High-throughput sequencing (HTS) of antibody repertoire libraries has become a powerful tool in the field of systems immunology. However, numerous sources of bias in HTS workflows may affect the ...obtained antibody repertoire data. A crucial step in antibody library preparation is the addition of short platform-specific nucleotide adapter sequences. As of yet, the impact of the method of adapter addition on experimental library preparation and the resulting antibody repertoire HTS datasets has not been thoroughly investigated. Therefore, we compared three standard library preparation methods by performing Illumina HTS on antibody variable heavy genes from murine antibody-secreting cells. Clonal overlap and rank statistics demonstrated that the investigated methods produced equivalent HTS datasets. PCR-based methods were experimentally superior to ligation with respect to speed, efficiency, and practicality. Finally, using a two-step PCR based method we established a protocol for antibody repertoire library generation, beginning from inputs as low as 1 ng of total RNA. In summary, this study represents a major advance towards a standardized experimental framework for antibody HTS, thus opening up the potential for systems-based, cross-experiment meta-analyses of antibody repertoires.
Full text
Available for:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Receptor tyrosine kinase-like orphan receptor 2 (ROR2) has been identified as a highly relevant tumor-associated antigen in a variety of cancer indications of high unmet medical need, including renal ...cell carcinoma and osteosarcoma, making it an attractive target for targeted cancer therapy. Here, we describe the
discovery of fully human ROR2-specific antibodies and potent antibody drug conjugates (ADCs) derived thereof by combining antibody discovery from immune libraries of human immunoglobulin transgenic animals using the Transpo-mAb mammalian cell-based IgG display platform with functional screening for internalizing antibodies using a secondary ADC assay. The discovery strategy entailed immunization of transgenic mice with the cancer antigen ROR2, harboring transgenic IgH and IgL chain gene loci with limited number of fully human V, D, and J gene segments. This was followed by recovering antibody repertoires from the immunized animals, expressing and screening them as full-length human IgG libraries by transposon-mediated display in progenitor B lymphocytes ("Transpo-mAb Display") for ROR2 binding. Individual cellular "Transpo-mAb" clones isolated by single cell sorting and capable of expressing membrane-bound as well as secreted human IgG were directly screened during antibody discovery, not only for high affinity binding to human ROR2, but also functionally as ADCs using a cytotoxicity assay with a secondary anti-human IgG-toxin-conjugate. Using this strategy, we identified and validated 12 fully human, monoclonal anti-human ROR2 antibodies with nanomolar affinities that are highly potent as ADCs and could be promising candidates for the therapy of human cancer. The screening for functional and internalizing antibodies during the early phase of antibody discovery demonstrates the utility of the mammalian cell-based Transpo-mAb Display platform to select for functional binders and as a powerful tool to improve the efficiency for the development of therapeutically relevant ADCs.
Next-generation sequencing (NGS) of antibody variable regions has emerged as a powerful tool in systems immunology by providing quantitative molecular information on polyclonal humoral immune ...responses. Reproducible and robust information on antibody repertoires is valuable for basic and applied immunology studies: thus, it is essential to establish the reliability of antibody NGS data.
We isolated RNA from antibody-secreting cells (ASCs) from either 1 mouse or a pool of 9 immunized mice in order to simulate both normal and high diversity populations. Next, we prepared three technical replicates of antibody libraries by RT-PCR from each diversity scenario, which were sequenced using the Illumina MiSeq platform resulting in >106 250 bp paired-end reads per replicate. We then assessed the robustness of antibody repertoire data based on clonal identification defined by amino acid sequence of either full-length VDJ region or the complementarity determining region 3 (CDR3). Leveraging modeling approaches adapted from mathematical ecology, we found that in either diversity scenario both CDR3 and VDJ detection nears completeness indicating deep coverage of ASC repertoires. Additionally, we defined reliability thresholds for accurate quantification and ranking of CDR3s and VDJs. Importantly, we show that both factors-(i) replicate sequencing and (ii) sequencing depth-are crucial for robust CDR3 and VDJ detection and ranking.
In summary, we established widely applicable experimental and computational guidelines for robust antibody NGS and analysis, which will help advance systems immunology studies related to the quantitative profiling of antibody responses following infection and vaccination.
Full text
Available for:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Mucosal transmission of the human immunodeficiency virus (HIV) results in a bottleneck in viral genetic diversity. Gnanakaran and colleagues used a computational strategy to identify signature amino ...acids at particular positions in Envelope that were associated either with transmitted sequences sampled very early in infection, or sequences sampled during chronic infection. Among the strongest signatures observed was an enrichment for the stable presence of histidine at position 12 at transmission and in early infection, and a recurrent loss of histidine at position 12 in chronic infection. This amino acid lies within the leader peptide of Envelope, a region of the protein that has been shown to influence envelope glycoprotein expression and virion infectivity. We show a strong association between a positively charged amino acid like histidine at position 12 in transmitted/founder viruses with more efficient trafficking of the nascent envelope polypeptide to the endoplasmic reticulum and higher steady-state glycoprotein expression compared to viruses that have a non-basic position 12 residue, a substitution that was enriched among viruses sampled from chronically infected individuals. When expressed in the context of other viral proteins, transmitted envelopes with a basic amino acid position 12 were incorporated at higher density into the virus and exhibited higher infectious titers than did non-signature envelopes. These results support the potential utility of using a computational approach to examine large viral sequence data sets for functional signatures and indicate the importance of Envelope expression levels for efficient HIV transmission.
Full text
Available for:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
While the contribution of CD8⁺ cytotoxic T lymphocytes to early containment of HIV-1 spread is well established, a role for NK cells in controlling HIV-1 replication during primary infection has been ...uncertain. The highly polymorphic family of KIR molecules expressed on NK cells can inhibit or activate these effector cells and might therefore modulate their activity against HIV-1-infected cells. In the present study, we investigated copy number variation in KIR3DH loci encoding the only activating KIR receptor family in rhesus monkeys and its effect on simian immunodeficiency virus (SIV) replication during primary infection in rhesus monkeys. We observed an association between copy numbers of KIR3DH genes and control of SIV replication in Mamu-A*01⁻ rhesus monkeys that express restrictive TRIM5 alleles. These findings provide further evidence for an association between NK cells and the early containment of SIV replication, and underscore the potential importance of activating KIRs in stimulating NK cell responses to control SIV spread.
Full text
Available for:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Abstract only
TPS1108
Background: The receptor tyrosine kinase-like orphan receptor 1 (ROR1) is highly expressed during embryonic development, but is minimally present or absent on post-partum ...healthy tissues. ROR1 is expressed in a variety of hematological and solid tumors and is associated with aggressive cancer phenotype and poor clinical outcomes. NBE-002 is an ADC targeting ROR1, obtained by site-specific, enzymatic conjugation of the anthracycline-derivative PNU-159682, modified with a non-cleavable linker to a humanized recombinant IgG1 monoclonal antibody, based on a novel anti-human ROR1 monoclonal antibody XBR1-402 (Peng et al. (2017) J. Mol. Biol. 429: 2954-73). Direct anti-tumor activity of NBE-002 was evaluated in immunodeficient, ROR1 expression-low/-intermediate/-high PDX models of several carcinoma and sarcoma subtypes. The most pronounced anti-tumor effect was achieved in triple-negative breast cancer (TNBC), at doses as low as 0.033 mg/kg, suggesting a best-in-class therapeutic index in light of the high tolerability in preclinical toxicology models. Administration in a fully immune competent setting (EMT6/ROR1 orthotopic breast cancer model) led to a strong anti-tumor response and a long-lasting anti-tumor immune protection dependent on CD8 T cells. Methods: NBE-002-01 (NCT04441099) is a first-in-human, open-label, multi-center, phase (Ph) 1/2 study of NBE-002 in adult patients with advanced solid tumors. Ph 1 of the study consists of a Dose Escalation Cohort (DEC), utilizing an accelerated titration design, followed by a traditional 3+3 design, and an optional Safety Expansion Cohort (SEC). Ph 2 will include two parallel Expansion Cohorts (EC), enrolling patients with advanced TNBC (EC1) or other solid tumors (EC2), with Simon’s two-stage design. Key eligibility criteria include Eastern Cooperative Oncology Group performance status of 0-2 (Ph 1) or 0-1 (Ph 2), adequate organ function defined as: hemoglobin ≥9.0 g/dL, neutrophils ≥1500 /µL, platelets ≥100000/µL, aspartate and alanine aminotransferases ≤2.5x the upper limit of normal (ULN), total bilirubin ≤1.5x ULN, creatinine ≤1.5x ULN, left ventricular ejection fraction ≥50%. The primary objectives are to assess safety and tolerability and to establish the recommended dose for further development (Ph 1), and to evaluate anti-tumor activity of (Ph 2). Secondary and exploratory objectives include characterization of immunogenicity as well as pharmacokinetic and pharmacodynamic profiles. NBE-002 is given intravenously once every three weeks until disease progression, unacceptable toxicity, withdrawal of consent, or other protocol-specific criteria are met. Ph 1 dose escalation was initiated on 17 JULY 2020 and is still recruiting in the US. Ph 2 is planned to be initiated in 2022. Clinical trial information: NCT04441099 .
ABSTRACT
Here, we demonstrate that
KIR2DL4
copy number variation (CNV) is associated with CD4
+
T-cell decline and functionality of cytokine-producing NK cells during primary simian immunodeficiency ...virus (SIV) infection in
Mamu-A
*
01
−
Indian-origin rhesus macaques, with higher
KIR2DL4
copy numbers being associated with a better preservation of CD4
+
T cells and an increased gamma interferon (IFN-γ) production from stimulated cytokine-producing NK cell subsets during acute SIVmac251 infection. These findings underscore the crucial role of activating killer-cell immunoglobulin-like receptors (KIRs) in NK cell-mediated SIV responses during early SIV infection.
PDF
