Pauline M. van Helden

IgG subclasses of anti-FVIII antibodies during immune tolerance induction in patients with hemophilia A

The eradication of inhibitory antibodies in patients with haemophilia A can be accomplished by frequent administration of high or intermediate doses of factor VIII (FVIII), so‐called immune tolerance induction (ITI). This study monitored the distribution of IgG subclasses of anti‐FVIII antibodies during ITI. FVIII‐specific antibodies of subclass IgG1 were detected in all inhibitor patients tested, anti‐FVIII IgG4 in 16, IgG2 in 10 and IgG3 in one of 20 patients analysed. Levels of anti‐FVIII IgG1 and IgG4 correlated well with inhibitor titres as measured by Bethesda assay. In low‐titre inhibitor patients, anti‐FVIII antibodies consisted primarily of subclass IgG1 whereas, anti‐FVIII antibodies of subclass IgG4 were more prominent in patients with high titre inhibitors who needed prolonged treatment or who failed ITI. Longitudinal analysis of 14 patients undergoing ITI revealed that the relative contribution of IgG subclasses was constant for most of the patients analysed. In two patients, the relative contribution of IgG4 increased during ITI. Overall, our findings document the distribution and dynamics of anti‐FVIII IgG subclasses during ITI. Future studies will need to address whether monitoring the relative contribution of anti‐FVIII subclasses IgG1 and IgG4 may be useful for the identification of patients who are at risk of failing ITI.

Tolerance to factor VIII in a transgenic mouse expressing human factor VIII cDNA carrying an Arg593 to Cys substitution

Inhibitory antibodies develop in approximately 25% of patients with severe hemophilia. A following treatment with factorVIII. In E-16KO or E-17KO mice, in which the factor VIII gene has been inactivated by insertion of a neo cassette, inhibitors develop following administration of factor VIII. Here, we describe the generation of transgenic mice expressing human factor VIII-R593C (huFVIII-R593C). Human factor VIII-R593C cDNA under control of a mouse albumin enhancer/promoter was injected into fertilized oocytes. Analysis of transgenic mice revealed that human factor VIII-R593C was expressed in the liver. Transgenic mice were crossed with factor VIII-deficient mice (E-16KO mice). In plasma of E-16KO mice antibodies were detected after five serial intravenous injections of factor VIII, while plasma of huFVIII-R593C/E-16KO mice did not contain detectable levels of antibodies. No antibody secreting cells were observed in either spleen or bone marrow of huFVIII-R593C/E-16KO mice. Also, factor VIII-specific memory B cells were not observed in the spleen of huFVIII-R593C/E-16KO mice. Analysis of T cell responses revealed that splenocytes derived of E-16KO mice secreted IL-10 and IFN-gamma following restimulation with factor VIII in vitro. In contrast, no factor VIII-specific T cell responses were observed in huFVIII-R593C/E-16KO mice. These results indicate that huFVIII-R593C/E-16KO mice are tolerant to intravenously administered factor VIII. It is anticipated that this model may prove useful for studying immune responses in the context of factor VIII gene therapy.

Stable long‐term cultures of self‐renewing B cells and their applications

Monoclonal antibodies are essential therapeutics and diagnostics in a large number of diseases. Moreover, they are essential tools in all sectors of life sciences. Although the great majority of monoclonal antibodies currently in use are of mouse origin, the use of human B cells to generate monoclonal antibodies is increasing as new techniques to tap the human B cell repertoire are rapidly emerging. Cloned lines of immortalized human B cells are ideal sources of monoclonal antibodies. In this review, we summarize our studies to the regulation of the replicative life span, differentiation, and maturation of B cells that led to the development of a platform that uses immortalization of human B cells by in vitro genetic modification for antibody development. We describe a number of human antibodies that were isolated using this platform and the application of the technique in other species. We also discuss the use of immortalized B cells as antigen‐presenting cells for the discovery of tumor neoantigens.

Patient-derived antibody recognizes a unique CD43 epitope expressed on all AML and has antileukemia activity in mice

Immunotherapy has proven beneficial in many hematologic and nonhematologic malignancies, but immunotherapy for acute myeloid leukemia (AML) and myelodysplastic syndrome (MDS) is hampered by the lack of tumor-specific targets. We took advantage of the tumor-immunotherapeutic effect of allogeneic hematopoietic stem cell transplantation and searched the B-cell repertoire of a patient with a lasting and potent graft-versus-AML response for the presence of AML-specific antibodies. We identified an antibody, AT1413, that was of donor origin and that specifically recognizes a novel sialylated epitope on CD43 (CD43s). Strikingly, CD43s is expressed on all World Health Organization 2008 types of AML and MDS. AT1413 induced antibody-dependent cell-mediated cytotoxicity and complement-dependent cytotoxicity of AML cells in vitro. Of note, AT1413 was highly efficacious against AML cells in a humanized mouse model without affecting nonmalignant human myeloid cells, suggesting AT1413 has potential as a therapeutic antibody.

AML-specific cytotoxic antibodies in patients with durable graft versus leukemia responses

Most patients with acute myeloid leukemia (AML) can only be cured when allogeneic hematopoietic stem-cell transplantation induces a graft-versus-leukemia immune response (GVL). Although the role of T cells and natural killer cells in tumor immunology has been established, less is known about the contribution of B cells. From B cells of high-risk patients with AML with potent and lasting GVL responses, we isolated monoclonal antibodies directed against antigens expressed on the cell surface of AML cells but not on normal hematopoietic and nonhematopoietic cells. A number of these donor-derived antibodies recognized the U5 snRNP200 complex, a component of the spliceosome that in normal cells is found in the cell. In AML however, the U5 snRNP200 complex is exposed on the cell membrane of leukemic blasts. U5 snRNP200 complex-specific antibodies induced death of AML cells in an Fc receptor-dependent way in the absence of cytotoxic leukocytes or complement. In an AML mouse model, treatment with U5 snRNP200 complex-specific antibodies led to significant tumor growth inhibition. Thus, donor-derived U5 snRNP200 complex-recognizing AML-specific antibodies may contribute to antitumor responses.

Abstract LB-200: A patient derived antibody targeting the tetraspanin CD9 synergistically inhibits tumor growth with an anti PD1 antibody

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

Abstract B052: Donor derived cytotoxic antibodies from transplanted AML patients targeting a cell surface expressed nuclear antigen

Unleashing the tumor-specific immune response by immunotherapies such as checkpoint inhibitors or allogeneic stem cell transplantation can result in long lasting tumor regressions. It is thought that T cell responses are responsible for tumor rejection. Here we tested the hypothesis that B cells contribute to tumor regression following immunotherapy by analysing the tumor-specific antibody repertoire in patients suffering from acute myeloid leukemia (AML), a high-risk malignancy with a poor prognosis. These patients receive an allogeneic hematopoietic stem cell transplantation (HSCT) which resets the immune system and can lead to potent graft versus leukemia (GvL) responses. We selected three patients with high-risk AML who mounted potent GvL responses after allogeneic HSCT. Of these patients we established antibody-producing clonal B cell lines following transduction of memory B cells from peripheral blood with BCL-6 and Bcl-xL and screened those for producing antibodies specifically binding to surface antigens on AML cell lines and AML blasts. A number of antibodies were identified that recognized primary AML blasts isolated from newly diagnosed AML patients, but did not bind to healthy bone marrow, peripheral blood mononuclear cells or tissues such as liver, skin and colon. Strikingly, 40% of these AML-specific antibodies induced direct cell death of cultured AML cell lines and of primary AML blasts. Cytotoxicity of the antibodies was rapid, occurred both at 37°C and 4°C and could be prevented by Cytochalasin D, an actin polymerization inhibitor that stabilizes the cytoskeleton. This, in combination with the observation that cell death could not be prevented by apoptosis inhibitors indicated that the tumor cells were killed through a necrotic pathway like oncosis. Interestingly, most cytotoxic antibodies recognized an antigen, which in normal cells is expressed in the nucleus but in AML cells is present on the membrane. Together these data indicate that tumor selective antibodies can be elicited following allogeneic HSCT in AML patients with a strong GvL response. The direct cytotoxic activities against tumor cells of a proportion of these antibodies suggest that they contribute to the GvL response. Citation Format: Marijn Gillissen, Martijn Kedde, Etsuko Yasuda, Sophie Levie, Arjen Bakker, Yvonne Claassen, Martino Bohne, Dave Speijer, Daisy Picavet, Jan Stap, Pauline van Helden, Tim Beaumont, Hergen Spits, Mette Hazenberg. Donor derived cytotoxic antibodies from transplanted AML patients targeting a cell surface expressed nuclear antigen. [abstract]. In: Proceedings of the CRI-CIMT-EATI-AACR Inaugural International Cancer Immunotherapy Conference: Translating Science into Survival; September 16-19, 2015; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(1 Suppl):Abstract nr B052.

Abstract A061: Acute myeloid leukemia patients cured after allogeneic hematopoietic stem cell transplantation generate tumor-specific cytotoxic antibodies that kill AML blasts

Background: Allogeneic hematopoietic stem cell transplantation (HSCT) can cure acute myeloid leukemia (AML) when the donor immune system generates a potent graft versus leukemia (GvL) response. While the role of T cells and NK cells in GvL immune responses has been established, the contribution of B cells to GvL responses is less clear. Using SEREX and other techniques, the presence of antibodies directed against established tumor antigens following allogeneic HSCT has been demonstrated, but because these antibodies were not obtained in monoclonal format, the function of these antibodies could not be analyzed. Aim: To investigate the role of antibodies produced by donor-derived B cells in GvL responses. Methods: We selected five patients with high-risk AML who remained disease-free for more than 5 years after allogeneic HSCT and thus have mounted a potent GvL response. From the peripheral blood of these patients we isolated memory B cells that we transduced with Bcl-6 and Bcl-xL, to establish antibody-producing clonal B cell lines. Blood was obtained 2 years after allogeneic HSCT. B cell lines were screened for the production of antibodies that specifically bound to surface antigens on AML cell lines and AML blasts isolated from patients in our clinic. Target identification was performed by immunoprecipitation and mass-spectometry. Results: We identified patient derived clonal B cell lines producing antibodies that recognized antigens expressed on the cell surface of AML cells, but not on normal hematopoietic and non-hematopoietic cells. Antibodies were donor-derived, and a number of these antibodies recognized the U5 snRNP200 complex. The U5 snRNP200 complex is a component of the spliceosome that in normal cells is located in the nucleus but that is exposed on the cell membrane of AML cells. U5 snRNP200 complex-specific antibodies were specific for allogeneic HSCT recipients with AML, as they were found in in 4 out of 5 AML patients screened, but were not found in multiple myeloma patients who received an allogeneic HSCT or in healthy individuals. Strikingly, U5 snRNP200 complex-specific antibodies induced death of AML cells in vitro, and, in a human AML mouse model, in vivo. Cell death was induced in the absence of cytotoxic leukocytes or of complement, through a non-apoptotic process that depended on destabilization of the cytoskeleton as cell death could be blocked by incubation of the target cells with cytochalasin D, an actin polymerization inhibitor. Cytotoxicity of the U5 snRNP200 antibodies was present at 4°C and 37°C, suggesting that cell death was induced by a passive process. Indeed, interaction of the antibodies with their target cells did not induce a calcium flux. Cytotoxicity of the antibodies depended on the Fc region of the antibody, since recombinant U5 snRNP200 complex-specific antibodies with a defective Fc region were not cytotoxic. Summary and Conclusions: Allogeneic HSCT recipients with robust donor anti-AML immunity generate antibodies against a component of the spliceosome, the U5 snRNP200 complex, that is expressed on the membrane of AML blasts. U5 snRNP200 antibodies are cytotoxic in vivo and in vitro, demonstrating the potency of the humoral immune system in tumor immunology. Citation Format: Marijn Gillissen, Martijn Kedde, Greta De Jong, Etsuko Yasuda, Sophie Levie, Arjen Bakker, Yvonne Claassen, Koen Wagner, Julien Villaudy, Martino Bohne, Dave Speijer, Paul Hensbergen, Pauline van Helden, Tim Beaumont, Hergen Spits, Mette D. Hazenberg. Acute myeloid leukemia patients cured after allogeneic hematopoietic stem cell transplantation generate tumor-specific cytotoxic antibodies that kill AML blasts [abstract]. In: Proceedings of the Second CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; 2016 Sept 25-28; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(11 Suppl):Abstract nr A061.

Abstract A026: Tumor-specific glycosylated CD43 is a novel and highly specific target for acute myeloid leukemia and myelodysplastic syndrome

Background: Acute myeloid leukemia (AML) and myelodysplastic syndrome (MDS) are high-risk diseases with a poor prognosis. Even with intensive treatment regimens less than 50% of patients can be cured, and for the majority of patients - those over 65 years of age and/or patients with comorbidities - such intensive regimens are not feasible. Novel therapeutic approaches such as immunotherapy directed against a highly specific tumor target are highly needed. Aims: The aim of our study was to identify novel therapeutic antibodies that are highly specific for AML and to discover novel tumor-specific antigens, widely expressed on AML and MDS but not on healthy hematopoietic and non-hematopoietic cells. Methods: For this we made use of the oldest human tumor immunology model with proven efficacy available: an allogeneic HSCT patient with a potent graft versus AML allo-immune response. From this patient we isolated CD27+ IgG+ memory B lymphocytes and transduced these cells with Bcl-6 and Bcl-xL, thereby generating pre-plasmablast B cell clones that produce abundant antibodies. Supernatants of these B cell clones were used to screen for binding to surface antigens on the AML cell line THP-1. Results: We identified an IgG1 antibody, AT14-013, that specifically interacted with AML cell lines THP-1, MOLM-13, SH-2 and others, and with leukemic blasts isolated from newly diagnosed AML and MDS patients from our clinic. AT14-013 did not interact with healthy hematopoietic and non-hematopoietic cells. This antibody was of donor origin and was antigen experienced as it contained 26/11 somatic hypermutations in the heavy and light chains, respectively. Target identification using mass spectrometry analysis and epitope mapping strategies with FLAG-tagged truncated variants of CD43 expressed by THP-1 that we created revealed CD43 as the target. CD43 is expressed by all hematopoietic cells, but AT14-013 targeted a specific, sialylated epitope on CD43 that is uniquely and widely expressed on all types of AML, as illustrated by its reactivity with blasts of each of 48 randomly selected AML and MDS patients in our clinic. AT14-013 induced antibody-dependent cell-mediated cytotoxicity and complement dependent cytotoxicity of AML cell lines and primary blasts. Summary and conclusion: We have identified onco-sialylated CD43 (CD43os) as a novel tumor-specific target that is widely expressed on AML and MDS blasts. Antibodies against this target have high potential as therapeutic antibodies, either as a naked antibody or manufactured into an antibody-drug conjugate, bispecific T cell engager or CAR (chimeric antigen receptor) T cell. Citation Format: Marijn Gillissen, Martijn Kedde, Etsuko Yasuda, Greta De Jong, Sophie Levie, Arjen Bakker, Paul Hensbergen, Julien Villaudy, Tim Beaumont, Pauline van Helden, Hergen Spits, Mette D. Hazenberg. Tumor-specific glycosylated CD43 is a novel and highly specific target for acute myeloid leukemia and myelodysplastic syndrome [abstract]. In: Proceedings of the Second CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; 2016 Sept 25-28; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(11 Suppl):Abstract nr A026.