Preliminary crystallographic study of the Fab fragments of two monoclonal anti-2-phenyloxazolone antibodies.

We report on the preparation, crystallization, and preliminary x-ray crystallographic study of the Fab fragments of two monoclonal anti-2-phenyloxazolone antibodies obtained from the secondary response to this hapten. The Fab fragment from one of these (NQ10/12.5) has been crystallized from polyethylene glycol 8000 solutions in a form suitable for high-resolution x-ray crystallographic studies. These crystals are monoclinic, space group C2, with a = 129.2 A, b = 79.4 A, c = 57.7 A, beta = 96.2 degrees, and one Fab/asymmetric unit. Determination of the three-dimensional structure of Fab NQ10/12.5 should help clarify the role of somatic mutation in the maturation of an immune response. This antibody and an anti-lysozyme antibody also under study apparently use the same germ-line encoded VK and a similar VH gene, respectively, as the idiotypic anti-oxazolone antibodies characteristic of the primary response. A comparative study of the two structures should shed light on the role of the pairing of heavy and light chains in the antigen-binding function of antibodies.

The introduction of techniques to obtain lymphocyte hybrids secreting antibodies of predetermined specificity (1) has made possible a crystallographic approach to the precise role of the nucleotide (and amino acid) sequence diversities generated by combinatorial diversification, imprecise joining of gene segments, and somatic mutation (reviewed in Ref. 2) in the antigen-binding functiov of antibodies. Thus, we have recently determined the 6-A resolution, three-dimensional structure of an antigen-antibody complex, one between the Fab fragment of a monoclonal anti-hen egg white lysozyme antibody and lysozyme (3), and a high-resolution study is in progress. In addition, other crystalline monoclonal antibodies of various hapten-and antigen-binding specificites are under study in our laboratory (4) and elsewhere (5-9).
We now report the crystallization of the Fab fragment of a monoclonal anti-2-phenyloxazolone antibody in a form suitable for a high-resolution x-ray diffraction analysis. Extensive sequence analysis of monoclonal antibodies to this hapten has been performed in order to clarify the way in which the various genetic mechanisms responsible for the generation of diversity operate during the onset and maturation of an immune re-* This work was supported by grants from Institut Pasteur and the Centre National de la Recherche Scientifique. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "aduertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§ To whom correspondence should be addressed.
sponse. The initial response (7 days after immunization) is dominated by very similar or identical antibodies expressing a pair of L' and H chain germ-line genes, V,-Ox1 and VH-0x1, respectively (10). Later in the response, the same germline gene combination is often used but the sequences are slightly different, indicating the importance of somatic point mutations in the maturation of the response (11). The mutants show a significant increase in the affinity for hapten. The two monoclonal antibodies used in this study are of the secondary response when new germ-line gene combinations become dominant. The affinity of NQ11/7.22.2 is not particularly high, but the antibody is related to the most commonly used germ-line gene combination of the secondary response, a combination rarely seen in primary responses.' On the other hand, NQ10/12.5 is a high-affinity antibody which includes an L chain most likely originating through several point mutations from the germ-line gene V,-0x1, which is the dominant germ-line gene in primary responses and is also commonly expressed in secondary responses. As often seen in secondary responses, the H chain of the antibody does not originate from the germ-line gene VH-Oxl. It is of interest that the anti-lysozyme antibody, whose crystal structure determination is already at an advanced stage, expresses an H chain of the VH-0x1 type.' Therefore, the study of this antibody and of NQ10/12.5 should provide considerable information on the structures of the H and L chains encoded by VH-0x1 and V,-0x1, respectively, and help in the understanding of the primary response to phenyloxazolone and the role of somatic mutation in its maturation.

MATERIALS AND METHODS
Preparation and Purification of Monoclonal Anti-phenyloxazolone Antibodies-The anti-phenyloxazolone hybridomas NQ10/12.5 and NQ11/7.22.2 are derived from a secondary response in BALB/c mice (11). Both cloned lines secrete antibodies of the IgGl class with K L chains.
The antibody was purified either from ascites of tumor-bearing mice or from spent fluid of high-density cultures as described (12). In the latter case, the antibody was precipitated with 50% (NH&SO,, redissolved in a minimal volume of phosphate-buffered saline, and dialyzed extensively against the same buffer. From this point, ascites or concentrated supernatants were processed in a similar fashion. The purification was performed at 4 "C by affinity chromatography on Protein A-Sepharose (13). The samples were adjusted to pH 8 by addition of 1/5 volumes of 0.5 M phosphate buffer, pH 8.0, clarified by passage through a small column of Sepharose 4B and then passed through a Protein A-Sepharose CL-4B column (Pharmacia). The effluent was discarded and the column was washed with 0.

Fab Fragments
of Anti-2-phenyloxazolone Antibodies phate, pH 8.0, while monitoring the absorption a t 280 nm. When the haseline returned to background values, batch elutions with 0.1 M sodium citrate at pH 6.0, 5.0, 4.0, and 3.0 were performed and the effluents were collected. The bulk of NQ10/12.5 eluted at pH 6.0, and NQ11/7.22.2 at pH 5.0 and 4.0, which is low for an IgG1 (13). The eluted proteins were neutralized immediately and either frozen a t -30 "C or stored in 50% (NH4),S04 with 0.024 azide.
Preparation of NQ10/12.5 and NQI 1/7.22.2 Fab Fragments-Purified NQ10/12.5 and NQ11/7.22.2 Fab fragments were digested with papain (14) after dialysis against 0.1 M potassium phosphate buffer, pH 5.2, to ohtain Fab fragments. Digestion conditions were as previously described for monoclonal anti-p-azophenylarsonate antibodies (4), with several modifications. In a typical preparation, 19.7 mg of NQ10/12.5 a t 8.8 mg/ml was digested a t 37 "C for 5 h with papain (Worthingon) at an enzymelsuhstrate ratio of 1:100 in the presence of 1.25 mM EDTA and 1.5 mM 2-mercaptoethanol. The course of the digestion was monitored by chromatographing 25-pl aliquots on an LKB UltroPac TSK-G30.000 SW 7.5 X 300-mm gel filtration column mounted on a Perkin-Elmer high performance liquid chromatography system. The reaction was terminated by the addition of 4 mM iodoacetamide at the point at which the peak corresponding to the Fab fragment reached its maximum height. Following dialysis against 40 mM sodium phosphate, pH 8.0, the reaction mixture was applied to a 4.0-ml diethylaminoethyl-cellulose (Serva) column equilibrated with the same buffer. The Fab fragments eluted in the run-through and were concentrated by vacuum dialysis against 0.01 M potassium phosphate, pH 7.0, in a Micro-ProDiCon concentrating apparatus (Bioblock). Undigested and partially digested antibody was removed in a final purification step by gel filtration on high performance liquid chromatography in 0.2 M sodium phosphate, pH 6.8, as described above. Fractions containing Fab NQ10/12.5 were pooled and concentrated by vacuum dialysis against 0.01 M potassium phosphate, pH 7.0. The Fab fragment of NQlll7.22.2 was prepared in a similar manner.
Crystallization of Fab NQIO/I2..5 and Fab NQ11/7.22.2-The Fab fragments of 17 monoclonal anti-phenyloxazolone antibodies were subjected to crystallization trials by vapor diffusion using the hanging drop technique (15). Small crystals of Fab NQ10/12.5 form I were initially obtained a t room temperature at PEG 8000 (Sigma) concentrations of 1.5-17.5% (w/v), 0.1 M potassium phosphate, 0.04% NaNs, pH 5.5; the crystals grew as elongated prisms terminated by bipyramids (Fig. 1). Under these conditions, however, crystals were obtained only sporadically. Large (up to 1.0 X 0.25 X 0.25 mm) Fab NQ10/12.5 form I crystals were subsequently grown in a reproducible fashion by "macroseeding," essentially as described by Thaller et al. (16). Briefly, hanging drops were prepared, containing 7 111 of Fab NQ10/12.5 solution (10-15 mg/ml in 0.01 M potassium phosphate, pH 7.0) and 7 pl of 13.75% PEG 8000, 0.1 M potassium phosphate, 0.04% NaN3, pH 5.5, and allowed to equilibrate overnight against 13.55% PEG 8000. A small, single, form I crystal of about 0.15 X 0.05 X 0.05 mm which had been previously washed in 12.5% PEG was then transferred into each of these drops using a siliconized glass capillary (0.50-mm internal diameter) adapted to a 1.0-ml Cornwall syringe pipette (Thomas). The seed crystal began growing within a day; in addition, other medium-sized form I crystals generally grew ab initio in the seeded drops.
Fab NQlll7.22.2 crystallized from solutions of 17.5-20'i PEG 8000, 0.10 M potassium phosphate, pH 6-7 as thin plates of up to 0.3 X 0.2 X 0.05 mm. Again, attempts to grow more suitable crystals by macroseeding were unsuccessful.
X-ray Analysis-Space groups and unit cell dimensions were determined using precession cameras and Ni-filtered CuKn radiation obtained from microfocus sealed-tube or rotating anode x-ray generators.

RESULTS AND DISCUSSION
X-ray diffraction patterns (Fig. 2)  Fab Fragments of Anti-2-phenyloxazolone Antibodies it unsuitable for a high-resolution x-ray study.