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Background Adaptive immunity to cancer cells has been shown to form a crucial part of cancer immunotherapy. Recently, the importance of tumor B-cell signatures were shown to correlate with melanoma survival. We investigated whether anti-tumor antibodies could be isolated from a patient with metastatic melanoma that was tumor-free for 6 years following adoptive transfer of ex vivo expanded autologous T cells (Verdegaal 2011). Methods Peripheral blood memory B cells were immortalized using AIMM's immmortalisation technology (ectopic Bcl-6 and Bcl-xL; Kwakkenbos 2010) expression and analyzed for the presence of tumor-reactive B cells. Results AT1412 antibody was identified by virtue of its differential binding to melanoma cells as compared to healthy melanocytes. AT1412 was found to bind the tetraspanin CD9, a broadly expressed protein involved in multiple cellular activities and disease progression. The crystal structure of the CD9 large extracellular loop in complex with an AT1412 Fab fragment revealed that AT1412 binds the CD9 epitope in an extended and unfolded conformation. In addition to melanoma, AT1412 binds other tumor types including gastric, colon- and pancreatic cancer. AT1412 was shown to induce antibody dependent cellular cytotoxicity (ADCC) and antibody dependent cellular phagocytosis (ADCP) of cancer cells. In addition, AT1412 demonstrated activation of monocytes, most likely via activation of CD9 on platelets-bound monocytes. In mice carrying a human immune system (HIS-mice; van Lent 2010) AT1412 strongly enhanced tumor rejection of A375 and SKMEL-5 tumor cells. AT1412 treatment was shown to enhance tumor infiltration of CD8 T cells and macrophages. AT1412 efficacy was further synergistically enhanced when combined with an anti-PD1 antibody (nivolumab). Other groups have shown that anti-CD9 antibodies can induce tumor rejection in mice. However, these antibodies could not be advanced due to induction of platelet aggregation. We show that AT1412 does not induce aggregation of both human and cynomolgus platelets. Also affinity matured versions of AT1412 do not induce platelet aggregation, supporting that AT1412 binds a unique epitope on CD9. In addition, we studied the safety of AT1412 during a one month exposure in a weekly administration in cynomolgus monkeys. Besides a transient thrombocytopenia AT1412 was well tolerated up to 10 mg/kg antibody (highest dose tested) and did not lead to other adverse events nor were any coagulation factors affected Conclusions Taken together, applying AIMM's proprietary B-cell immortalization technology we isolated antibody AT1412 that targets a unique epitope on CD9. AT1412 was shown to induce tumor rejection as a single agent and enhances the activity of anti-PD-1 antibodies, while not inducing platelet aggregation. One month exposure of AT1412 in monkeys indicated that AT1412 can be safely administered. Preclinical development of AT1412 is ongoing to initiate clinical evaluation in 2020. Funding Dutch Cancer Society, grant UVA 2010-4822 Citation Format: Remko Schotte, Julien Villaudy, Martijn Kedde, Wouter Pos, Koen Wagner, Viviana Neviani, Daniel Go, Etsuko Yasuda, Christien Fatmawati, Els Verdegaal, Susan van Hal, Yvonne Claasen, Hans van Eenennaam, Pauline van Helden, Piet Gros, Sjoerd van der Burg, Hergen Spits. A patient-derived anti-CD9 antibody induces tumor rejection and synergistically enhances anti-PD1 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 532.

Introduction Despite rapid advances in immunotherapeutic options for precursor B-acute lymphoblastic leukemia (BCP-ALL), outcomes remain poor especially for adult ALL and relapsed pediatric ALL. With conventional chemotherapy remission percentages in adult ALL range from 75 to 90%, but relapse rates are high and long-term leukemia-free survival ranges between 35-70% depending on age and risk group. The introduction of CD19-targeting immunotherapy has significantly improved patient outcomes in (relapsed) BCP-ALL. However, tumor escape via downregulation of CD19 occurs in a significant number of patients. An ongoing medical need remains for the identification of alternative immunotherapeutic targets for treatment of ALL. Methods CD9 is expressed in 60-80% of BCP-ALL, is correlated with adverse prognosis and has been proposed as therapeutic target for BCP-ALL. AT1412 is a fully human CD9-targeting antibody, that was identified from a patient cured of metastatic melanoma after adoptive T-cell therapy, using our B-cell immortalization technology (AIMSelect) [Kwakkenbos et al. Nat. Med. 2010]. The antibody was selected based on differential binding to melanoma cells as compared to healthy melanocytes and was shown to be successful in killing melanoma cells in vitro and in vivo. Results Binding of AT1412 to BCP-ALL cell lines SUP-B15, MHH-CALL-2 and CCRF-SB varied as expected based on CD9 levels that we detected using a commercial CD9 antibody. AT1412-induced antibody dependent cellular cytotoxicity (ADCC) and antibody dependent cellular phagocytosis (ADCP) correlated with the level of AT1412 binding. No binding was seen to the T-ALL cell line Jurkat. These findings were confirmed in primary ALL samples, obtained prospectively at diagnosis from a cohort of patients with T- or B-ALL (n=38). AT1412 showed binding to the majority of B-ALL samples but not to T-ALL samples. AT1412 induced ADCC of nearly all B-ALL samples it bound to (10/11) and of none of the non-binding B-ALL or T-ALL samples. Cytotoxicity significantly correlated with the level of AT1412 binding. These findings were corroborated by the observation that AT1412 induced specific B-ALL cell death when a bone marrow sample from a newly diagnosed BCP-ALL patient was incubated with AT1412. Remarkably, AT1412 induced cell death in the absence of added effector cells or other (chemo)therapeutic agents, while the sample contained over 80% blasts and as little as 3% lymphocytes. We are currently investigating the in vivo efficacy of the antibody in a humanized immune system mouse model with human BCP-ALL. Conclusion Taken together, the majority of BCP-ALL express CD9 and this is associated with poor prognosis. Our data demonstrate that CD9 can be successfully targeted by the human CD9 antibody AT1412, suggesting that AT1412 has the potential to be developed as a therapeutic antibody for B-ALL. AT1412 is currently being advanced through preclinical development. Citation Format: Greta de Jong, Sophie E. Levie, Remko Schotte, Wouter Pos, Daniel Go, Etsuko Yasuda, Madalina Cercel, Susan E. van Hal, Esmay Frankin, Aniko Szabo, Martijn Kedde, Els Verdegaal, Julien Villaudy, Sjoerd van der Burg, Pauline M. van Helden, Hans van Eenennaam, Hergen Spits, Anita van Rijneveld, Mette D. Hazenberg. AT1412, a patient-derived antibody in development for the treatment of CD9-positive precursor B-acute lymphoblastic leukemia [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 531.

Introduction CD43s, a unique sialylated form of CD43 expressed by myeloid malignancies, is a novel target and AT1413, an antibody targeting CD43s was shown to have therapeutic potential against acute myeloid leukemia (AML) and myelodysplastic syndrome. CD43s is recognized by the human antibody AT1413, that was isolated from a high-risk AML patient who successfully cleared the leukemia after allogeneic stem cell transplantation. Because CD43 is also expressed in non-hematopoietic cells we studied whether CD43s is also present and can also be targeted on non-hematopoietic tumors. Materials and methods AT1413 binding on a panel of tumor cell lines was analyzed by flow cytometry. AT1413 was constructed into a bispecific T-cell engaging format (AT1413 bTCE) by linking the full-length AT1413 IgG to two single chain variable fragments against CD3ϵ with a combination of site-specific enzymatic and chemical coupling. A monovalent T-cell engager was produced as heterotrimer consisting of one AT1413 monovalent chain and one anti-CD3 scFV fused to AT1413 monovalent chain. Point mutations in the IgG heavy chain were introduced to prevent interactions between AT1413 T-cell engagers and Fc-receptors. The cytotoxicity-inducing activities were established using PBMCs as effector and tumor cells as target cells using standard cytotoxic assays in vitro and in vivo in a mouse model carrying human immune cells. Results We observed that AT1413 binds to non-hematopoietic tumor cells, such as melanoma and breast cancer. AT1413 immune precipitated CD43s from melanoma cells confirming that it recognizes the same target on melanoma as on AML. AT1413 induced antibody dependent cellular cytotoxicity against melanoma cell lines and primary melanoma samples. However, AT1413 was unable to affect growth of melanoma cells in vivo. To increase the efficacy of AT1413 it was formatted as a bispecific T-cell engaging antibody (TCE): one binding bivalently (bTCE) to CD43s and monovalently to CD3ϵ and the other monovalently (KiH) to both CD43s and CD3ϵ. In vitro, these TCEs redirected T-cell cytotoxicity against melanoma cells with different potencies. To investigate their effects in vivo, we grafted mice that harbor a human immune system with the melanoma cell line A375. Treatment with both AT1413 bTCE and AT1413 KiH significantly reduced tumor outgrowth. Conclusion AT1413 recognizes a sialylated epitope on CD43 shared by melanoma, AML and MDS cells. Two different bispecific TCE forms of AT1413 induce strong anti-tumor cytotoxic activities in vitro and in vivo. These data indicate a broad therapeutic potential of AT1413. Citation Format: Greta de Jong, Lina Bartels, Martijn Kedde, Els Verdegaal, Marijn A. Gillissen, Sophie E. Levie, Madalina G. Cercel, Susan E. van Hal-van Veen, Christien Fatmawati, Dorien van de Berg, Etsuko Yasuda, Yvonne Claassen, Arjen Q. Bakker, Remko Schotte, Julien Villaudy, Koen Wagner, Hergen Spits, Mette D. Hazenberg, Pauline M. van Helden. T-cell engager bispecific formats of an AML patient-derived antibody targeting a unique sialylated CD43 epitope induce kill of melanoma cells in vitro and in vivo [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 542.

Background: Adoptive cell therapy (ACT) with tumor-reactive T cells has shown consistent clinical efficacy. We evaluated the response to ACT in combination with interferon alpha (IFNa) preconditioning in stage IV metastatic melanoma patients, most of which were progressive on CTLA-4 and/or PD-1 checkpoint blockade therapy. Methods: Thirty-four patients were treated with ex vivo expanded tumor reactive T cells, derived from mixed-lymphocyte-autologous tumor cultures, or with autologous tumor-infiltrating lymphocytes and evaluated for clinical response. Clinical and immunological parameters associated with response were also evaluated. Results: Best overall response defined as clinical benefit, comprising either complete response, partial response or stable disease for more than 6 months, was observed in 29% of the patients. Six of the 14 (43%) immunotherapy naïve patients and 4 of the 20 (20%) patients progressive on prior immunotherapy benefited from ACT. The overall survival (OS) was 90% versus 28.6% at 1 year and 46.7% versus 0% at 3 years follow-up, of clinical responder and non-responder patients, respectively. Median OS was 36 versus 7 months, respectively. IFNa pre-treatment resulted in leuko-, neutro- and lymphopenia, which was sustained during the treatment in clinical responders and associated with response. Differences in antigen-specificity, but not in phenotype, cytokine profile, or CD8+ T cell number of the ACT products correlated with clinical response. Cross-reactivity of the ACT products to one or more allogeneic HLA-matched melanoma cell lines was associated with short OS after treatment while the ACT products of very long-term survivors showed solely recognition of patient-specific neo-antigens. Conclusion: This study demonstrates that ACT in combination with a mild IFNa preconditioning regimen can induce clinical benefit even in immunotherapy pre-treated patients, albeit with lower success than in immunotherapy naïve patients. ACT products comprising neo-antigen reactivity may be more effective. Future Perspectives: As a substantial percentage of the infused T cells expressed one or more inhibitory checkpoint molecules (CTLA-4, PD-1, or TIM-3), we started a new trial combining ACT and anti-PD-1 (ACTME trial; NCT03638375). The phase Ia of this trial was just completed with nine immunotherapy pre-treated patients showing that the combined treatment is feasible and safe. Updated and accumulated disease response data will be presented. Citation Format: Monique K. van der Kooij, Els M. Verdegaal, Marten Visser, Carolien E. van der Minne, Linda de Bruin, Pauline Meij, Anton G. Terwisscha van Scheltinga, Marij J. Welters, Saskia J. Santegoets, Noel F. de Miranda, Inge C. Roozen, Gerrit-Jan Liefers, Ellen Kapiteijn, Sjoerd H. van der Burg. Low-dose Interferon-alpha pre-conditioning and adoptive cell therapy in metastatic melanoma patients refractory to standard (immune) therapies - a phase 1/2 study [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 CT194.

Background. It is generally accepted that immune reactions against cancer cells can be induced by immunotherapy. Here, we investigated the possibility that an antibody response has contributed to the success of the immunotherapy of a cancer patient. Methods. A patient with metastatic melanoma was successfully treated by adoptive transfer of ex vivo expanded autologous tumor reactive T cells1. This patient is still tumor free 9 years after treatment. Peripheral blood memory B cells were immortalized by ectopic Bcl-6 and Bcl-xL expression2 and analyzed for the presence of tumor-reactive B cells. Results. We isolated one B cell clone that produced an antibody, named AT1412, recognizing a novel cell surface epitope on the tetraspanin CD9 which is broadly expressed and involved in multiple cellular activities including proliferation and adherence. AT1412 bound more strongly to melanoma cells than to melanocytes and other healthy tissues indicating that the AT1412 epitope is overexpressed on tumor cells. In addition, AT1412 strongly reacted with other tumor types including colon, pancreas and, breast cancer. Further analysis revealed that AT1412 favors binding to a clustered state of CD9. CD9 clusters are dependent on palmitoylation and known to be present on metastatic cells3. Previously published high affinity mouse antibodies targeting human CD9 were found to trigger aggregation of platelets and thus unfit for therapeutic use4. In sharp contrast, AT1412 does not induce platelet aggregation. To address whether AT1412 fails to aggregate platelets because of its low affinity for CD9 we generated high affinity variants of AT1412 using our highly efficient affinity maturation platform. We obtained AT1412 variants with affinities up to 250 fold higher than the wild type AT1412 comparable to that of mouse anti-human CD9 antibodies. These high affinity variants still did not aggregate platelets indicating that the absence of platelet aggregation by AT1412 is due to the spatial arrangement of the AT1412 epitope present on CD9 and not its low affinity. The antibody was able to reduce growth of melanoma tumors and block metastasis in melanoma xenografted mice. A striking synergistic inhibition of tumor growth was observed in mice that were treated with our CD9 antibody in the presence of a clinically successful anti-PD1 checkpoint inhibitor antibody. Discussion. These data suggest that the antibody contributed to the success of the immunotherapy in this patient. This antibody could act together with tumor-reactive T cells in eradicating circulating tumor cells and/or preventing settlement of metastatic tumor cells. Importantly, no antibody-related adverse effects were observed during and after treatment of this patient indicating that the antibody is safe for use in humans. 1. Verdegaal, Cancer Immunol Immunother, 2011 2. Kwakkenbos, Nat Med, 2010 3. Yang, JBC, 2006 4. Boucheix, FEBS Letter, 1983 Citation Format: Remko Schotte, Pauline van Helden, Daniel Go, Christien Fatmawati, Els Verdegaal, Camile Bru, Julien Villaudy, Koen Wagner, Sjoerd van der Burg, Hergen Spits, Wouter Pos. A patient derived antibody targeting the tetraspanin CD9 synergistically inhibits tumor growth with an anti PD1 antibody [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 LB-200. doi:10.1158/1538-7445.AM2017-LB-200