Engineered allogeneic T cells decoupling T-cell-receptor and CD3 signalling enhance the antitumour activity of bispecific antibodies.

Bispecific antibodies (biAbs) used in cancer immunotherapies rely on functional autologous T cells, which are often damaged and depleted in patients with haematological malignancies and in other immunocompromised patients. The adoptive transfer of allogeneic T cells from healthy donors can enhance the efficacy of biAbs, but donor T cells binding to host-cell antigens cause an unwanted alloreactive response. Here we show that allogeneic T cells engineered with a T-cell receptor that does not convert antigen binding into cluster of differentiation 3 (CD3) signalling decouples antigen-mediated T-cell activation from T-cell cytotoxicity while preserving the surface expression of the T-cell-receptor-CD3 signalling complex as well as biAb-mediated CD3 signalling and T-cell activation.

Advances in antibody-based therapy in oncology.

Monoclonal antibodies are a growing class of targeted cancer therapeutics, characterized by exquisite specificity, long serum half-life, high affinity and immune effector functions. In this review, we outline key advances in the field with a particular focus on recent and emerging classes of engineered antibody therapeutic candidates, discuss molecular structure and mechanisms of action and provide updates on clinical development and practice.

High-throughput T cell receptor engineering by functional screening identifies candidates with enhanced potency and specificity.

A major challenge in adoptive T cell immunotherapy is the discovery of natural T cell receptors (TCRs) with high activity and specificity to tumor antigens. Engineering synthetic TCRs for increased tumor antigen recognition is complicated by the risk of introducing cross-reactivity and by the poor correlation that can exist between binding affinity and activity of TCRs in response to antigen (peptide-MHC). Here, we developed TCR-Engine, a method combining genome editing, computational design, and deep sequencing to engineer the functional activity and specificity of TCRs on the surface of a human T cell line at high throughput.

SARS-CoV-2 reactive and neutralizing antibodies discovered by single-cell sequencing of plasma cells and mammalian display.

Characterization of COVID-19 antibodies has largely focused on memory B cells; however, it is the antibody-secreting plasma cells that are directly responsible for the production of serum antibodies, which play a critical role in resolving SARS-CoV-2 infection. Little is known about the specificity of plasma cells, largely because plasma cells lack surface antibody expression, thereby complicating their screening. Here, we describe a technology pipeline that integrates single-cell antibody repertoire sequencing and mammalian display to interrogate the specificity of plasma cells from 16 convalescent patients.

A Single-Cell Atlas of Lymphocyte Adaptive Immune Repertoires and Transcriptomes Reveals Age-Related Differences in Convalescent COVID-19 Patients.

COVID-19 disease outcome is highly dependent on adaptive immunity from T and B lymphocytes, which play a critical role in the control, clearance and long-term protection against SARS-CoV-2. To date, there is limited knowledge on the composition of the T and B cell immune receptor repertoires [T cell receptors (TCRs) and B cell receptors (BCRs)] and transcriptomes in convalescent COVID-19 patients of different age groups. Here, we utilize single-cell sequencing (scSeq) of lymphocyte immune repertoires and transcriptomes to quantitatively profile the adaptive immune response in COVID-19 patients of varying age.

Platypus: an open-access software for integrating lymphocyte single-cell immune repertoires with transcriptomes.

High-throughput single-cell sequencing (scSeq) technologies are revolutionizing the ability to molecularly profile B and T lymphocytes by offering the opportunity to simultaneously obtain information on adaptive immune receptor repertoires (VDJ repertoires) and transcriptomes. An integrated quantification of immune repertoire parameters, such as germline gene usage, clonal expansion, somatic hypermutation and transcriptional states opens up new possibilities for the high-resolution analysis of lymphocytes and the inference of antigen-specificity. While multiple tools now exist to investigate gene expression profiles from scSeq of transcriptomes, there is a lack of software dedicated to single-cell immune repertoires.

A Functional Screening Strategy for Engineering Chimeric Antigen Receptors with Reduced On-Target, Off-Tumor Activation.

In recent years, chimeric antigen receptor (CAR) T cell cancer immunotherapies have advanced substantially in the clinic. However, challenges related to safety persist; one major concern occurs when CARs trigger a response to antigen present on healthy cells (on-target, off-tumor response). A strategy to ameliorate this relies on the complex relationship between receptor affinity and signaling, such that one can engineer a CAR that is only activated by tumor cells expressing high antigen levels.

Expression of IgG Monoclonals with Engineered Immune Effector Functions.

The therapeutic development of monoclonal antibodies requires robust and reliable methods for their recombinant expression and characterization. In this context, an increasingly important aspect in the antibody development process is to determine the contribution of Fc-mediated immune effector functions to therapeutic activity. Here we describe steps for the cloning and mammalian expression of mouse and human IgG monoclonals with reduced immune effector functions, based on mutation of Fc-gamma receptor and complement-binding sites.

Sequencing and Affinity Determination of Antigen-Specific B Lymphocytes from Peripheral Blood.

Here we describe methods for screening human blood to isolate peripheral blood mononuclear cells (PBMCs) capable of binding fluorescently labeled antigen, as well as methods for the amplification and sequencing of B cell receptor (BCR) heavy and light chain genes. Detailed protocols are provided for transient mammalian expression in a hexahistidine-tagged Fab format, purification by immobilized metal affinity chromatography (IMAC), and affinity determination by BioLayer interferometry (BLI).

Transient expression of human antibodies in mammalian cells.

Recombinant expression of antibody molecules in mammalian cells offers important advantages over traditionally utilized bacterial expression, including glycosylation required for antibody functionality and markedly reduced levels of endotoxin contamination. Advances in transient mammalian expression systems enable high yields (>100 mg/liter) that now allow for effective recombinant antibody production at a reasonable cost. Here, we provide step-by-step protocols for the design and recombinant expression of full-length IgG antibodies and antibody-derived constructs (including Fab, Fc-fusions and bispecifics) in mammalian cells.

Cytosolic Recognition of RNA Drives the Immune Response to Heterologous Erythrocytes.

The archetypal T cell-dependent antigen is sheep red blood cells (SRBCs), which have defined much of what we know about humoral immunity. Early studies using solubilized or sonicated SRBCs argued that the intact structure of SRBCs was important for optimal antibody responses. However, the reason for the requirement of intact SRBCs for the response to polyvalent protein antigen remained unknown.

IL-2 Shapes the Survival and Plasticity of IL-17-Producing γδ T Cells.

IL-17-producing γδ T (γδT-17) cells have proved to be an important early source of IL-17 in many inflammatory settings and are emerging as an important participant in protumor immune responses. Considering that their peripheral activation depends largely on innate signals rather than TCR ligation, it is important to understand what mechanisms exist to curb unwanted activation. Expression of the high-affinity IL-2R on γδT-17 cells prompted us to investigate a role for this cytokine.

Potent antitumour activity of interleukin-2-Fc fusion proteins requires Fc-mediated depletion of regulatory T-cells.

Interleukin-2 (IL-2) is an established therapeutic agent used for cancer immunotherapy. Since treatment efficacy is mediated by CD8 and NK cell activity at the tumour site, considerable efforts have focused on generating variants that expand these subsets systemically, as exemplified by IL-2/antibody complexes and 'superkines'. Here we describe a novel determinant of antitumour activity using fusion proteins consisting of IL-2 and the antibody fragment crystallizable (Fc) region.

Circumventing the stability-function trade-off in an engineered FN3 domain.

The favorable biophysical attributes of non-antibody scaffolds make them attractive alternatives to monoclonal antibodies. However, due to the well-known stability-function trade-off, these gains tend to be marginal after functional selection. A notable example is the fibronectin Type III (FN3) domain, FNfn10, which has been previously evolved to bind lysozyme with 1 pM affinity (FNfn10-α-lys), but suffers from poor thermodynamic and kinetic stability.

Challenges and opportunities for non-antibody scaffold drugs.

The first candidates from the promising class of small non-antibody protein scaffolds are now moving into clinical development and practice. Challenges remain, and scaffolds will need to be further tailored toward applications where they provide real advantages over established therapeutics to succeed in a rapidly evolving drug development landscape.

Redemption of autoantibodies on anergic B cells by variable-region glycosylation and mutation away from self-reactivity.

The best-understood mechanisms for achieving antibody self/non-self discrimination discard self-reactive antibodies before they can be tested for binding microbial antigens, potentially creating holes in the repertoire. Here we provide evidence for a complementary mechanism: retaining autoantibodies in the repertoire displayed as low levels of IgM and high IgD on anergic B cells, masking a varying proportion of autoantibody-binding sites with carbohydrates, and removing their self-reactivity by somatic hypermutation and selection in germinal centers (GCs). Analysis of human antibody sequences by deep sequencing of isotype-switched memory B cells or in IgG antibodies elicited against allogeneic RhD+ erythrocytes, vaccinia virus, rotavirus, or tetanus toxoid provides evidence for reactivation of anergic IgM(low) IgD+ IGHV4-34+ B cells and removal of cold agglutinin self-reactivity by hypermutation, often accompanied by mutations that inactivated an N-linked glycosylation sequon in complementarity-determining region 2 (CDR2).