A second generation human haplotype map of over 3.1 million SNPs.

We describe the Phase II HapMap, which characterizes over 3.1 million human single nucleotide polymorphisms (SNPs) genotyped in 270 individuals from four geographically diverse populations and includes 25-35% of common SNP variation in the populations surveyed. The map is estimated to capture untyped common variation with an average maximum r2 of between 0.9 and 0.96 depending on population. We demonstrate that the current generation of commercial genome-wide genotyping products captures common Phase II SNPs with an average maximum r2 of up to 0.8 in African and up to 0.95 in non-African populations, and that potential gains in power in association studies can be obtained through imputation. These data also reveal novel aspects of the structure of linkage disequilibrium. We show that 10-30% of pairs of individuals within a population share at least one region of extended genetic identity arising from recent ancestry and that up to 1% of all common variants are untaggable, primarily because they lie within recombination hotspots. We show that recombination rates vary systematically around genes and between genes of different function. Finally, we demonstrate increased differentiation at non-synonymous, compared to synonymous, SNPs, resulting from systematic differences in the strength or efficacy of natural selection between populations.

Genome-wide detection and characterization of positive selection in human populations.

With the advent of dense maps of human genetic variation, it is now possible to detect positive natural selection across the human genome. Here we report an analysis of over 3 million polymorphisms from the International HapMap Project Phase 2 (HapMap2). We used 'long-range haplotype' methods, which were developed to identify alleles segregating in a population that have undergone recent selection, and we also developed new methods that are based on cross-population comparisons to discover alleles that have swept to near-fixation within a population. The analysis reveals more than 300 strong candidate regions. Focusing on the strongest 22 regions, we develop a heuristic for scrutinizing these regions to identify candidate targets of selection. In a complementary analysis, we identify 26 non-synonymous, coding, single nucleotide polymorphisms showing regional evidence of positive selection. Examination of these candidates highlights three cases in which two genes in a common biological process have apparently undergone positive selection in the same population:LARGE and DMD, both related to infection by the Lassa virus, in West Africa;SLC24A5 and SLC45A2, both involved in skin pigmentation, in Europe; and EDAR and EDA2R, both involved in development of hair follicles, in Asia.

A T-cell engaging bispecific antibody with a tumor-selective bivalent folate receptor alpha binding arm for the treatment of ovarian cancer.

The use of T-cell engagers (TCEs) to treat solid tumors is challenging, and several have been limited by narrow therapeutic windows due to substantial on-target, off-tumor toxicities due to the expression of low levels of target antigens on healthy tissues. Here, we describe TNB-928B, a fully human TCE that has a bivalent binding arm for folate receptor alpha (FRα) to selectively target FRα overexpressing tumor cells while avoiding the lysis of cells with low levels of FRα expression. The bivalent design of the FRα binding arm confers tumor selectivity due to low-affinity but high-avidity binding to high FRα antigen density cells. TNB-928B induces preferential effector T-cell activation, proliferation, and selective cytotoxic activity on high FRα expressing cells while sparing low FRα expressing cells. In addition, TNB-928B induces minimal cytokine release compared to a positive control TCE containing OKT3. Moreover, TNB-928B exhibits substantial ex vivo tumor cell lysis using endogenous T-cells and robust tumor clearance in vivo, promoting T-cell infiltration and antitumor activity in mouse models of ovarian cancer. TNB-928B exhibits pharmacokinetics similar to conventional antibodies, which are projected to enable favorable administration in humans. TNB-928B is a novel TCE with enhanced safety and specificity for the treatment of ovarian cancer.

TNB-486 induces potent tumor cell cytotoxicity coupled with low cytokine release in preclinical models of B-NHL.

The therapeutic potential of targeting CD19 in B cell malignancies has garnered attention in the past decade, resulting in the introduction of novel immunotherapy agents. Encouraging clinical data have been reported for T cell-based targeting agents, such as anti-CD19/CD3 bispecific T-cell engager blinatumomab and chimeric antigen receptor (CAR)-T therapies, for acute lymphoblastic leukemia and B cell non-Hodgkin lymphoma (B-NHL). However, clinical use of both blinatumomab and CAR-T therapies has been limited due to unfavorable pharmacokinetics (PK), significant toxicity associated with cytokine release syndrome and neurotoxicity, and manufacturing challenges. We present here a fully human CD19xCD3 bispecific antibody (TNB-486) for the treatment of B-NHL that could address the limitations of the current approved treatments. In the presence of CD19+ target cells and T cells, TNB-486 induces tumor cell lysis with minimal cytokine release, when compared to a positive control. In vivo, TNB-486 clears CD19+ tumor cells in immunocompromised mice in the presence of human peripheral blood mononuclear cells in multiple models. Additionally, the PK of TNB-486 in mice or cynomolgus monkeys is similar to conventional antibodies. This new T cell engaging bispecific antibody targeting CD19 represents a novel therapeutic that induces potent T cell-mediated tumor-cell cytotoxicity uncoupled from high levels of cytokine release, making it an attractive candidate for B-NHL therapy.

A bispecific antibody agonist of the IL-2 heterodimeric receptor preferentially promotes in vivo expansion of CD8 and NK cells.

The use of recombinant interleukin-2 (IL-2) as a therapeutic protein has been limited by significant toxicities despite its demonstrated ability to induce durable tumor-regression in cancer patients. The adverse events and limited efficacy of IL-2 treatment are due to the preferential binding of IL-2 to cells that express the high-affinity, trimeric receptor, IL-2Rαßγ such as endothelial cells and T-regulatory cells, respectively. Here, we describe a novel bispecific heavy-chain only antibody which binds to and activates signaling through the heterodimeric IL-2Rßγ receptor complex that is expressed on resting T-cells and NK cells. By avoiding binding to IL-2Rα, this molecule circumvents the preferential T-reg activation of native IL-2, while maintaining the robust stimulatory effects on T-cells and NK-cells in vitro. In vivo studies in both mice and cynomolgus monkeys confirm the molecule's in vivo biological activity, extended pharmacodynamics due to the Fc portion of the molecule, and enhanced safety profile. Together, these results demonstrate that the bispecific antibody is a safe and effective IL-2R agonist that harnesses the benefits of the IL-2 signaling pathway as a potential anti-cancer therapy.

Attenuating CD3 affinity in a PSMAxCD3 bispecific antibody enables killing of prostate tumor cells with reduced cytokine release.

BACKGROUND: Therapeutic options currently available for metastatic castration-resistant prostate cancer (mCRPC) do not extend median overall survival >6 months. Therefore, the development of novel and effective therapies for mCRPC represents an urgent medical need. T cell engagers (TCEs) have emerged as a promising approach for the treatment of mCRPC due to their targeted mechanism of action. However, challenges remain in the clinic due to the limited efficacy of TCEs observed thus far in solid tumors as well as the toxicities associated with cytokine release syndrome (CRS) due to the usage of high-affinity anti-CD3 moieties such as OKT3. METHODS: Using genetically engineered transgenic rats (UniRat and OmniFlic) that express fully human IgG antibodies together with an NGS-based antibody discovery pipeline, we developed TNB-585, an anti-CD3xPSMA TCE for the treatment of mCRPC. TNB-585 pairs a tumor-targeting anti-PSMA arm together with a unique, low-affinity anti-CD3 arm in bispecific format. We tested TNB-585 in T cell-redirected cytotoxicity assays against PSMA+ tumor cells in both two-dimensional (2D) cultures and three-dimensional (3D) spheroids as well as against patient-derived prostate tumor cells. Cytokines were measured in culture supernatants to assess the ability of TNB-585 to induce tumor killing with low cytokine release. TNB-585-mediated T cell activation, proliferation, and cytotoxic granule formation were measured to investigate the mechanism of action. Additionally, TNB-585 efficacy was evaluated in vivo against C4-2 tumor-bearing NCG mice. RESULTS: In vitro, TNB-585 induced activation and proliferation of human T cells resulting in the killing of PSMA+ prostate tumor cells in both 2D cultures and 3D spheroids with minimal cytokine release and reduced regulatory T cell activation compared with a positive control antibody that contains the same anti-PSMA arm but a higher affinity anti-CD3 arm (comparable with OKT3). In addition, TNB-585 demonstrated potent efficacy against patient-derived prostate tumors ex vivo and induced immune cell infiltration and dose-dependent tumor regression in vivo. CONCLUSIONS: Our data suggest that TNB-585, with its low-affinity anti-CD3, may be efficacious while inducing a lower incidence and severity of CRS in patients with prostate cancer compared with TCEs that incorporate high-affinity anti-CD3 domains.

Vibrational Spectroscopy Reveals Effects of Electron Push-Pull and Solvent Polarity on Electron Delocalization in Radical Anions of Donor-Acceptor Compounds.

The nature of excess electrons has been studied in donor-acceptor (D-A) compounds based on substituted triarylamines and a nitrile-functionalized fluorene by changing the substituents on the triarylamines and also the solvent polarity. We observed that both electron push-pull capability at the distant location in the amine donor unit and solvation in solvents of varying polarity significantly affect the nitrile ν(C≡N) vibrations of the fluorene acceptor unit in radical anions of these D-A compounds. Quantum calculations show that the push-pull capability translates the position of an excess electron while keeping its width relatively constant. On the other hand, solvation changes both, making an electron more compact in polar media. The current study points to the idea that solvation plays a more significant role in controlling the nature of excess electrons, while synthetic modification that influences electron push-pull capability enables further tuning.

Efficient tumor killing and minimal cytokine release with novel T-cell agonist bispecific antibodies.

T-cell-recruiting bispecific antibodies (T-BsAbs) have shown potent tumor killing activity in humans, but cytokine release-related toxicities have affected their clinical utility. The use of novel anti-CD3 binding domains with more favorable properties could aid in the creation of T-BsAbs with improved therapeutic windows. Using a sequence-based discovery platform, we identified new anti-CD3 antibodies from humanized rats that bind to multiple epitopes and elicit varying levels of T-cell activation. In T-BsAb format, 12 different anti-CD3 arms induce equivalent levels of tumor cell lysis by primary T-cells, but potency varies by a thousand-fold. Our lead CD3-targeting arm stimulates very low levels of cytokine release, but drives robust tumor antigen-specific killing in vitro and in a mouse xenograft model. This new CD3-targeting antibody underpins a next-generation T-BsAb platform in which potent cytotoxicity is uncoupled from high levels of cytokine release, which may lead to a wider therapeutic window in the clinic.

Sequence-Based Discovery Demonstrates That Fixed Light Chain Human Transgenic Rats Produce a Diverse Repertoire of Antigen-Specific Antibodies.

We created a novel transgenic rat that expresses human antibodies comprising a diverse repertoire of heavy chains with a single common rearranged kappa light chain (IgKV3-15-JK1). This fixed light chain animal, called OmniFlic, presents a unique system for human therapeutic antibody discovery and a model to study heavy chain repertoire diversity in the context of a constant light chain. The purpose of this study was to analyze heavy chain variable gene usage, clonotype diversity, and to describe the sequence characteristics of antigen-specific monoclonal antibodies (mAbs) isolated from immunized OmniFlic animals. Using next-generation sequencing antibody repertoire analysis, we measured heavy chain variable gene usage and the diversity of clonotypes present in the lymph node germinal centers of 75 OmniFlic rats immunized with 9 different protein antigens. Furthermore, we expressed 2,560 unique heavy chain sequences sampled from a diverse set of clonotypes as fixed light chain antibody proteins and measured their binding to antigen by ELISA. Finally, we measured patterns and overall levels of somatic hypermutation in the full B-cell repertoire and in the 2,560 mAbs tested for binding. The results demonstrate that OmniFlic animals produce an abundance of antigen-specific antibodies with heavy chain clonotype diversity that is similar to what has been described with unrestricted light chain use in mammals. In addition, we show that sequence-based discovery is a highly effective and efficient way to identify a large number of diverse monoclonal antibodies to a protein target of interest.

Multispecific Antibody Development Platform Based on Human Heavy Chain Antibodies.

Heavy chain-only antibodies (HCAbs) do not associate with light chains and their VH regions are functional as single domains, forming the smallest active antibody fragment. These VH regions are ideal building blocks for a variety of antibody-based biologics because they tolerate fusion to other molecules and may also be attached in series to construct multispecific antibodies without the need for protein engineering to ensure proper heavy and light chain pairing. Production of human HCAbs has been impeded by the fact that natural human VH regions require light chain association and display poor biophysical characteristics when expressed in the absence of light chains. Here, we present an innovative platform for the rapid development of diverse sets of human HCAbs that have been selected in vivo. Our unique approach combines antibody repertoire analysis with immunization of transgenic rats, called UniRats, that produce chimeric HCAbs with fully human VH domains in response to an antigen challenge. UniRats express HCAbs from large transgenic loci representing the entire productive human heavy chain V(D)J repertoire, mount robust immune responses to a wide array of antigens, exhibit diverse V gene usage and generate large panels of stable, high affinity, antigen-specific molecules.

Multiplexed variation scanning for 1,000 amplicons in hundreds of patients using mismatch repair detection (MRD) on tag arrays.

Identification of the genetic basis of common disease may require comprehensive sequence analysis of coding regions and regulatory elements in patients and controls to find genetic effects caused by rare or heterogeneous mutations. In this study, we demonstrate how mismatch repair detection on tag arrays can be applied in a case-control study. Mismatch repair detection allows >1,000 amplicons to be screened for variations in a single laboratory reaction. Variation scanning in 939 amplicons, mostly in coding regions within a linkage peak, was done for 372 patients and 404 controls. In total, >180 Mb of DNA was scanned. Several variants more prevalent in patients than in controls were identified. This study demonstrates an approach to the discovery of susceptibility genes for common disease: large-scale direct sequence comparison between patients and controls. We believe this approach can be scaled up to allow sequence comparison in the whole-genome coding regions among large sets of cases and controls at a reasonable cost in the near future.

Highly multiplexed molecular inversion probe genotyping: over 10,000 targeted SNPs genotyped in a single tube assay.

Large-scale genetic studies are highly dependent on efficient and scalable multiplex SNP assays. In this study, we report the development of Molecular Inversion Probe technology with four-color, single array detection, applied to large-scale genotyping of up to 12,000 SNPs per reaction. While generating 38,429 SNP assays using this technology in a population of 30 trios from the Centre d'Etude Polymorphisme Humain family panel as part of the International HapMap project, we established SNP conversion rates of approximately 90% with concordance rates >99.6% and completeness levels >98% for assays multiplexed up to 12,000plex levels. Furthermore, these individual metrics can be "traded off" and, by sacrificing a small fraction of the conversion rate, the accuracy can be increased to very high levels. No loss of performance is seen when scaling from 6,000plex to 12,000plex assays, strongly validating the ability of the technology to suppress cross-reactivity at high multiplex levels. The results of this study demonstrate the suitability of this technology for comprehensive association studies that use targeted SNPs in indirect linkage disequilibrium studies or that directly screen for causative mutations.