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To link to full-text access for this article, visit this link: http://dx.doi.org/10.1016/j.cell.2006.08.044 Byline: Matthew E. Call (1)(2), Jason R. Schnell (3), Chenqi Xu (1), Regina A. Lutz (1), James J. Chou (3), Kai W. Wucherpfennig (1)(2) Abstract: The T cell receptor (TCR) [alpha][beta] heterodimer communicates ligand binding to the cell interior via noncovalently associated CD3[gamma]E, CD3[delta]E, and I[paragraph]I[paragraph] dimers. While structures of extracellular components of the TCR-CD3 complex are known, the transmembrane (TM) domains that mediate assembly have eluded structural characterization. Incorporation of the I[paragraph]I[paragraph] signaling module is known to require one basic TCR[alpha] and two I[paragraph]I[paragraph] aspartic acid TM residues. We report the NMR structure of the I[paragraph]I[paragraph].sub.TM dimer, a left-handed coiled coil with substantial polar contacts. Mutagenesis experiments demonstrate that three polar positions are critical for I[paragraph]I[paragraph] dimerization and assembly with TCR. The two aspartic acids create a single structural unit at the I[paragraph]I[paragraph] interface stabilized by extensive hydrogen bonding, and there is evidence for a structural water molecule (or molecules) within close proximity. This structural unit, representing only the second transmembrane dimer interface solved to date, serves as a paradigm for the assembly of all modules involved in TCR signaling. Author Affiliation: (1) Department of Cancer Immunology and AIDS, Dana-Farber Cancer Institute, Harvard Medical School, Boston, MA 02115, USA (2) Program in Immunology, Harvard Medical School, Boston, MA 02115, USA (3) Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, MA 02115, USA Article History: Received 13 June 2006; Revised 23 July 2006; Accepted 11 August 2006 Article Note: (miscellaneous) Published: October 19, 2006

Assembly of the I cell receptor (ICR) with its dimeric signaling modules, CD3δϵ, CD3γϵ, and ζζ is organized by transmembrane (TM) interactions. Each of the three assembly steps requires formation of a three-helix interface involving one particular basic TCR TM residue and two acidic TM residues of the respective signaling dimer. The extracellular domains of CD3δϵ and CD3γϵ contribute to assembly, but TCR interaction sites on CD3 dimers have not been defined. The structures of the extracellular domains of CD3δϵ and CD3γϵ demonstrated parallel β-strands ending at the first cysteine in the CXXCXEXXX motif present in the stalk segment of each CD3 chain. Mutation of the membrane-proximal cysteines impaired assembly of either CD3 dimer with TCR, and little complex was isolated when all four membrane-proximal cysteines were mutated to alanine. These mutations had, however, no discernable effect on CD3δϵ or CD3γϵ dimerization. CD3δϵ assembled with a TCRα mutant that lacked both immunoglobulin domains, but shortening of the TCRα connecting peptide reduced assembly, consistent with membrane-proximal TCRα-CD3δϵ interactions. Chelation of divalent cations did not affect assembly, indicating that coordination of a cation by the tetracysteine motif was not required. The membrane-proximal cysteines were within close proximity but only formed covalent CD3 dimers when one cysteine was mutated. The four cysteines may thus form two intrachain disulfide bonds integral to the secondary structure of CD3 stalk regions. The three-chain interaction theme first established for the TM domains thus extends into the membrane- proximal domains of TCRα-CD3δϵ and TCRβ-CD3γϵ. [ABSTRACT FROM AUTHOR]

Simple Summary: The development and refinement chimeric antigen receptor (CAR)-T cell immunotherapy has significantly improved the prognosis of patients with B cell malignancies. Severe treatment related toxicities however remain a significant challenge for the field. This perspective reviews 17 clinical trials of the most widely used anti-CD19 (FMC63) CAR-T cell therapies, with the aim of dissecting the contribution of the structural and costimulatory domains of the CAR to clinical outcomes and toxicities. The CD28 structural hinge and transmembrane CAR domains are highlighted as strongly associated with both clinical efficacy and severe toxicity. This perspective supports further investigation into the structural CAR domains for improved CAR design and safer CAR-T cell therapies. Chimeric antigen receptor (CAR)-T cell therapy has transformed the treatment of B cell malignancies, improving patient survival and long-term remission. Nonetheless, over 50% of patients experience severe treatment-related toxicities including cytokine release syndrome (CRS) and neurotoxicity. Differences in severity of toxic side-effects among anti-CD19 CARs suggest that the choice of costimulatory domain makes a significant contribution to toxicity, but comparisons are complicated by additional differences in the hinge and transmembrane (TM) domains of the most commonly used CARs in the clinic, segments that have long been considered to perform purely structural roles. In this perspective, we examine clinical and preclinical data for anti-CD19 CARs with identical antigen-binding (FMC63) and signalling (CD3ζ) domains to unravel the contributions of different hinge-TM and costimulatory domains. Analysis of clinical trials highlights an association of the CD28 hinge-TM with higher incidence of CRS and neurotoxicity than the corresponding sequences from CD8, regardless of whether the CD28 or the 4-1BB costimulatory domain is used. The few preclinical studies that have systematically varied these domains similarly support a strong and independent role for the CD28 hinge-TM sequence in high cytokine production. These observations highlight the value that a comprehensive and systematic interrogation of each of these structural domains could provide toward developing fundamental principles for rational design of safer CAR-T cell therapies. [ABSTRACT FROM AUTHOR]

The impressive success of chimeric antigen receptor (CAR)-T cell therapies in treating advanced B-cell malignancies has spurred a frenzy of activity aimed at developing CAR-T therapies for other cancers, particularly solid tumors, and optimizing engineered T cells for maximum clinical benefit in many different disease contexts. A rapidly growing body of design work is examining every modular component of traditional single-chain CARs as well as expanding out into many new and innovative engineered immunoreceptor designs that depart from this template. New approaches to immune cell and receptor engineering are being reported with rapidly increasing frequency, and many recent high-quality reviews (including one in this special issue) provide comprehensive coverage of the history and current state of the art in CAR-T and related cellular immunotherapies. In this review, we step back to examine our current understanding of the structure-function relationships in natural and engineered lymphocyte-activating receptors, with an eye towards evaluating how well the current-generation CAR designs recapitulate the most desirable features of their natural counterparts. We identify key areas that we believe are under-studied and therefore represent opportunities to further improve our grasp of form and function in natural and engineered receptors and to rationally design better therapeutics. [ABSTRACT FROM AUTHOR]

De novo-designed receptor transmembrane domains (TMDs) present opportunities for precise control of cellular receptor functions. We developed a de novo design strategy for generating programmed membrane proteins (proMPs): single-pass α-helical TMDs that self-assemble through computationally defined and crystallographically validated interfaces. We used these proMPs to program specific oligomeric interactions into a chimeric antigen receptor (CAR) that we expressed in mouse primary T cells and found that both in vitro CAR T cell cytokine release and in vivo antitumor activity scaled linearly with the oligomeric state encoded by the receptor TMD, from monomers up to tetramers. All programmed CARs stimulated substantially lower T cell cytokine release relative to the commonly used CD28 TMD, which we show elevated cytokine release through lateral recruitment of the endogenous T cell costimulatory receptor CD28. Precise design using orthogonal and modular TMDs thus provides a new way to program receptor structure and predictably tune activity for basic or applied synthetic biology. [ABSTRACT FROM AUTHOR]