Engineering multi-specific antibodies against HIV-1.

As increasing numbers of broadly neutralizing monoclonal antibodies (mAbs) against HIV-1 enter clinical trials, it is becoming evident that combinations of mAbs are necessary to block infection by the diverse array of globally circulating HIV-1 strains and to limit the emergence of resistant viruses. Multi-specific antibodies, in which two or more HIV-1 entry-targeting moieties are engineered into a single molecule, have expanded rapidly in recent years and offer an attractive solution that can improve neutralization breadth and erect a higher barrier against viral resistance. In some unique cases, multi-specific HIV-1 antibodies have demonstrated vastly improved antiviral potency due to increased avidity or enhanced spatiotemporal functional activity. This review will describe the recent advancements in the HIV-1 field in engineering monoclonal, bispecific and trispecific antibodies with enhanced breadth and potency against HIV-1. A case study will also be presented as an example of the developmental challenges these multi-specific antibodies may face on their path to the clinic. The tremendous potential of multi-specific antibodies against the HIV-1 epidemic is readily evident. Creativity in their discovery and engineering, and acumen during their development, will be the true determinant of their success in reducing HIV-1 infection and disease.

"Stapling" scFv for multispecific biotherapeutics of superior properties.

Single-chain fragment variable (scFv) domains play an important role in antibody-based therapeutic modalities, such as bispecifics, multispecifics and chimeric antigen receptor T cells or natural killer cells. However, scFv domains exhibit lower stability and increased risk of aggregation due to transient dissociation ("breathing") and inter-molecular reassociation of the two domains (VL and VH). We designed a novel strategy, referred to as stapling, that introduces two disulfide bonds between the scFv linker and the two variable domains to minimize scFv breathing. We named the resulting molecules stapled scFv (spFv). Stapling increased thermal stability (Tm) by an average of 10°C. In multiple scFv/spFv multispecifics, the spFv molecules display significantly improved stability, minimal aggregation and superior product quality. These spFv multispecifics retain binding affinity and functionality. Our stapling design was compatible with all antibody variable regions we evaluated and may be widely applicable to stabilize scFv molecules for designing biotherapeutics with superior biophysical properties.

Multi-targeted immunotherapeutics to treat B cell malignancies.

The concept of multi-targeted immunotherapeutic systems has propelled the field of cancer immunotherapy into an exciting new era. Multi-effector molecules can be designed to engage with, and alter, the patient's immune system in a plethora of ways. The outcomes can vary from effector cell recruitment and activation upon recognition of a cancer cell, to a multipronged immune checkpoint blockade strategy disallowing evasion of the cancer cells by immune cells, or to direct cancer cell death upon engaging multiple cell surface receptors simultaneously. Here, we review the field of multi-specific immunotherapeutics implemented to treat B cell malignancies. The mechanistically diverse strategies are outlined and discussed; common B cell receptor antigen targeting strategies are outlined and summarized; and the challenges of the field are presented along with optimistic insights for the future.

Tripokin: A multi-specific immunocytokine for cancer immunotherapy.

Antibodies that target the tumor microenvironment can be used to deliver pro-inflammatory payloads, such as cytokines. Cytokines are small proteins able to modulate the activity of the immune system, and antibody-cytokine fusion proteins have been tested in preclinical and clinical settings. In this study, we describe Tripokin, a novel multi-specific fusion protein that combines interleukin-2 and a single amino acid mutant of tumor necrosis factor. The two pro-inflammatory payloads were fused to the L19 antibody, a clinical-grade antibody against the extradomain B of fibronectin. The human payloads were used for clinical applications, while the corresponding murine cytokines were used for preclinical studies. The resulting fusion proteins were produced in mammalian cells and purified to homogeneity. The murine Tripokin product was well tolerated in tumor-bearing mice at three doses of 30 µg in a 2-day interval and promoted rapid tumor eradication in murine models, more efficiently than single-agent immunocytokines. Tripokin induced rapid tumor necrosis and stimulated a robust immune response, impacting innate and adaptive immune pathways. In addition, the combination with immune checkpoint inhibitors further boosted the therapeutic efficacy of our molecule. Tripokin represents a promising clinical candidate for the simultaneous delivery of interleukin-2 and tumor necrosis factor to neoplastic sites.

Monoclonal antibody therapeutics with up to five specificities: functional enhancement through fusion of target-specific peptides.

The recognition that few human diseases are thoroughly addressed by mono-specific, monoclonal antibodies (mAbs) continues to drive the development of antibody therapeutics with additional specificities and enhanced activity. Historically, efforts to engineer additional antigen recognition into molecules have relied predominantly on the reformatting of immunoglobulin domains. In this report we describe a series of fully functional mAbs to which additional specificities have been imparted through the recombinant fusion of relatively short polypeptides sequences. The sequences are selected for binding to a particular target from combinatorial libraries that express linear, disulfide-constrained, or domain-based structures. The potential for fusion of peptides to the N- and C- termini of both the heavy and light chains affords the bivalent expression of up to four different peptides. The resulting molecules, called zybodies, can gain up to four additional specificities, while retaining the original functionality and specificity of the scaffold antibody. We explore the use of two clinically significant oncology antibodies, trastuzumab and cetuximab, as zybody scaffolds and demonstrate functional enhancements in each case. The affect of fusion position on both peptide and scaffold function is explored, and penta-specific zybodies are demonstrated to simultaneously engage five targets (ErbB2, EGFR, IGF-1R, Ang2 and integrin αvß3). Bispecific, trastuzumab-based zybodies targeting ErbB2 and Ang2 are shown to exhibit superior efficacy to trastuzumab in an angiogenesis-dependent xenograft tumor model. A cetuximab-based bispecific zybody that targeting EGFR and ErbB3 simultaneously disrupted multiple intracellular signaling pathways; inhibited tumor cell proliferation; and showed efficacy superior to that of cetuximab in a xenograft tumor model.