ALBERT

Description: Introduction: Chronic antigenic stimulation may play an important role in the pathogenesis of malignant lymphomas. Although most MCL cases are believed to have an antigen-naive B cell as cell of origin, overrepresentation of certain VH genes has been reported. Therefore we screened BCRs from MCLs for possible antigens. Methods: BCRs were expressed as recombinant Fabs based on corresponding pairs of functional variable region heavy and light chain genes, which had been amplified from isolated genomic DNA of snap-frozen MCL specimens and established MCL cell lines. The purified BCR-Fabs were checked for binding to proteins expressed on macroarrays of human cDNA expression libraries and on bacterial lysates. In addition, sera from patients with MCL were screened for antibodies against respective BCR antigens. Results: The recombinant MCL-BCR derived Fabs from 9 patients and of four established MCL cell lines were tested on protein arrays. Recombinant lymphoma-BCR-derived Fabs from 4/9 patients and from 1/4 MCL cell lines reacted with human low density lipoprotein receptor-related protein associated protein 1 (LRPAP1). Specific secondary modifications of LRPAP1 explaining its autoimmunogenicity were not found. 8/30 patients with MCL had anti-LRPAP1-antibodies in their serum, which was the case in only 1/200 healthy controls. Finally, LRPAP1 specifically induced proliferation of Maver1 cells that express a BCR with specificity for LRPAP1. Conclusions: LRPAP1 is the first molecularly defined antigenic target of MCL-BCRs. The high frequency of LRPAP1-reactive BCRs in MCL suggests a role of LRPAP1 in the pathogenesis of MCL, even in cases with unmutated VH genes. The prevalence of LRPAP1-antibodies in MCL patients and healthy controls identifies LRPAP1-antibody as the first serologic risk factor for MCL (odds ratio: 72.36) Supported by Wilhelm-Sander-Stiftung Disclosures No relevant conflicts of interest to declare.

Description: Introduction: The major task of a B-cell receptor is the binding and internalization of its antigenic target, and its processing for antigen presentation to T-cells. Chronic antigenic stimulation has been discussed to play a role in the pathogenesis of malignant B-cell lymphomas. We therefore systematically searched for the antigenic targets of BCRs from various B-cell neoplasms. Methods: Recombinant BCRs were expressed as recombinant Fabs (rFabs) based on corresponding pairs of functional variable region heavy and light chain genes, which had been amplified from isolated genomic DNA lymphoma biopsies. Whenever possible, "natural" Fabs (nFabs) were also obtained by papain digestion of fresh or cultured lymphoma cells. Both nFab and rFab were used to screen for binding to proteins expressed on macroarrays derived from human cDNA expression libraries and identical binding pattern of nFabs and rFabs was demonstrated by an antigen competition assay. Results: Two antigens (paratarg-7 and sumoylated HSP-90 which are hyperphosphorylated and sumoylated, respectively, in patients compared to healthy controls) are the targets of paraproteins from (depending on ethnicity) 30-50% of all multiple myeloma patients; the BCR from 67% of patients with primary CNS lymphoma target hyperglycosylated neurabin, 26% of the BCR from ABC-type DLBCL target hypophosphorylated ARS2 and 45% of all mantle cell BCR target LRPAP1; optineurin is the BCR target of 12% follicular lymphomas and various autoantigens have been identified as the targets of roughly 30% of all CLL cases. For all autoantigens binding to its specific BCR, rapid internalization and induction of proliferation was demonstrated, indicating partial dependence on antigenic stimulation even in cell lines that had been in culture for years. Most importantly, BCR-specific cytotoxicity of recombinant pseudomonas-exotoxin conjugated ARS2 against an ABC-DLBCL cell line with BCR specific for ARS2 (OCI-Ly3) was demonstrated in vitro and in vivo after establishment of OCI-Ly3 lymphomas in SCID beige mice. Conclusions: Assuming that only a minority of BCR targets have been identified to date, the prevalence of posttranslationally modified autoantigens strongly supports a role of chronic antigenic stimulation in many B-cell neoplasms. Due to the predominance of a single or few BCR antigens in each malignant B-cell entity studied, BARs represent an attractive and novel therapeutic concept for a broad spectrum of B-cell neoplasms and are the first therapeutic approach in oncology that targets exclusively the malignant cells. BARs can be used for conjugation with toxins, radionuclides and small molecules as well as for bispecific constructs (e. g. with CD3 or CD16) and CAR T-cells, the toxicity of which should be drastically reduced due to the ultimate specificity of BARs that spares normal B-cells. Supported by Wilhelm-Sander-Stiftung Disclosures Pfreundschuh: Roche, Janssen, Celgene: Honoraria, Research Funding.

Description: Background: Sequence analyses of variable immunoglobulin gene fragments of PCNSL from immunocompetent patients have shown a VH4-34 restriction raising speculations on a selection/stimulation of a functional BCR by an autoantigen in the central nervous system. The present study focused on the search for these hypothetic autoantigens presumably driving the malignant transformation of B-cells into PCNSL cells by chronic BCR stimulation in immunocompetent patients. Methods: BCRs were expressed as recombinant Fabs based on corresponding pairs of functional variable region heavy and light chain genes, which had been amplified from isolated genomic DNA of snap-frozen lymphoma specimens and checked for binding to proteins expressed on macroarrays of human cDNA expression libraries. Results: Recombinant BCR expression was attempted in 21 and successful in twelve PCNSL cases. The VH4-34 family was overrepresented, but was found in less than a quarter of the PCNSL patients (4/21). Screening of the recombinant BCRs on protein arrays revealed that 8 of 12 recombinant PCNSL-BCRs reacted with SAMD14 and the SAM domain of neurabin-1, two proteins with high homology and preferential expression in the CNS. Subsequent proteomic analysis of cryoconserved lymphoma specimens showed that SAMD14 and the SAM domain of neurabin-1 were alternatively N-glycosylated in patients with a PCNSL-BCR specific for SAMD14 and neurabin-1, but not in the remaining PCNSL patients with BCR specificities other than for SAMD14/neurabin-1. Compared to SAMD14 and neurabin-1 from healthy controls, Asn 339 of SAMD14 and Asn 1277 of neurabin-1 were shown to carry additionally glycosylated Asn residues. Of interest, both additional glycosylation sites belonged to atypical, non-canonical Asn-Leu-Glu-Gln (N-L-E-Q) sites instead of the Asn-X-Ser/Thre consensus sequence (N-X-S/T; where X can be any amino acid except proline), which is reported to constitute 97% of N-glycosylation sites under physiologic circumstances. These atypical N-hyperglycosylations were shown for every case with sufficient biopsy material for this proteomic analysis and a PCNSL-BCR specific for SAMD14 and neurabin-1 in their PCNSL and CNS, and to a lesser degree in their peripheral blood mononuclear cells and patient-derived EBV-transformed lymphoblastoid cell lines (LCLs). Of the recombinant BCRs of all cases with sufficient material to test for hyperglycosylation, only the BCRs of the 6 cases with hyperglycosylated SAMD14/neurabin-1 reacted against SAMD14/neurabin-1. Of note, glycosylation status of 2/8 cases with recombinant SAMD14/neurabin-1 reactive BCRs could not be analyzed due to insufficient cryomaterial left after variable region gene PCRs. No hyperglycosylation of SAMD14 and neurabin-1 was found in the peripheral blood of 400 healthy controls, 100 newborns and 50 nursery residents. Moreover, antibodies against SAMD14 and neurabin-1 were detected in the sera and cerebrospinal fluids of an independent second cohort of patients with PCNSL (8/22), but not in sera of patients with secondary CNS manifestations of systemic DLBCL (0/17) or of healthy controls (0/92). Conclusion: Our results strongly suggest that the atypical (NLEQ) glycosylation of the highly homologous SAMD14 and the SAM domain of neurabin-1 maintains a chronic autoimmunogenic stimulation in the CNS, ultimately leading to the malignant transformation of B-cells with a BCR specific for these atypically N-hyperglycosylated proteins into an aggressive B-cell lymphoma in the CNS in a majority of patients with PCNSL. The fact that the VH4-34 family represents only a minority of VH families recognizing SAMD14/neurabin-1 underlines the extraordinary autoimmunogenicity of these posttranslationally modified proteins in a broad range of individuals. Our results provide the first and strongest experimental evidence for the role of chronic autoantigenic stimulation as a first step in the pathogenesis of aggressive B-cell lymphomas. Supported by Deutsche Forschungsgemeinschaft DFG, Deutsche José Carreras Leukämie Stiftung, Wilhelm-Sander-Stiftung, Deutsche Krebshilfe e.V. Disclosures No relevant conflicts of interest to declare.

Description: Chronic antigenic stimulation of the B-cell receptor (BCR) seems to play a critical role in the pathogenesis of B-cell lymphomas. We recently identified ARS2, LRPAP1 and Neurabin-I as the autoantigenic targets of the B-cell receptors of approximately 25% of diffuse large B cell lymphomas (DLBCLs) of the ABC type, 45% of mantle cell lymphomas (MCLs) and 66% of primary CNS lymphomas, respectively. These BCR antigens can be used to target lymphoma cells in an approach we designated as BAR (B-cell receptor antigens for reverse targeting). Since the most established approach to deliver therapeutic payloads to specific targets are antibodies which have well-defined pharmacokinetics, we constructed an antibody like construct (BAR-body) incorporating the DLBCL-BAR ARS2 in substitution for the variable domains of the heavy and light chains. This ARS2 containing BAR-body showed promising efficacy in in-vitro experiments. Here, we report the results of the initial in-vivo experiments using lymphoma xenograft mouse models. To create the ARS2 BAR-body, we exchanged the heavy and light chain variable region sequences of an IgG1 antibody with a sequence of similar length (approximately 120 amino acids) of the ARS2 protein (aa 343 - 466) containing the DLBCL reactive epitope (aa 343 - 375). The construct was assembled in a pCR2.1 vector, then transferred to a pSfi FLAG Tag vector and transfected into HEK293 cells for production. Flow cytometry was used for binding experiments of the ARS2 BAR-body to lymphoma cell lines. For mouse experiments we inoculated 1x107 cells of the human DLBCL cell line U2932 (expresses a BCR with reactivity for ARS2) subcutaneously into the left flank of 12 NOD SCID mice. Tumor volume was calculated from day 14 measuring the long and short diameters of the tumor mass in millimeters. 6 mice were treated with 60 mg/kg ARS2 BAR-body intraperitoneally on days 23, 25, 30, 32, 37, 39, 44 and 46 after tumor inoculation. 6 control mice were mock-treated with PBS following the same time schedule. Tumor growth was evaluated by calculating the change in tumor volume from the first measurement at day 14. We cloned, expressed and characterized an ARS2 containing BAR-body incorporating 4 molecules of the lymphoma-reactive epitope of ARS2 resulting in an antibody like construct using a BAR (ARS2) as binding moiety instead of normal variable regions. The ARS2 BAR-body could successfully be cloned and expressed as confirmed by western blot analysis. Flow cytometric binding assays confirmed specific binding to the DLBCL cell line U2932 which expresses a BCR receptor with reactivity for ARS2 while control cell lines could not be stained by the ARS2 BAR-body. U2932 cells could successfully be transplanted subcutaneously into the flank of NOD SCID mice to generate a xenograft mouse model. Tumors in the treatment group increased their mean volume 14.1 times while tumors in the control group grew by a factor of 18.3 as compared to the initial mean tumor volume analyzed at day 14. Approaches using the cognate antigen of B-cell receptors to target malignant B cells have an exclusive specificity for the BCR of the malignant clone and can be expected to be less toxic than the currently available antibody derived therapies targeting B-cells. The ARS2 BAR-body shows promising activity against ARS2 reactive B-cell lymphoma cells in in-vivo mouse experiments. Toxic effects using the ARS2 BAR-body for the first time in-vivo were not observed. Further studies are necessary to reproduce and optimize the current experiments to form the basis for the translational development of BAR-bodies. Disclosures Stilgenbauer: AbbVie, AstraZeneca, Celgene, Gilead Sciences, Inc., GSK, Hoffmann La-Roche, Janssen, Novartis: Consultancy, Honoraria, Research Funding, Speakers Bureau.

Description: Background: 6 cycles CHOP-like chemotherapy plus rituximab (6x R-CHOP) are the standard treatment for young patients with DLBCL. The MInT trial established a subgroup with favourable prognosis as defined as aaIPI=0 and no bulky disease [Pfreundschuh et al., Lancet Oncol 2006; 7: 379-391] with a 3-year EFS of 89%, PFS of 95% and OS of 98%. We hypothesized that 4 cycles of CHOP plus 6 applications of rituximab are non-inferior to the standard treatment of 6x R-CHOP in this population. Patients and Methods: 18 to 60 year-old patients, aaIPI =0 without bulky (≥7.5 cm) disease were randomized to receive 6x R-CHOP or 4x R-CHOP+2xR at 21-day cycles. Radiotherapy was not planned to be given except for prophylactic radiotherapy of the contralateral testis in patients with testicular lymphoma. The primary endpoint was progression free survival (PFS) with events defined as progressive disease, relapse or death. Assuming a 93% 3-years PFS for the 6x R-CHOP arm, it was planned to tolerate an impairment of 5.5% by reducing the number of courses to 4x R-CHOP+2xR to prove non-inferiority with a power of 80% and an alpha-error of 5% (one-sided). Results: Between 12/2005 and 10/2016, 592 patients were randomized in the international multi-center FLYER trial and 588 patients were evaluable for this final analysis. 295 patients were assigned to receive 6x R-CHOP and 293 were assigned to receive 4x R-CHOP+2xR. There were no relevant differences in demographics (median age: 48 years, 99% aaIPI=0, 1% aaIPI=1, 0.3% bulky disease), protocol adherence and toxicity between the two arms. PFS, EFS and OS after 4x R-CHOP+2xR were as good as after 6x R-CHOP. After 66 months median observation, the 3-year PFS rate of the patients receiving 4x R-CHOP+2xR was 96% vs. 94% of patients receiving 6x R-CHOP (p=0.760). The lower limit of the 95% CI of the difference between treatment arms was 0% and excludes -5.5% demonstrating the non-inferiority. The 3-year EFS was identical (89%) in both treatment arms. The 3-years OS was 99% in patients receiving 4x R-CHOP+2xR and 98% in patients receiving 6x R-CHOP. In a multivariable analysis adjusting for strata (stage and E-involvement), the hazard ratio of 4x R-CHOP+2xR compared to 6x R-CHOP was 1.0 (95% CI: 0.7-1.6; p=0.896) for EFS, 0.9 (95% CI: 0.5-1.6; p=0.797) for PFS, and 0.8 (95% CI: 0.4-1.9; p=0.671) for OS. With respect to relapse rate there was also no significant difference between the two treatment arms. 4% (95% CI 2-7%) of the patients in the 4x R-CHOP+2xR arm relapsed vs. 5% (95% CI 3-8%) of the patients in the 6x R-CHOP arm. 33% of relapses occurred in the first two years after study inclusion but continue to be seen with longer follow-up in both arms. Conclusion: In young patients with favourable prognosis DLBCL outcome after 4x R-CHOP+ 2xR is non-inferior compared to the previous standard 6x R-CHOP. Thus, chemotherapy can be spared without compromising prognosis in this population. Supported by Deutsche Krebshilfe Figure. Figure. Disclosures Poeschel: Roche: Other: Travel grants; Amgen: Other: Travel grants. Held:BMS: Consultancy, Other: Travel grants, Research Funding; Amgen: Research Funding; Roche: Consultancy, Other: Travel grants, Research Funding; MSD: Consultancy; Spectrum: Research Funding. Holte:Roche, Norway: Research Funding; Novartis Pharmaceuticals Corporation: Membership on an entity's Board of Directors or advisory committees. Viardot:Roche: Consultancy, Honoraria; Amgen: Consultancy; Gilead Kite: Consultancy, Honoraria; BMS: Consultancy, Honoraria; Pfizer: Consultancy, Honoraria. Borchmann:Novartis: Consultancy, Honoraria. Keller:Celgene: Research Funding; BMS: Consultancy; Takeda: Consultancy, Research Funding; Janssen-Cilag: Consultancy, Equity Ownership; Roche: Consultancy; MSD: Consultancy. Schmidt:Gilead: Honoraria, Other: Travel Grants; Celgene: Honoraria; Takeda: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Travel Grants. Marks:Merck: Honoraria; BMS: Honoraria; Servier: Honoraria. Stilgenbauer:Boehringer-Ingelheim: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Novartis: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Genentech: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Celgene: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Janssen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Amgen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Pharmcyclics: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Genzyme: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Sanofi: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; AbbVie: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Gilead: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; GSK: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Hoffmann La-Roche: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Mundipharma: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding. Schmitz:Riemser: Honoraria, Other: Travel grants; Kite/Gilead: Honoraria, Other: Travel grants; Novartis: Honoraria, Other: Travel grants; Celgene: Other: Travel grants; Roche: Honoraria. Murawski:Takeda: Consultancy; Janssen: Other: Travel grants.

Description: In patients with insufficient T-cell responses to the human cytomegalovirus (HCMV), reactivation can occur which significantly contributes to morbidity and mortality after allogeneic stem cell transplantation (HSCT). Despite the event of new therapeutics like letermovir, HCMV infection remains a major complication after stem cell and solid organ transplantation. HCMV infected cells process HCMV proteins intracellularly and present HCMV-peptides on the cell surface in the context of HLA class I complexes. Antibodies naturally do not bind to peptides that are presented in HLA complexes and immunity against HCMV is mostly dependent on T-cells recognizing HLA I/HCMV-peptide complexes via their T-cell receptor. Antibodies that are generated to bind to peptides that are presented in HLA complexes are termed T-cell receptor like (TCR-like) antibodies. It has recently been shown that the T-cell response against an HLA-C*07:02 presented HCMV-peptide (CRVLCCYVL) derived from the IE-1 antigen is particularly strong. Also, HLA-C*07:02 is less susceptible to viral immune evasion than HLA-B and HLA-A molecules making it an attractive target for TCR-like antibodies. Here, we set out to obtain and characterize HLA-C*07:02 restricted, HCMV-specific antibodies. Antibody Fab fragments (Fabs) specific for the HLA-C*07:02 restricted HCMV-peptide CRVLCCYVL were identified using phage display technology. For selection, phages were incubated for 1 hour with biotinylated HLA-C*07:02/CRVLCCYVL complexes at decreasing concentrations (15, 5 and 1 µg/ml). Before each round a pre-absorption using HLA-C*07:02 complexes folded with control-peptide was performed. Selected phages were eluted and used to infect E. coli bacteria of the strain TG1 to produce soluble Fabs, which were then tested for binding to HCMV-peptide (CRVLCCYVL) and control-peptide pulsed lymphoblastoid cell lines (LCLs) and lymphocytes expressing HLA-C*07:02. As control, selected Fabs were tested for binding to HCMV-peptide (CRVLCCYVL) and control-peptide loaded LCLs and lymphocytes expressing different HLA-C alleles. By this, we were able to identify 3 different Fabs (4/4, 7/3 and 13/2) that bound to HLA-C*07:02 expressing LCLs and blood lymphocytes loaded with the HCMV-peptide CRVLCCYVL. All Fabs proofed to be highly specific since they showed no binding properties towards HLA-C*07:02 positive LCLs and lymphocytes that were loaded with no peptide or a control peptide. Furthermore, only LCLs and lymphocytes expressing the HLA-C*07:02 allele could be stained by our Fabs after HCMV-peptide loading. LCLs and lymphocytes of different HLA-C alleles that were pulsed with the HCMV-peptide CRVLCCYVL or a control peptide were not bound by the selected Fabs. Using phage display technology, we identified 3 TCR-like antibody fragments that target specifically HLA-C*07:02 positive LCLs and lymphocytes when loaded with the IE-1 derived HCMV-peptide CRVLCCYVL. The Fabs 4/4, 7/3 and 13/2 seem to be highly specific and to our knowledge the described Fabs are the first TCR-like antibodies that target an HLA-C presented peptide. Further studies are underway to determine binding properties to HCMV infected fibroblasts and affinity of the identified HLA-C*07:02 restricted and HCMV-specific Fabs and to explore their therapeutic potential. Disclosures Held: Bristol-Myers Squibb: Consultancy, Other: Travel support, Research Funding; Roche: Consultancy, Other: Travel support, Research Funding; Amgen: Research Funding; Acrotech: Research Funding; MSD: Consultancy. Stilgenbauer:AbbVie, AstraZeneca, Celgene, Gilead Sciences, Inc., GSK, Hoffmann La-Roche, Janssen, Novartis: Consultancy, Honoraria, Research Funding, Speakers Bureau.

Description: Introduction: Burkitt lymphoma represents the most aggressive neoplasm of mature sIg+ B cells. Besides the characterisitc translocation of the MYC gene with an immunoglobulin heavy or light chain gene locus, activating mutations in the TCF3 gene and inactivating mutations in the ID3 gene represent the key events in the pathogenesis of Burkitt lymphoma. These TCF3/ID3 mutations result in tonic and antigen-independent B cell receptor (BCR) pathway activation. Additionally, chronic BCR activation by antigens might play a role in Burkitt lymphoma pathogenesis and we set out to identify such potential target antigens of BCRs of Burkitt lymphoma. Methods: BCRs were expressed as recombinant Fabs in TG1 E.coli based on corresponding pairs of functional variable region heavy and light chain genes, which had been amplified from isolated genomic DNA of snap-frozen sporadic Burkitt lymphoma specimens. Additionally, natural Fabs and recombinant Fabs were produced of 8 established Burkitt lymphoma cell lines by Papain digestion and BCR expression cloning. The purified pooled Fabs were screened for reactivities against non-modified and differently posttranslationally modified human protein macroarrays. Reactivities were verified by ELISA with coated N-terminally FLAG-tagged candidate antigens, each separately for the posttranslationally modified and non-modified isoforms. Recombinant Fabs derived of mantle cell lymphoma, diffuse large B cell lymphoma and primary central nervous system lymphoma served as controls. Moreover the functional effects on proliferation and BCR pathway activation after addition of the identified target antigens to Burkitt lymphoma cell lines with and without reactive BCRs were analyzed by proliferation assays and western blots. Finally, mutation status, methylation status and expression level of identified target antigens were analyzed in ICGC MMML-Seq lymphoma databases for differences in Burkitt lymphoma versus distinct lymphoma entities. Results: The Burkitt lymphoma derived Fabs were tested on posttranslationally modified protein arrays. The BCR of CA46 line showed specific reactivity against sumoylated Bystin, the Fab of the BL41 line reacted specifically against acetylated HSP40. Recombinant Fabs of diffuse large B cell lymphoma, primary central nervous system lymphoma and mantle cell lymphoma did neither bind sumoylated Bystin nor acetylated HSP40. Addition of the posttranslationally modified cognate antigens to respective Burkitt lymphoma cell line with the reactive BCR induced proliferaton. The analysis of the ICGC MMML-Seq lymphoma databases (representing different cohorts) showed a higher expression of Bystin in Burkitt lymphoma compared to other aggressive B-cell lymphomas. However, the mutation and methylation status of Bystin and HSP40 in the ICGC MMML-seq cohort did not provide any direct indication of the origin of their immunogenic post-translational modifications. Conclusions: A subgroup of sporadic Burkitt lymphoma has autoreactive BCRs with specific affinity against posttranslationally modified self antigens, demonstrating a new aspect in the pathogenesis of Burkitt lymphoma. Specific secondary modifications, as sumolytation of Bystin or acetylation of HSP40 appear to evoke the immunogenicity. Future studies will focus on the functional consequences of the antigen/BCR interaction on Burkitt cells. Furthermore, the causes of these posttranslationally modified neoantigens will be investigated in more detail. Disclosures Stilgenbauer: Mundipharma: Consultancy, Honoraria, Other: travel support, Research Funding; Pharmacyclics: Consultancy, Honoraria, Other: travel support, Research Funding; Celgene: Consultancy, Honoraria, Other: travel support, Research Funding; Roche: Consultancy, Honoraria, Other: travel support, Research Funding; Genetech: Consultancy, Honoraria, Other: travel support, Research Funding; Amgen: Consultancy, Honoraria, Other: travel support, Research Funding; AbbVie: Consultancy, Honoraria, Other: travel support, Research Funding; Novartis: Consultancy, Honoraria, Other: travel support, Research Funding; GlaxoSmithKline: Consultancy, Honoraria, Other: travel support, Research Funding; Janssen: Consultancy, Honoraria, Other: travel support, Research Funding; Gilead: Consultancy, Honoraria, Other: travel support, Research Funding.

Description: To address the role of chronic antigenic stimulation in primary central nervous system lymphoma (PCNSL), we searched for autoantigens and identified sterile α-motif domain containing protein 14 (SAMD14) and neural tissue-specific F-actin binding protein I (neurabin-I) as autoantigenic targets of the B-cell receptors (BCRs) from 8/12 PCNSLs. In the respective cases, SAMD14 and neurabin-I were atypically hyper-N-glycosylated (SAMD14 at ASN339 and neurabin-I at ASN1277), explaining their autoimmunogenicity. SAMD14 and neurabin-I induced BCR pathway activation and proliferation of aggressive lymphoma cell lines transfected with SAMD14- and neurabin-I-reactive BCRs. Moreover, the BCR binding epitope of neurabin-I conjugated to truncated Pseudomonas exotoxin-killed lymphoma cells expressing the respective BCRs. These results support the role of chronic antigenic stimulation by posttranslationally modified central nervous system (CNS) driver autoantigens in the pathogenesis of PCNSL, serve as an explanation for their CNS tropism, and provide the basis for a novel specific treatment approach.

Description: Background Chronic antigenic stimulation of the B-cell receptor (BCR) seems to play a critical role in the pathogenesis of B-cell lymphomas. We recently identified ARS2 and LRPAP1 as the autoantigenic targets of the B-cell receptors of approximately 25% of diffuse large B cell lymphomas (DLBCLs) of the ABC type and 45% of mantle cell lymphomas (MCLs), respectively. These BCR antigens can be used to target lymphoma cells in an approach we designated as BAR (B-cell receptor antigens for reverse targeting). The optimal therapeutic format BARs can be integrated in has yet to be found. Since the most established approach to deliver therapeutic payloads to specific targets are antibodies which have well-defined pharmacokinetics, we constructed and tested an antibody like construct (BAR-body) incorporating the DLBCL-BAR ARS2 in substitution for the variable domains of the heavy and light chains. Material and methods To create the ARS2 BAR-body, we exchanged the heavy and light chain variable region sequences of an IgG1 antibody with a sequence of similar length (approximately 120 amino acids) of the ARS2 protein (aa 343 - 466) containing the DLBCL reactive epitope (aa 343 - 375). The construct was assembled in a pCR2.1 vector, then transferred to a pSfi FLAG Tag vector for fusion with the FLAG tag and transfected into HEK293 cells for production. Purification of the BAR-body was performed via anti-FLAG antibody affinity chromatography. The BAR-body was detected by western blot analysis and binding capacity to the ARS2-reactive lymphoma cell lines U2932 and OCI-Ly3 and the not ARS2-reactive control DLBCL cell line TMD8 was assessed by flow cytometry. ARS2 BAR-body induced cytotoxicity of lymphoma cells with an ARS2 reactive BCR was measured by LDH release assays with human PBMCs as effector cells at an E:T ratio of 10:1. Results We cloned, expressed and characterized an ARS2 containing BAR-body incorporating 4 molecules of the lymphoma-reactive epitope of ARS2 resulting in an antibody like construct using a BAR (ARS2) as binding moiety instead of normal variable regions. The ARS2 BAR-body could successfully be cloned and expressed as confirmed by western blot analysis, which showed the construct at approximately 150 kD as was to be expected. The BAR-body bound specifically to the ARS2-reactive lymphoma cell lines U2932 and OCI-Ly3 and did not bind to the DLBCL cell line TMD8, which has a B-cell receptor of different specificity or to lymphoma cell lines of different entities. In LDH release assays with 5 x 104 PBMCs and 5 x 103 lymphoma cells (E:T ratio of 10:1) the ARS2 BAR-body induced PBMC mediated specific lysis of the ARS2 reactive lymphoma cell lines U2932 and OCI-Ly3 but not the control DLBCL cell line TMD8 starting at a concentration of 0,1µg/ml. Cytotoxic effects were dose dependent, reached a maximum of 50% specific lysis at a concentration of 1µg/ml and did not increase at concentrations of 10µg/ml. Conclusion Here, we show that BARs can substitute for the variable domains as binding moiety in antibody like constructs to target the BCR of B-cell lymphomas. Because approaches using their specific cognate antigen for targeting the malignant B cells have an exclusive specificity for the BCR of the malignant clone, they can be expected to be less toxic than the currently available antibody derived therapies targeting B-cells, because they leave normal B-lymphocytes unaffected. By incorporating BARs into the well-known format of an antibody we hope to capitalize on years of experience with this therapeutic format from conducting and interpreting in vivo experiments to the translation of the BAR approach into the clinic. Disclosures Stilgenbauer: Genentech: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Novartis: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Hoffmann La-Roche: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Genzyme: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Janssen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Gilead: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; GSK: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Mundipharma: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Celgene: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Amgen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Boehringer-Ingelheim: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; AbbVie: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Pharmcyclics: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Sanofi: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding.