Pre-existing antibodies that bind polyethylene glycol are present in about 40% of healthy individuals. It is currently unknown if pre-existing anti-polyethylene glycol (PEG) antibodies can alter the bioactivity of pegylated drugs with a single long PEG chain, which represents the majority of newly developed pegylated medicines. Methoxy polyethylene glycol-epoetin beta (PEG-EPO) contains a single 30 kDa PEG chain and is used to treat patients suffering from anemia. We find that the pre-existing human anti-PEG IgM and IgG antibodies from normal donors can bind to PEG-EPO. The prevalence and concentrations of anti-PEG IgM and IgG antibodies were also higher in patients that responded poorly to PEG-EPO. Monoclonal anti-PEG IgM and IgG antibodies at concentrations found in normal donors blocked the biological activity of PEG-EPO to stimulate the production of new erythrocytes in mice and accelerated the clearance of ¹²⁵I-PEG-EPO, resulting in PEG-EPO accumulation primarily in the liver and spleen. Accelerated clearance by the anti-PEG IgG antibody was mediated by the Fc portion of the antibody. Importantly, infusing higher doses of PEG-EPO could compensate for the inhibitory effects of anti-PEG antibodies, suggesting that pre-existing anti-PEG antibodies can be “dosed through.” Our study indicates that the bioactivity and therapeutic activity of PEG-EPO may be reduced in patients with elevated levels of pre-existing anti-PEG antibodies. New pegylated medicines with a single long PEG chain may also be affected in patients with high levels of anti-PEG antibodies.

Nearly all chimeric antigen receptors (CARs) signal in the absence of antigen, referred to as ‘tonic signaling’. Tonic signaling of CARs containing 41BB domains enhances T cell fitness and function, in contrast to the exhaustion driven by CD28-containing CARs. Here we show that 41BB induces BACH2, a transcriptional regulator that directs stem and memory programs. Overexpression of BACH2 successfully prevented exhaustion but locked CAR T cells in a quiescent state. We linked BACH2 to a degradation domain to tune BACH2, enabling us to prevent exhaustion while enabling potent effector function that broadly enhanced the long-term efficacy of CAR T cells targeting liquid and solid tumors. Through interrogation of clinical CAR products, we further found an association between BACH2 activity and clinical outcomes in patients with leukemia. These data identify a central function for BACH2 in regulating CAR T cell efficacy.

Chimeric antigen receptor (CAR)–based therapies have pioneered synthetic cellular immunity but remain limited in their long-term efficacy. Emerging data suggest that dysregulated CAR-driven T-cell activation causes T-cell dysfunction and therapeutic failure. To re-engage the precision of the endogenous T-cell response, we designed MHC-independent T-cell receptors (miTCR) by linking antibody variable domains to T-cell receptor constant chains. Using predictive modeling, we observed that this standard “cut and paste” approach to synthetic protein design resulted in myriad biochemical conflicts at the hybrid variable–constant domain interface. Through iterative modeling and sequence modifications, we developed structure-enhanced miTCRs which significantly improved receptor-driven T-cell function across multiple tumor models. We found that 41BB costimulation specifically prolonged miTCR T-cell persistence and enabled improved leukemic control in vivo compared with classic CAR T cells. Collectively, we have identified core features of hybrid receptor structure responsible for regulating function. Significance: Improving the durability of engineered T-cell immunotherapies is critical to enhancing efficacy. We used a structure-informed design to evolve improved miTCR function across several models. This work underscores the central role of synthetic receptor structure in T-cell function and provides a framework for improved receptor engineering.

Supplementary Figure 7 | a, Overlay of predicted mut031 α chain variable region and resolved FMC63 variable chain structure. Modeling performed using AlphaFold2. b, Memory lineages of human T cells engineered to express miTCR1 WT or mut035 at the conclusion of manufacturing cultures. c-d, Proportion of human T cells that were early lineage (naïve or central memory) c, throughout 6 day stimulation manufacturing cultures or d, one day after clearance of Nalm6. e, Expansion of human T cells expressing miTCR1 WT, mut035 or 19/BBζ CAR from two donors. b-d, data from one donor.

Supplementary Figure 5 | a-b, Expression of TCR constant chains on the surface of a, Jurkat and b, human T cells transduced with miTCR variants. c-d, Comparison of surface TCR expression in miTCR1 and miTCR2 variant engineered c, Jurkats and d, human T cells. e, Change in surface expression of TCR constant chains based on presence or absence of PD insertion in the miTCRα chain. f, Expression of NFAT and g, NFκB in reporter Jurkat cells expressing 1G4 or miTCR variants in response to Nalm6. h, Expansion of human T cells expressing miTCR1 and miTCR2 variants upon exposure to Nalm6. d, cumulative data from three independent donors. b,e, representative data from three independent donors. Screening studies in h performed using two independent donors. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 by Student’s t-test (c-e).

Supplementary Figure 8 | a, Cytotoxicity, as measured by fold change in Nalm6 growth during co-culture, and b, expansion of human T cells expressing transgenic TCR 1G4, 19/BBζ or 19/28ζ CAR against Nalm6WT or Nalm6triple. c-d, Endogenous expression of 41BBL, CD80 and CD86 in c, Raji and d, K562 cell lines. e, Growth rate of engineered Nalm6 cell lines. f-g, Cytotoxicity of 19/BBζ CAR and mut035 T cells over time during f, acute stimulation and g, re-stimulation. 19/BBζ CAR were initially co-cultured with Nalm6WT and re-stimulated with Nalm6WT; mut035 T cells were initially co-cultured with Nalm641BBL and re-stimulated with Nalm6WT. h, Percent of cells expressing either 19/BBζ CAR or mut035 miTCR on day 0 or after five days of chronic stimulation with various Nalm6 cell lines. i, Expansion of 19/BBζ CAR or mut035 miTCR T cells over the course of chronic stimulation with various Nalm6 cell lines. a-b and f-g Representative data from three independent donors; i, Representative of five independent donors. Statistical significance determined using two-way ANOVA.