Geometric parameters that affect the behavior of logic-gated CAR T cells.

Clinical applications of CAR-T cells are limited by the scarcity of tumor-specific targets and are often afflicted with the same on-target/off-tumor toxicities that plague other cancer treatments. A new promising strategy to enforce tumor selectivity is the use of logic-gated, two-receptor systems. One well-described application is termed Tmod™, which originally utilized a blocking inhibitory receptor directed towards HLA-I target antigens to create a protective NOT gate. Here we show that the function of Tmod blockers targeting non-HLA-I antigens is dependent on the height of the blocker antigen and is generally compatible with small, membrane-proximal targets. We compensate for this apparent limitation by incorporating modular hinge units to artificially extend or retract the ligand-binding domains relative to the effector cell surface, thereby modulating Tmod activator and blocker function. By accounting for structural differences between activator and blocker targets, we developed a set of simple geometric parameters for Tmod receptor design that enables targeting of blocker antigens beyond HLA-I, thereby broadening the applications of logic-gated cell therapies.

HLA-A∗02-gated safety switch for cancer therapy has exquisite specificity for its allelic target antigen.

Innovative cell-based therapies are important new weapons in the fight against difficult-to-treat cancers. One promising strategy involves cell therapies equipped with multiple receptors to integrate signals from more than one antigen. We developed a specific embodiment of this approach called Tmod, a two-receptor system that combines activating and inhibitory inputs to distinguish between tumor and normal cells. The selectivity of Tmod is enforced by the inhibitory receptor (blocker) that recognizes an antigen, such as an HLA allele, whose expression is absent from tumors because of loss of heterozygosity. Although unwanted cross-reactivity of the blocker likely reduces efficacy rather than safety, it is important to verify the blocker's specificity. We have tested an A∗02-directed blocker derived from the PA2.1 mouse antibody as a safety mechanism paired with a mesothelin-specific activating CAR in our Tmod construct. We solved the crystal structure of humanized PA2.1 Fab in complex with HLA-A∗02 to determine its binding epitope, which was used to bioinformatically select specific class I HLA alleles to test the blocker's functional specificity in vitro. We found that this A∗02-directed blocker is highly specific for its cognate antigen, with only one cross-reactive allele (A∗69) capable of triggering comparable function.

The serum protein transthyretin as a platform for dimerization and tetramerization of antibodies and Fab fragments to enable target clustering.

Transthyretin (TTR) is an abundant homotetrameric serum protein and was selected here for engineering higher-valency molecules because of its compact size, simple structure, and natural propensity to tetramerize. To demonstrate this utility, we fused TTR to the C terminus of conatumumab, an antibody that targets tumor necrosis factor-related apoptosis-inducing ligand receptor 2, as heavy chains to form antibody dimers and Fab heavy chains to form Fab tetramers. Moreover, we used constant heavy domain 3 heterodimerization substitutions to create TTR-mediated conatumumab tetramers. The conatumumab-TTR fusions displayed substantially enhanced potency in cell-based assays, as well as in murine tumor xenograft models. We conclude that antibody-TTR fusions may provide a powerful platform for multimerizing antibody and Fab fragments to enhance the capabilities of human therapeutics that benefit from target clustering and higher-order antigen-binding valency.

Culture temperature modulates half antibody and aggregate formation in a Chinese hamster ovary cell line expressing a bispecific antibody.

Therapeutic bispecific antibodies are formed by assembly of multichain polypeptides. In general, a bispecific antibody has two different light chains and two different heavy chains that fold and correctly pair via engineered interchain interactions. Because of some incorrect assembly, product-related impurities can be prevalent (e.g., half molecules, mispaired light chains, homodimers), requiring its removal during subsequent purification. In this study, we investigated the modulation of impurity levels in a stable Chinese hamster ovary cell line X expressing a bispecific antibody A formed by two light chains (LC1 and LC2) and two heavy chains (HC1 and HC2) that assembled intracellularly into a heterodimer (LC1-HC1 + LC2-HC2) via engineered charged residues. Cell line X exhibited the best volumetric productivity, growth, and viability in culture compared with other clones but also showed higher levels of half antibody species (>10%); therefore, to minimize process yield loss, better understanding, and control of impurity formation was pursued. We found this cell line decreased half antibody levels from 16% to 1% when temperature changed from 36°C to 32.5°C or 31.5°C. However, lower temperature also increased high-molecular-weight (HMW) species from 4% to 12%. To determine the impurity species composition, we characterized enriched fractions with half antibody or HMW. Intact mass spectrometry analysis revealed half antibody was LC2-HC2, whereas HMW was a mixture with ~50% as LC1-HC1 homodimer. Results suggested LC2-HC2 was easily folded and could be secreted as half antibody, especially at 36°C. On the contrary, LC1-HC1 was more susceptible to misfold or aggregate, a phenomenon more acute for cell line X at lower culture temperature because of 60% increased LC1 and HC1 messenger RNA levels. Although temperature modulation was cell line X-specific, the propensity of LC2-HC2 to form half antibodies and LC1-HC1 to aggregate appeared in other cell lines also expressing bispecific antibody A, suggesting an amino-acid sequence-dependent mechanism. In summary, impurity formation in cell line X was temperature-dependent and was influenced by different molecule characteristics between the LC1-HC1 and LC2-HC2 parts. Ultimately, we selected a biphasic cell culture process with a growth phase followed by a lower temperature phase to improve product quality and purification yield.