The term bispecific antibody (bsAb) is used to describe a large family of molecules designed to recognize two different epitopes or antigens. BsAbs come in many formats, ranging from relatively small proteins, merely consisting of two linked antigen-binding fragments, to large immunoglobulin G (IgG)-like molecules with additional domains attached. An attractive bsAb feature is their potential for novel functionalities - that is, activities that do not exist in mixtures of the parental or reference antibodies. In these so-called obligate bsAbs, the physical linkage of the two binding specificities creates a dependency that can be temporal, with binding events occurring sequentially, or spatial, with binding events occurring simultaneously, such as in linking an effector to a target cell. To date, more than 20 different commercialized technology platforms are available for bsAb creation and development, 2 bsAbs are marketed and over 85 are in clinical development. Here, we review the current bsAb landscape from a mechanistic perspective, including a comprehensive overview of the pipeline.

Bispecific antibodies: a mechanistic review of the pipeline.

Decreased NAD+ levels have been shown to contribute to metabolic dysfunction during aging. NAD+ decline can be partially prevented by knockout of the enzyme CD38. However, it is not known how CD38 is regulated during aging, and how its ecto-enzymatic activity impacts NAD+ homeostasis. Here we show that an increase in CD38 in white adipose tissue (WAT) and the liver during aging is mediated by accumulation of CD38+ immune cells. Inflammation increases CD38 and decreases NAD+. In addition, senescent cells and their secreted signals promote accumulation of CD38+ cells in WAT, and ablation of senescent cells or their secretory phenotype decreases CD38, partially reversing NAD+ decline. Finally, blocking the ecto-enzymatic activity of CD38 can increase NAD+ through a nicotinamide mononucleotide (NMN)-dependent process. Our findings demonstrate that senescence-induced inflammation promotes accumulation of CD38 in immune cells that, through its ecto-enzymatic activity, decreases levels of NMN and NAD+.

CD38 ecto-enzyme in immune cells is induced during aging and regulates NAD+ and NMN levels.

Despite several therapeutic advances over the past decade, multiple myeloma (MM) remains largely incurable, indicating a need for new treatment approaches. Chimeric antigen receptor (CAR) T cell therapy works by mechanisms distinct from those of other MM therapies and involves the modification of patient or donor T cells to target specific cell-surface antigens. B cell maturation antigen (BCMA) is expressed only on plasma cells, a small subset of B cells and MM cells, which makes it a suitable target antigen for such therapies. At the time of writing, data from >20 clinical trials involving anti-BCMA CAR T cells have demonstrated that patients with relapsed and/or refractory MM can achieve objective responses. These early investigations have been instrumental in demonstrating short-term safety and efficacy; however, most patients do not have disease remission lasting >18 months. Attempts to reduce or delay the onset of relapsed disease are underway and include identifying additional CAR T cell target antigens and methods of enhancing BCMA expression on MM cells. Engineering CAR T cells to enhance both the activity and safety of treatment continues to be a promising avenue for improvement. In this Review we summarize data from clinical trials that have been carried out to date, describe novel antigens that could be targeted in the future, and highlight potential future innovations that could enhance the efficacy and/or reduce the toxicities associated with CAR T cell therapies.

CAR T cell therapies for patients with multiple myeloma

Chimeric antigen receptors (CARs) are engineered proteins designed to target T cells to cancer cells. To effectively activate the T cells in which they are expressed, CARs must contain a costimulatory domain. The CAR T cell products approved for the treatment of B cell lymphomas and/or acute lymphoblastic leukaemia or multiple myeloma incorporate either a CD28-derived or a 4-1BB-derived costimulatory domain. Almost all other clinically tested CARs also use costimulatory domains from CD28 or 4-1BB. In preclinical experiments, cytokine release is usually greater with CARs containing CD28 versus 4-1BB costimulatory domains; however, constructs with either domain confer similar anticancer activity in mouse models. T cell products expressing CARs with either CD28 or 4-1BB costimulatory domains have been highly efficacious in patients with relapsed haematological malignancies, with anti-CD19 products having similar activity regardless of the source of the costimulatory domain. In large-cohort clinical trials, the rates of neurological toxicities have been higher with CD28-costimulated CARs, although this finding is probably the result of a combination of factors rather than due to CD28 signalling alone. Future preclinical and clinical research should aim to compare different costimulatory domains while controlling for confounding variables. Herein, we provide an overview of T cell costimulation by CD28 and 4-1BB and, using the available preclinical and clinical data, compare the efficacy and toxicity profiles associated with CARs containing either costimulatory domain.

A comparison of chimeric antigen receptors containing CD28 versus 4-1BB costimulatory domains.

Patient-derived T cells genetically reprogrammed to express CD19-specific chimeric antigen receptors (CARs) have shown remarkable clinical responses and are commercially available for the treatment of patients with certain advanced-stage B cell malignancies. Nonetheless, several trials have revealed pre-existing and/or treatment-induced immune responses to the mouse-derived single-chain variable fragments included in these constructs. These responses might have contributed to both treatment failure and the limited success of redosing strategies observed in some patients. Data from early phase clinical trials suggest that CAR T cells are also associated with immunogenicity-related events in patients with solid tumours. Generally, the clinical implications of anti-CAR immune responses are poorly understood and highly variable between different CAR constructs and malignancies. These observations highlight an urgent need to uncover the mechanisms of immunogenicity in patients receiving CAR T cells and develop validated assays to enable clinical detection. In this Review, we describe the current clinical evidence of anti-CAR immune responses and discuss how new CAR T cell technologies might impact the risk of immunogenicity. We then suggest ways to reduce the risks of anti-CAR immune responses to CAR T cell products that are advancing towards the clinic. Finally, we summarize measures that investigators could consider in order to systematically monitor and better comprehend the possible effects of immunogenicity during trials involving CAR T cells as well as in routine clinical practice.

Immunogenicity of CAR T cells in cancer therapy.

Chimeric antigen receptor (CAR)-expressing T cells targeting B-cell maturation antigen (BCMA) have activity against multiple myeloma, but improvements in anti-BCMA CARs are needed. We demonstrated recipient anti-CAR T-cell responses against a murine single-chain variable fragment (scFv) used clinically in anti-BCMA CARs. To bypass potential anti-CAR immunogenicity and to reduce CAR binding domain size, here we designed CARs with antigen-recognition domains consisting of only a fully human heavy-chain variable domain without a light-chain domain. A CAR designated FHVH33-CD8BBZ contains a fully human heavy-chain variable domain (FHVH) plus 4-1BB and CD3ζ domains. T cells expressing FHVH33-CD8BBZ exhibit similar cytokine release, degranulation, and mouse tumor eradication as a CAR that is identical except for substitution of a scFv for FHVH33. Inclusion of 4-1BB is critical for reducing activation-induced cell death and promoting survival of T cells expressing FHVH33-containing CARs. Our results indicate that heavy-chain-only anti-BCMA CARs are suitable for evaluation in a clinical trial.

/pdf/anti-bcma-chimeric-antigen-receptors-with-fully-human-heavy-2eat57ayje.pdf

Anti-BCMA chimeric antigen receptors with fully human heavy-chain-only antigen recognition domains.

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.

/pdf/sequence-based-discovery-demonstrates-that-fixed-light-chain-5fi3387sk7.pdf

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

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 

https://www.tandfonline.com/doi/pdf/10.1080/19420862.2021.1890411

Benjamin Buelow

Papers

Anti-BCMA chimeric antigen receptors with fully human heavy-chain-only antigen recognition domains.

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

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