Modular basis for potent SARS-CoV-2 neutralization by a prevalent VH1-2-derived antibody class

SUMMARYAntibodies with heavy chains that derive from the VH1-2 gene constitute some of the most potent SARS-CoV-2-neutralizing antibodies yet identified. To provide insight into whether these genetic similarities inform common modes of recognition, we determined structures of the SARS-CoV-2 spike in complex with three VH1-2-derived antibodies: 2-15, 2-43, and H4. All three utilized VH1-2-encoded motifs to recognize the receptor-binding domain (RBD), with heavy chain N53I enhancing binding and light chain tyrosines recognizing F486RBD. Despite these similarities, class members bound both RBD-up and -down conformations of the spike, with a subset of antibodies utilizing elongated CDRH3s to recognize glycan N343 on a neighboring RBD – a quaternary interaction accommodated by an increase in RBD separation of up to 12 Å. The VH1-2-antibody class thus utilizes modular recognition encoded by modular genetic elements to effect potent neutralization, with VH-gene component specifying recognition of RBD and CDRH3 component specifying quaternary interactions.HighlightsDetermine structures of VH1-2-derived antibodies 2-43, 2-15, and H4 in complex with SARS-CoV-2 spikeDefine a multi-donor VH1-2-antibody class with modular components for RBD and quaternary recognitionReveal structural basis of RBD-up and RBD-down recognition within the classShow somatic hypermutations and avidity to be critical for potencyDelineate changes in spike conformation induced by CDRH3-mediated quaternary recognition

RNA-Seq De Novo Assembly of Clonal Immunoglobulin Rearrangements Identifies Interesting Biology and Uncovers Prognostic Features in Multiple Myeloma

Introduction Although monoclonal immunoglobulin (Ig) production by myeloma cells is one of the central features of the disease, genotypic identification of the clonal Ig sequence remains understudied in multiple myeloma (MM). Here, using extensive RNA-seq data, we study molecular features of clonal Ig rearrangements, as well as their association with other MM markers and patient outcome. Methods We performed deep RNA-seq on purified CD138+ MM cells from 429 newly-diagnosed uniformly-treated patients with long clinical follow-up. For each sample, we performed de novo assembly using sequences that appeared in the library with a frequency of at least one in a million. Germline V and J genes were then BLASTed against the assembled contigs to determine the clonal germline genes and pinpoint mutations. Using the sequences reconstructed from the Ig contigs and the BLAST output, we ran IgBLAST to fully characterize the predominant Ig V(D)J sequence. Results We tested the accuracy of our approach by looking at 24 technical duplicates and one triplicate. In all cases, the predicted gene and gene allele were consistent across replicates. Next, we evaluated our large patient cohort, identifying IGHV3 as the most common clonal VH gene subgroup (53.3%), followed by IGHV4 (17.8%) and IGHV1 (15.6%). Importantly, we observed a significant association between poorer prognosis and IGHV3, both for progression-free survival (PFS) (p=0.0019) and overall survival (OS) (p=0.012). IGHV3-30 (11%, the most commonly rearranged VH gene) and IGHV3-9 (4.8%) were the drivers behind this poor prognosis (IGHV3-30: PFS p=0.021; OS p=0.013) (IGHV3-9: PFS p=0.002). IGHV3-30 was even more preferentially rearranged than in normal B-cell VH repertoires from previous studies (8.5%, 6.3%) and ours (2%). Remarkably, these results sharply contrast with what has been observed in CLL. In this malignancy, IGHV3-30 use has been seen to be underrepresented and usually characterizes an indolent clinical course, while IGHV3-21 and possibly IGHV3-23 carry poor prognosis. We predicted light chain usage through the presence of clonal VL sequences. The most frequent VL genes were from the κ locus (69.4% total): IGKV1-33 (12.4%), IGKV1-5 (11.3%), IGKV3-20 (9.9%) and IGKV1-39 (8.0%). Del(22q) was observed more frequently in patients with IGλ (OR=10.0, p=6e-15) and, within this group, del(22q) was more frequent if Vλ belonged to the more centromeric V-clusters C or B, in contrast to cluster A (OR=8.4, p=5e-4). Remarkably, patients with Vλ gene from cluster A presented worse OS (vs. Vk: p=0.0079; vs. Vλ B,C: p=0.067). The proportion of mutated bases was higher in the heavy chain than in the light chain (mean 7.0% vs. 4.8%, max 14.6% vs. 14.3%), and it was associated with OS (heavy p=0.0020, light p=0.036, both=0.0056), but not PFS. Interestingly, mutated Ig in CLL results in a more benign clinical course. We further found that 24.9% and 22.7% of the mutations lay within WRCY or RGYW AID motifs in the light and heavy chains respectively (enrichment p<1e-16), while AID mutations in a TW or WA context accounted for 22.9% and 25.7% (p=0.14, p=0.64). Higher ratios of mutations in WRCY vs. RGYW motifs within the light chain were highly predictive of poor prognosis (PFS p=0.0019, OS p=6.3e-4). Strikingly, IGλ usage was linked to higher ratios (p=3e-6), an association not explained by germline sequence variability (p=0.24). The usage of IGHV3 genes and the AID WRCY/RGYW motif ratio were independent markers of each other (p=1) and of other markers of poor prognosis in MM, such as presence of either t(4;14) or del(17p) (IGHV3 p=0.10; motif ratio p=0.49). In conclusion, de novo Ig heavy and light chain assembly using RNA-seq identifies interesting biology, may provide MM markers and highlights a novel application of high-throughput genomics. Disclosures Anderson: OncoPep Inc.: Equity Ownership, Membership on an entity's Board of Directors or advisory committees. Avet-Loiseau:sanofi: Consultancy; celgene: Consultancy; amgen: Consultancy; janssen: Consultancy.

Detection of the Malignant B Cell Clone in Multiple Myeloma Via High Throughput Sequencing Is Robust to Significant Levels of Somatic Hypermutation

Background: High-throughput sequencing (HTS) of antibody gene rearrangements is an emerging tool for minimal residual disease (MRD) monitoring in B cell malignancies in which the malignant clone harbors a monoclonal Ig heavy chain (IgH) and/or light chain (κ or λ) rearrangement. This approach has shown promise in B-ALL and CLL, but experience with this technique applied to samples from multiple myeloma patients is limited. Approach: We conducted HTS of PCR-amplified IgH (VDJ and DJ) rearrangements from bone marrow aspirates of 21 patients with various plasma cell dyscrasias (MM, MGUS, LPL) and peripheral blood of a patient with plasma cell leukemia. In 17/21 samples, an aliquot was enriched for CD138+ cells by immunomagnetic separation and analyzed separately. Dominant clones from enriched and un-enriched aliquots were compared to verify the malignant clonotype sequence(s). Disease burden in un-enriched samples was also evaluated by microscopy of the bone marrow aspirate smear and ranged from 0 (hemodilute) to 37% plasma cells. Results: In 19 out of 21 samples, a clearly dominant IgH gene rearrangement (>2.5% of total sequences, range 2.9-99.9%) was identified with clear separation from background frequency (at least 2.7-fold higher frequency than next most common clone). In 17/17 cases with paired CD138-enriched samples, the dominant sequences in the enriched and un-enriched samples were identical, indicating successful identification of the malignant clonal Ig rearrangements in the un-enriched sample. More than one IgH rearrangement suitable for longitudinally tracking the malignant clone was identified in 8 of 21 cases. The two cases without an expected, productive IgH rearrangement were IgG-κ and IgG-λ. This suggested that somatic hypermutation (SHM) in the primer binding sites might interfere with some clonal amplifications, so we investigated the degree of SHM in the VH segment of the 19 cases with at least one detected dominant Ig rearrangement. A total of 18 productive VDJ rearrangements were identified, and had SHM frequencies ranging from 2% to 19% in the sequenced portions of the rearranged VH gene. 8 myeloma clones harbored an identifiable DJ rearrangement, none of which showed evidence of SHM. Finally, 3 myeloma clones harbored nonproductive VDJ rearrangements, two with no SHM, and one with 2 SHM in 84 bp of the sequenced VH gene. Conclusion: HTS of Ig heavy and light chain rearrangements can successfully identify the malignant plasma cell clone in clinical specimens, including those with low disease burden and significant SHM. Application of this technique to MRD evaluation in multiple myeloma warrants further development. Disclosures Carlson: Adaptive Biotechnologies: Consultancy, Equity Ownership. Vogl:Celgene Corporation: Consultancy; Amgen: Consultancy; Millennium/Takeda: Research Funding; GSK: Research Funding; Acetylon: Research Funding. Stadtmauer:Janssen: Consultancy.

Chlamydia Psittaci negative MALT Lymphomas of the Ocular Adnexa Are Characterized by Biased Use of the VH4 Family and Evidence for Antigen Selection

Abstract 2644 Mucosa-associated lymphoid tissue (MALT) lymphomas are the most common lymphomas in the ocular adnexa. Similar to extranodular MALT lymphomas in other tissues, ocular adnexa MALT lymphomas (OAMALTL) are frequently characterized by an indolent clinical course and often remain localized for many years. While chronic infection (e. g. Helicobacter pylori) or autoimmune diseases (e.g. Hashimoto's thyroiditis and Sjogren's syndrome) frequently precede and predispose patients to develop MALT lymphomas in the stomach, thyroid and salivary glands, respectively, the etiology and pathogenesis of OAMALTL are still controversial. An Italian group and investigators from several other geographic regions demonstrated Chlamydia psittaci (C. psittaci) DNA present in OAMALTL, suggesting that this pathogen may be implicated in the development of these lymphomas. However, similar studies performed by us and other investigators on US-based patients have failed to corroborate this finding. Furthermore, DNA from other bacteria was also not detected by us, thus not supporting a bacterial etiology in US-based patients. Since non-bacterial antigens may predispose patients to OAMALTL, we have examined immunoglubulin (Ig) heavy chain variable region (VH) usage and mutations in the largest to date cohort of C psittaci negative OAMALTL originating from a single institution. DNA was extracted by standard techniques from 68 fresh OAMALTL tissues and used for direct PCR or PCR followed by cloning and sequencing using family specific VH leader and JH primers. To control for potential PCR error, all patient samples were evaluated by 2 independent PCR reactions and sequencing and only cases with identical CDR3 regions in the independent PCR runs were defined as clonal. Using this definition, clonal rearranged VHDJH sequences were identified in 44 (64.7%) tumors originating from the orbit (19), conjunctivae (19) and lacrimal gland (6). Forty seven clonal VH gene sequences were detected (3 of the patients had 2 clonal sequences) with 46 potentially functional and 1 harboring an out-of-frame junction with a stop codon. In 14 (31.8%) cases the PCR product could be sequenced directly, whereas in 30 (68.2%) cases PCR amplicons had to be subcloned to identify the VH gene. The 46 potentially functional VH were derived from 4 of the 7 human VH gene families with the following distribution: VH1, 13.1%; VH2, 2.2%; VH3, 39.1%; VH4, 45.6%, demonstrating a biased overrepresentation of the VH4 gene family (p=0.001). The most frequently encountered genes were VH4-34 (n=8), VH3-30 (n=6), VH3-23 (n=5) and VH4-30 (n=4). VH4-34 represented 17% of all the potentially functional VH genes identified in this study in contrast to its usage in 4–7% in adult peripheral B lymphocytes. Only 3 VH gene sequences were unmutated. The average percent homology to the germ line sequence in the 43 functional mutated sequences was 93.2% (range 71.5–99.6). Multinomial algorithm for antigen selection revealed evidence for positive selection in the CDR in 15 sequences, negative selection in the FR in 17 sequences, and selection in both CDR and FR (in the same sequence) in 7 sequences. Selection analysis using the focused binomial test demonstrated evidence of selection in the FR in 12 sequences and in the CDR in only 4 sequences. Selection was detected in 5 of the 8 functional VH4-34 sequences. Analysis of the tumor-derived CDR3 sequences revealed low similarity and an absence of stereotyped sequences with no homology to antibacterial and other previously published antibodies. The average CDR3 isoelectric point was 5.91±1.89 (SD). The average CDR3 length was 15.73±13.62 (SD) amino acids, with 19 sequences harboring 15–19 amino acids and 7 CDR3 sequences longer than 19 amino acids. Intraclonal variation was assessed by extensive molecular cloning in 8 potentially functional VH gene isolates from 8 randomly selected tumor specimens. Confirmed ongoing mutations were detected in the 6 of the 8 analyzed sequences. Overall our findings demonstrate that C. psittaci negative OAMALTL exhibit biased usage of VH families and genes with evidence for intraclonal heterogeneity and antigen selection in multiple tumors, implicating immunological stimulation in the pathogenesis of these lymphomas. The nature of the antigens potentially playing role in these processes is currently unknown and requires further studies. Disclosures: No relevant conflicts of interest to declare.