancreatic cancer. PMN-cells may play a role promoting immune suppression of T-cells, increase the risk of metastasis and interfering with the treatment. Trial Registration: NCT02907099, Ethics Approval: This study was approved by the M.D. Anderson Institutional Review Board, approval number 2016-0410. Citation Format: Carmelia M. Noia Barreto, Debora A. Ledesma, Swati Gite, Luisa M. Solis, Mei Jiang, Renganayaki K. Pandurengan, Robert Wolff, Milind Javle, Shubham Pant, Gauri Varadhachary, Rachna Shroff, Abi Vainstein-Haras, Ella Sorani, Tzipora M. Lustig, Osnat Kashtan, Yosi Gozlan, Steven M. Townson, Jeanne M. Fahey, James Yao, Ignacio I. Wistuba, Michael Overman, David Fogelman, Edwin R. Parra. High cytotoxic T-cell or polymorphonuclear cell infiltrates in the tumor microenvironment correlate with responses to BL8040 plus pembrolizumab combination therapy in metastatic pancreatic tumors: Scientific correlates of a phase II clinical trial [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 CT204.

73 Background: C-X-C chemokine receptor type 4 (CXCR4) helps to retain the hematopoietic stem cells (HSC) in the bone marrow (BM). CXCR4 binds to its ligand CXCL12/SDF1 which is constitutively expressed in the BM thereby inhibiting the mobilization of CXCR4 expressing immune progenitor cells. Moreover, increased numbers of effector cells in the tumor microenvironment (TME) are directly correlated to enhanced immunotherapeutic efficacy. Therefore, we hypothesized that CXCR4 antagonism will result in movement of immune progenitor cells from BM to periphery leading to increased availability of T-cells in the periphery and better infiltration of effector cells into the TME. Methods: In TC-1 mouse tumor-model, we tested the effects of CXCR4 antagonist (BL-8040; 4 doses; 24 h apart; 20 mg/kg) on the tumor growth and mice survival in the presence of tumor-specific antigen priming using E7 peptide vaccine (3 doses, one week apart). Anti-tumor immune responses were determined in the tumor tissues obtained 3-4 days after the second vaccination using flow cytometry. Results: We found that BL-8040 given with specific antigenic stimulation results in significantly enhanced anti-tumor immune response, leading to a decrease in tumor growth (p≤0.001 at day 21) and prolonged mice survival. At day 35 after tumor implantation, 80% of mice survived in the BL-8040 + vaccine treatment group compared to 0% survival following BL-8040 or vaccine treatments. Interestingly, we also found that BL-8040 leads to a significant increase in the numbers of antigen-specific CD8+ T-cells in the TME. We also found that starting BL-8040 prior to or together with the priming of the mice did not affect the outcome, suggesting that the scheduling of BL-8040 does not affect the therapeutic outcomes. Conclusions: These results suggest that BL-8040 treatment enhances anti-tumor immune response by potentially increasing the immune progenitor cells in the periphery leading to a better immune response. These results also suggest that BL-8040, a CXCR4 antagonist, is a promising immune-modulatory agent with potent anti-tumor effects.

276 Background: BL-8040 is a novel CXCR4 antagonist being developed for multiple oncology indications. Preclinical studies demonstrated that BL-8040 increases the number of immune cells in peripheral blood and promotes CD8+ T cell infiltration into orthotropic pancreatic mouse tumors, reducing tumor load. BL-8040 is being evaluated in a Phase 2a, multicenter, open label trial in patients with metastatic pancreatic cancer (the COMBAT study). Patients are undergoing a 5-day period of monotherapy in which they receive daily doses of BL-8040, followed by 21-day cycles in which patients receive one dose of pembrolizumab and 3 doses/week of BL-8040 until disease progression or discontinuation. To date, 32 patients have been enrolled. Methods: On Day 1 and Day 5, blood samples were taken at pre- and post-dosing, to evaluate peripheral immune cell subset frequency by flow cytometry. In addition, core biopsies were taken from liver metastases, where possible, to assess immune cell infiltration into tumors and the tumor microenvironment (TME). Results: Here we present interim PD biomarker data from the BL-8040 monotherapy portion of the trial. Flow cytometry shows that BL-8040 monotherapy caused an approximately two-fold reduction in frequency of peripheral T regulatory cells, but had no effect on the frequency of T cells, NKT cells or cell populations that contain B cells (CD3- CD56-). Additionally, BL-8040 remained bound to CXCR4 on peripheral immune cells throughout the period of monotherapy. Analysis of available biopsies (N = 7) shows an up to 15-fold increase in the CD3+ population, and up to two-fold increase of CD8+ cells, in the tumor periphery and TME of 43% (3/7) of the patients after five days of BL-8040 monotherapy compared to baseline. Conclusions: In summary, the PD biomarker results in humans support the proposed mechanism of action for BL-8040 that was based on preclinical mouse models. Analysis of tumor biopsies is ongoing, with an emphasis on investigating the effects of BL-8040 on tumor-resident immune cells and the TME. Clinical trial information: NCT02826486.

68 Background: AGI-134 is a fully synthetic alpha-Gal glycolipid for intratumoral (i.t.) treatment of solid tumors to induce a patient-specific anti-tumor immune response. AGI-134 recruits pre-existing anti-Gal antibodies to the injected lesion, leading to complement activation and enhanced tumor antigen processing. Using the B16.F10 murine melanoma model, we have previously demonstrated that AGI-134 evokes a robust abscopal anti-tumor effect, with treatment of a primary lesion protecting mice from the growth of distant lesions. In the current study, we investigated the response of injected tumors to AGI-134 administration. Methods: Tumors were induced by s.c. injection of B16.F10 or ovalbumin expressing B16 (B16.OVA) cells into the flank of α1,3galactosyltransferase knock out mice. After reaching a treatable size, the tumors were injected twice, 24 hrs apart, with PBS or 1-1.25 mg AGI-134, and tumor growth monitored for up to 32 days. In addition, to measure the activation of complement after treatment with AGI-134, tumors were processed 2 hours after treatment and the i.t. concentration of complement fragment C5a determined by ELISA. Results: Almost 50% of AGI-134-treated B16.F10 tumors fully regressed vs. 24% in the PBS controls. Moreover, AGI-134 treatment had a significant survival benefit, with 23% of AGI-134-treated mice dying or requiring euthanisia due to tumor mass by Day 27 post treatment vs. 43% in the PBS groups (p < 0.05, Mantel-Cox test). In the B16.OVA model, 67% of the AGI-134 treated tumors fully regressed vs. 0% in PBS-treated mice in two independent assays. Comparison of C5a levels in B16.F10 tumors 2 hrs after AGI-134 or PBS treatment demonstrated that AGI-134 induced significant complement activation within injected tumors. Conclusions: Having previously shown that AGI-134 induces an abscopal effect that protects mice from the development of secondary tumors, we now show that AGI-134 also induces regression of injected tumors: AGI-134-treatment induces the activation of complement within the tumor and leads to complete regression of the tumor in significantly more AGI-134-treated mice than PBS-treated. Clinical trials with AGI-134 are planned for 2018.

TPS533 Background: Checkpoint inhibitors such as pembrolizumab are active against a variety of tumor types; however, pancreatic cancer patients generally do not see improvements in their disease from single agent treatment. Blocking CXCR4 may potentiate the activity of checkpoint inhibitors by (1) increasing mobilization of lymphocytes from bone marrow, (2) blocking production of SDF-1, in turn allowing better penetration of immune cells into the tumor, (3) reducing the entry of suppressor T cells into tumor, and (4) upregulating CCL20 in the tumor microenvironment, which may help attract dendritic cells into the tumor. We have launched a study combining BL8040, a CXCR4 antagonist, with pembrolizumab in order to determine the efficacy of this combination. Methods: Major inclusion criteria require patients with pancreatic adenocarcinoma whose tumors have progressed through at least one line of therapy. There must be sufficient disease to allow for pre- and post-treatment biopsy as well as measurement of response by RECIST criteria. Patients must be age 18 or older and ECOG 0-1. Liver function, renal, and hematologic criteria are fairly rigorous. Major exclusion criteria include patients with viral infections or those who require supraphysiologic steroid use. Treatment includes a two week cycle of single agent BL-8040 given on days 1-5 and 8-12. Cycles 2 and beyond include treatment on day 1 with pembrolizumab, 200 mg flat dose, plus BL-8040 given SQ on days 1,4,8, and 11 of a two week cycle. The primary endpoint is response rate; other major clinical objectives include progression free and overall survival. Scientific correlates include assessments of immune cell infiltrates into tumor via histology and immunoflourescence, histological assessment of stromal components, and gene expression signatures. Blood is likewise being collected for serum cytokine analysis, circulating free DNA analysis, circulating tumor cells, and exosome analysis. Clinical trial information: NCT02907099.

e14544 Background: Cancer cells affect their micro-environment by recruiting immune cells that support tumor growth, metastasis and inhibition of anti-tumor effector T and NK cell recruitment. In this study, we investigated the role of BL-8040, a CXCR4 antagonist in cancer immunotherapy and its ability to modulate the immunosuppressive tumor micro-environment. Methods: The effect of BL8040 on tumor micro-environment was tested in 3 different cancer mouse models: lung cancer, pancreatic cancer and melanoma. The mobilization of immune cells to the periphery in response to BL8040 was tested, as well as the accumulation of immune cells both within and surrounding the tumor in the pancreatic cancer mouse model. Results: BL8040 was found to be a potent and robust mobilizer of immune cells. Immunophenotyping of the mobilized cells revealed that the mobilization of CD4 and CD8 T lymphocytes, as well as of dendritic cells (DC), was significantly increased in the cancer-bearing mice compared to their naïve counterparts. Importantly, a significant mobilization of effector CD8 T cells and activated CD8 T cells in the cancer-bearing mice was also detected following BL8040 treatment. Concomitantly, in the pancreatic cancer mouse model, treatment with BL8040 increased CD8 T cell accumulation within the tumor and inhibited tumor growth. Conclusions: The immune cell population that is mobilized in response to BL8040 treatment is different in cancer mouse models and naïve mice. The ability of BL8040 to induce mobilization of leukocytes, cytotoxic and activated CD8 T cells and DCs is affected by the presence of a tumor. In our models of pancreatic cancer, mobilization of immune cells from the bone marrow into the circulation and their accumulation within the tumor and tumor microenvironment resulted in inhibition of tumor growth. These results indicate that BL8040 may affect the tumor microenvironment and therefore can potentially synergize with immunomodulatory agents. In vivo pre-clinical studies as well as clinical studies are currently ongoing for testing the combination of BL8040 with immunomodulatory agents in different cancer models.