Complement component 5a receptor 1 (C5aR1) is a G-protein coupled receptor expressed on granulocytes, monocytes, mast cells, astrocytes, and microglia. The receptor is activated as a part of both the complement pathway and extrinsic pathway. It is known to play a role in oncology and in inflammation and is associated with certain diseases such as Alzheimer’s disease, asthma, and COVID-19 and is a therapeutic target of interest for these indications. For this project, we utilized a dual library approach to finding antibodies against C5aR1; by immunizing a mouse against the antigen, we were able to sequence its immune repertoire and generate both an immune library and synthetic library. We then conducted a biopanning campaign against the target using both cell-based and protein-based approaches. Using NGS, we selected clones which were produced as IgG antibodies and their binding and specificity assessed using Flow Cytometry. We were able to identify several specific binders to C5aR1, demonstrating Twist Biopharma’s ability to find binders to difficult GPCR targets using multiple library approaches.
B-cell maturation antigen (BCMA) is a protein of about 20KDa and belongs to the tumor necrosis factor-receptor (TNFR) superfamily. BCMA is overexpressed in neoplastic plasma cells of patients with multiple myeloma (MM). ABL101 is a BCMA and 4-1BB dual targeting bispecific antibody and augments T cell function through BCMA-dependent 4-1BB clustering and signaling. ABL101 activated 4-1BB signaling in a BCMA expression dependent manner. In T cell activation assay using PBMC or T cells from different donors ABL101 induced T-cell activation, leading to IFN-g secretion and cytolytic killing of BCMA expressing tumor cells. In h4-1BB transgenic mouse model bearing MC38-hBCMA, single dosing of ABL101 (8 mg/kg) significantly inhibited tumor growth with one mouse showing complete remission (CR). Mouse with CR was further protected from the re-challenge of previously exposed tumors, implicating that immunological memory might be generated. To determine the potential use of ABL101 in combination with CD3 T cell engagers, suboptimal dose of ABL101 and CD3 T cell engager were combined. ABL101 exhibited synergistic T cell activation and cytotoxicity with BCMAxCD3 T cell engager. Moreover, in H929 bearing hematopoietic stem cell (HSC) humanized mouse model, combination of ABL101 with BCMAxCD3 T cell engager showed synergistically potent anti-tumor effect. In conclusion, our data indicates that ABL101 (BCMAx4-1BB) induces T-cell activation and tumor cell killing directly in a BCMA expression dependent manner and has combination effect with BCMAxCD3 T cell engager. This strongly suggests that ABL101 is a promising therapeutic tool for the treatment of multiple myeloma patients with BCMA overexpression.
A subclass of Camelid antibodies, devoid of light chains, are a structurally unique family of immunoglobulins (IgGs) that carry a significant potential for biologics development given their small size, stability and high affinity. In particular, recombinant VHH regions of camelid IgGs (also referred to as nanobodies) have been at the focus of recent antibody engineering efforts, given their excellent tissue penetration compared to full size IgGs and monovalency. Although those properties make them easy to manufacture, the biggest challenge in realizing the full potential of nanobodies is the initial elucidation of the protein sequence. Standard sequencing approach involves next generation sequencing (NGS) from the circulating peripheral blood lymphocytes, or lymph or spleen, followed by generation of a phage display library used to perform affinity maturation in vitro.
In this study, we are using an alternative approach, by sequencing alpaca IgGs directly from serum proteins de novo, without the need for genomic analysis. This is the first successful study to sequence alpaca IgG proteome de novo, with a technology that directly sequenced IgGs from the immune repertoire, thus identifying more physiologically relevant, high affinity binders while not being limited by the availability of B-cells in circulation.
A total of 10 sequences were identified, from that pool of sequenced alpaca antibodies, six monoclonal IgGs representative were selected for synthesis of recombinant monoclonal antibodies and further development. Two targets were chosen based on the original antigen pool used for alpaca immunization.
VHH antibody is highlighted as the next generation antibody drug because of their unique characteristics such as small molecular weight (Approximately 15 kDa), ease of engineering, and different binding mode to antigens compared to conventional antibodies. However, the drug discovery process for VHH requires optimization such as humanization for reducing immunogenicity. To solve this issue, we developed a synthetic VHH library with humanized framework regions. Firstly, we analyzed the structure and sequences of VHHs in the database, and found that CDR3 of VHHs are classified into three major structures, and that there are several trends of amino acid sequences of VHHs for each structures.
CD3 T-cell engagers have the potential to be a cornerstone of immuno-oncology. Developing effective T-cell engagers requires discovery of two parental antibodies that work together: a CD3-binding arm that fine-tunes T-cell activation and a tumor-binding arm with high specificity for cancer cells. But pairs of parental antibodies that function effectively in concert with each other are rare, necessitating diverse panels of antibodies that can be paired and tested to find optimal clinical candidates. To streamline T-cell engager development, we generated a large and diverse panel of fully human CD3-binding antibodies. We will present data demonstrating how integrating this panel with our bispecific engineering and high-throughput antibody assessment capabilities enabled identification of developable, proof-of-concept CD3 T-cell engagers with potent tumor cell-killing activity and minimal cytokine release.
Antibodies are critical components of the immune response and act by engaging with components and effector molecules of the immune system via their Fc region thereby exerting various biological functions. As such, antibodies have proven to be successful as therapeutic molecules, e.g. for treatment of cancer and immune modulation. Depending on the application of the therapeutic antibody and whether the therapeutic effect is dependent on the interaction with the immune system's effector molecules (e.g. FcγRs), Fc engineering to inhibit or enhance Fc effector functions may be desired. The impact of Fc engineering on engagement of FcγRs is often assessed by investigating the direct 1:1 interaction (affinity) using assays such as SPR. However, the complexity and level of physiological relevance is low as e.g. the influence of antigen-binding, target-mediated antibody clustering and subsequent target-mediated clustering of the FcRs on the effector cells are absent. Furthermore, in a physiological setting, multiple cell types are present, each presenting a specific set of FcγRs on their surface, and other factors such as competing IgG and complement factors, present in circulation, may also impact the functional outcome of antibody-FcγR engagement.
Here we explored the impact of a non-activating Fc, IgG1-P329R, on various aspects of FcγR binding, activation, and function, to highlight how the level of complexity of the experimental set-up can impact the outcome. Although in vitro analysis showed no binding of IgG1-P329R to FcγRs (ELISA), substantial activation of T cells (CD69 upregulation) in a PBMC co-culture by IgG1-CD3-P329R was observed, which was dependent on the presence of monocytes but not B or NK cells.
Small-cell lung cancer (SCLC) is the most aggressive form of lung cancer and the leading cause of global cancer-related mortality. In this study, we first isolated MF-CADM1-specific fully human single-chain variable fragments (scFvs) from the human synthetic scFv antibody library using the phage display technology. Following the selected scFv conversion to human immunoglobulin G1 (IgG1) scFv-Fc antibodies (K103.1–4), multiple characterization studies, including antibody cross-species reactivity, purity, production yield, and binding affinity, were verified. Finally, via intensive in vitro efficacy and toxicity evaluation studies, we identified K103.3 as a lead antibody that potently promotes the death of human SCLC cell lines, including NCI-H69, NCI-H146, and NCIH187, by activated Jurkat T cells without severe endothelial toxicity. Taken together, these findings suggest that antibody-based targeting of MF-CADM1 may be an effective strategy to potentiate T cell-mediated SCLC death, and MF-CADM1 may be a novel potential therapeutic target in SCLC for antibody therapy.
The serum half-life of endogenous albumin is approximately 19 days in humans and 1.5-2.5 days in rodents. This extended half-life in humans is primarily due to effective recycling upon internalization mediated by the neonatal Fc receptor (FcRn). One limitation for many protein therapeutics smaller than 60 kD is short serum half-life. One way to address this limitation is to fuse the proteins of interest with an anti-albumin antibody. The fusion protein then binds to albumin taking advantage of FcRn-mediated recycling thus extending its in vivo half-life.
In this poster, we summarized the discovery of an anti-human/cyno/mouse serum albumin VHH lead from a proprietary high-quality large alpaca/llama native phage- displayed library, humanized and optimized the lead molecule with improved affinity. Furthermore, data from mouse PK and tumor-inhibition efficacy studies are presented.
