Early lymphocyte collection for anti-CD19 CART production improves T-cell fitness in patients with relapsed/refractory diffuse large B-cell lymphoma.

BACKGROUND: Chimeric antigen receptor (CAR) T cells targeted to the CD19 B-cell antigen form an approved treatment for patients with relapsed/refractory diffuse large B-cell lymphoma (r/r DLBCL). However, since this therapy is administered after multiple lines of treatment and exposure to lymphotoxic agents, there is an urgent need to optimize this modality of treatment. METHODS: To circumvent the difficulties of harvesting adequate and optimal T cells from DLBCL patients and improve CART therapy, we suggest an earlier lymphopheresis (i.e. at first relapse, before salvage treatment). We conducted a prospective study and evaluated the potential benefit of an earlier lymphopheresis (early group, n = 22) on the clinical outcome of CD19-CART infused DLBCL patients, in comparison with standard lymphopheresis (i.e. at second relapse and beyond; standard group, n = 23). RESULTS: An increased percentage of naïve T cells and increased in vitro T-cell functionality were observed in the early group. Additionally, these cells exhibit a lower exhaustion profile than T cells collected in the standard group. CONCLUSION: While improved T-cell phenotype and function in the lymphopheresis product did not translate into significantly improved clinical outcomes, a trend towards better overall survival (OS) and progression-free survival (PFS) was observed. Early lymphopheresis maximizes the potential of salvage therapies, without compromising CAR T-cell quality.

Enhanced crystallization of the Cys18 to Ser mutant of bovine gammaB crystallin.

The cysteine residues of the gamma crystallins, a family of ocular lens proteins, are involved in the aggregation and phase separation of these proteins. Both these phenomena are implicated in cataract formation. We have used bovine gammaB crystallin as a model system to study the role of the individual cysteine residues in the aggregation and phase separation of the gamma crystallins. Here, we compare the thermodynamic and kinetic behavior of the recombinant wild-type protein (WT) and the Cys18 to Ser (C18S) mutant. We find that the solubilities of the two proteins are similar. The kinetics of crystallization, however, are different. The WT crystallizes slowly enough for the metastable liquid-liquid coexistence to be easily observed. C18S, on the other hand, crystallizes rapidly; the metastable coexisting liquid phases of the pure mutant do not form. Nevertheless, the coexistence curve of C18S can be determined provided that crystallization is kinetically suppressed. In this way we found that the coexistence curve coincides with that of the WT. Despite the difference in the kinetics of crystallization, the two proteins were found to have the same crystal forms and almost identical X-ray structures. Our results demonstrate that even conservative point mutations can bring about dramatic changes in the kinetics of crystallization. The implications of our findings for cataract formation and protein crystallization are discussed.

Crystal cataracts: human genetic cataract caused by protein crystallization.

Several human genetic cataracts have been linked recently to point mutations in the gammaD crystallin gene. Here we provide a molecular basis for lens opacity in two genetic cataracts and suggest that the opacity occurs because of the spontaneous crystallization of the mutant proteins. Such crystallization of endogenous proteins leading to pathology is an unusual event. Measurements of the solubility curves of crystals of the Arg-58 to His and Arg-36 to Ser mutants of gammaD crystallin show that the mutations dramatically lower the solubility of the protein. Furthermore, the crystal nucleation rate of the mutants is enhanced considerably relative to that of the wild-type protein. It should be noted that, although there is a marked difference in phase behavior, there is no significant difference in protein conformation among the three proteins.

Molecular basis of a progressive juvenile-onset hereditary cataract.

In a recent paper, patients with a progressive juvenile-onset hereditary cataract have been reported to have a point mutation in the human gammaD crystallin gene (Stephan, D. A., Gillanders, E., Vanderveen, D., Freas-Lutz, D., Wistow, G., Baxevanis, A. D., Robbins, C. M., VanAuken, A., Quesenberry, M. I., Bailey-Wilson, J., et al. (1999) Proc. Natl. Acad. Sci. USA 96, 1008-1012). This mutation results in the substitution of Arg-14 in the native protein by a Cys residue. It is not understood how this mutation leads to cataract. We have expressed recombinant wild-type human gammaD crystallin (HGD) and its Arg-14 to Cys mutant (R14C) in Escherichia coli and show that R14C forms disulfide-linked oligomers, which markedly raise the phase separation temperature of the protein solution. Eventually, R14C precipitates. In contrast, HGD slowly forms only disulfide-linked dimers and no oligomers. These data strongly suggest that the observed cataract is triggered by the thiol-mediated aggregation of R14C. The aggregation profiles of HGD and R14C are consistent with our homology modeling studies that reveal that R14C contains two exposed cysteine residues, whereas HGD has only one. Our CD, fluorescence, and differential scanning calorimetric studies show that HGD and R14C have nearly identical secondary and tertiary structures and stabilities. Thus, contrary to current views, unfolding or destabilization of the protein is not necessary for cataractogenesis.

Aeolotopic interactions of globular proteins.

Protein crystallization, aggregation, liquid-liquid phase separation, and self-assembly are important in protein structure determination in the industrial processing of proteins and in the inhibition of protein condensation diseases. To fully describe such phase transformations in globular protein solutions, it is necessary to account for the strong spatial variation of the interactions on the protein surface. One difficulty is that each globular protein has its own unique surface, which is crucial for its biological function. However, the similarities amongst the macroscopic properties of different protein solutions suggest that there may exist a generic model that is capable of describing the nonuniform interactions between globular proteins. In this paper we present such a model, which includes the short-range interactions that vary from place to place on the surface of the protein. We show that this aeolotopic model [from the Greek aiolos ("variable") and topos ("place")] describes the phase diagram of globular proteins and provides insight into protein aggregation and crystallization.

Self-assembly of helical ribbons.

The self-assembly of helical ribbons is examined in a variety of multicomponent enantiomerically pure systems that contain a bile salt or a nonionic detergent, a phosphatidylcholine or a fatty acid, and a steroid analog of cholesterol. In almost all systems, two different pitch types of helical ribbons are observed: high pitch, with a pitch angle of 54 +/- 2 degrees, and low pitch, with a pitch angle of 11 +/- 2 degrees. Although the majority of these helices are right-handed, a small proportion of left-handed helices is observed. Additionally, a third type of helical ribbon, with a pitch angle in the range 30-47 degrees, is occasionally found. These experimental findings suggest that the helical ribbons are crystalline rather than liquid crystal in nature and also suggest that molecular chirality may not be the determining factor in helix formation. The large yields of helices produced will permit a systematic investigation of their individual kinetic evolution and their elastic moduli.

Oligomerization and phase separation in globular protein solutions.

We have chemically crosslinked a globular protein, gamma IIIb-crystallin, to produce a system of well-defined oligomers: monomers, dimers, trimers and a mixture of higher n-mers. Gel electrophoresis, size exclusion chromatography, quasielastic light scattering spectroscopy, and electrospray ionization mass spectrometry were used to characterize the oligomers formed. The liquid-liquid phase separation boundaries of the various oligomers were measured. We find that at a given concentration the phase separation temperature strongly increases with the molecular weight of the oligomers. This phase behavior is very similar to previous findings for gamma II-crystallin, for which oxidation-induced oligomerization is accompanied by an increase in the phase separation temperature. These findings imply that for phase separation, the detailed changes of the surface properties of the proteins are less important than the purely steric effects of oligomerization.

Phase separation in aqueous solutions of lens gamma-crystallins: special role of gamma s.

We have studied liquid-liquid phase separation in aqueous ternary solutions of calf lens gamma-crystallin proteins. Specifically, we have examined two ternary systems containing gamma s--namely, gamma IVa with gamma s in water and gamma II with gamma s in water. For each system, the phase-separation temperatures (Tph (phi)) alpha as a function of the overall protein volume fraction phi at various fixed compositions alpha (the "cloud-point curves") were measured. For the gamma IVa, gamma s, and water ternary solution, a binodal curve composed of pairs of coexisting points, (phi I, alpha 1) and (phi II, alpha II), at a fixed temperature (20 degrees C) was also determined. We observe that on the cloud-point curve the critical point is at a higher volume fraction than the maximum phase-separation temperature point. We also find that typically the difference in composition between the coexisting phases is at least as significant as the difference in volume fraction. We show that the asymmetric shape of the cloud-point curve is a consequence of this significant composition difference. Our observation that the phase-separation temperature of the mixtures in the high volume fraction region is strongly suppressed suggests that gamma s-crystallin may play an important role in maintaining the transparency of the lens.