1. A library of related monoclonal antibodies isolated from various points in the murine immune response has been developed. The antibodies were elicited using the same diketone hapten used to elicit the catalytic aldolase 38C2 antibody (Wagner, J., Lerner, R. A., Barbas, C. F., (1995) Science 270, 1797). The binding properties of two commercially available fully mature aldolase antibodies, four members of the 38C2 family from our lab, and a non-specific IgG were studied by fluorescence spectroscopy. Prodan (6-propionyl-2- (dimethylamino)naphthalene) is a fluorescent hapten analogue that binds reversibly to the 38C2 family of antibodies with micromolar affinity. Steady state fluorescence spectra, polarization, and lifetime distributions were measured for the hapten analogue in solution and bound to the various antibodies. Prodan exhibits a marked blue shift in its fluorescence emission and an increase in intensity and polarization upon binding. These data are analyzed to determine binding constants, and scale with fluorescence studies of hapten binding from tryptophan quenching. Prodan lifetime distributions as measured by multifrequency phase fluorometry show two types of antibody binding environments. These results are interpreted in terms of the model of antibody evolution in which higher affinity is achieved through binding site rigidification. Abstract

2. Antibody Structure Antibodies, or immunoglobulin (Ig) molecules, are composed of four chains, two light and two heavy The structure of Ig molecules can be divided into a constant region, in which the amino acid sequence is largely conserved, and a variable region, where the amino acid sequence for different Ig molecules has considerably more variation Within the variable region, there are three hypervariable regions, referred to as complementarity determining regions (CDR1-3), as they are located at the binding sites of the antibody molecule RasMol image of IgG molecule, PDB code 1IGT, Harris, L. J., Skaletsky, E., and McPherson, A. (1998) J. Mol. Biol. 275, 861-872. 1 Antigen-binding Sites Variable Regions Constant Regions Carbo- hydrates CDR3 CDR2 CDR1

3. Affinity Maturation Upon exposure to an unknown antigen, the immune system begins a process called affinity maturation The germline antibody that binds the antigen most tightly undergoes somatic hypermutation in the variable region to form antibodies that each have a new active site and, therefore, modified affinity for the antigen The immune system selects for the modified antibody that most tightly binds the antigen and somatic hypermutation occurs again to produce a more mature antibody with an even more specialized binding site Therefore, more mature antibodies should show both more rigid binding sites and also smaller dissociation constants 2

4. Aldolase Family of Antibodies 38C2 is a catalytic antibody, first developed at the Scripps Research Institute - Wagner, J., Lerner, R. A., Barbas, C. F., Science 270, 1797 (1995) This antibody was raised against a diketone hapten by the process of reactive immunization, and catalyzes the aldol reaction Reactive immunization involves an actual chemical reaction between the hapten and the antibody, rather than the typical non-covalent interaction between the hapten and the antibody The evidence for catalytic activity is the observation of an absorption band at 316 nm due to the vinylogous amide formed in the reaction of the diketone hapten with a lysine residue in the binding site of the antibody The antibodies in this study do not show an absorption peak at 316 nm, indicating that the interaction between the hapten and these antibodies is not covalent 3

5. Aldolase Family of Antibodies The antibodies used in this study were raised against the same diketone hapten used to generate the mature 38C2 antibody Antibodies were collected at different time points of the murine immune response Antibodies Examined: Primary (A3.1.1): collected 12 days after initial exposure to the hapten Secondary (2c26.1): collected 5 days after the first boost Tertiary (3.22): collected 5 days after the second boost Mature 38C2: collected 14 days after the second boost Mature 84G3: collected 14 days after the second boost 4

6. Ligand Molecules 1,3-Diketone Hapten all antibodies studied were raised against Prodan, or 6-propionyl-2- (dimethylamino)naphthalene Fluorescent molecule Structural similarities to the 1,3-diketone hapten indicated that prodan could bind to the antibodies studied 5

7. Questions Addressed Can Prodan, a highly fluorescent and environmentally sensitive small molecule, mimic hapten binding? What does Prodan tell us about the relative hydrophobicities of the antibody binding sites? Does Prodan fluorescence provide evidence for an increase in the rigidity of the antibody binding sites as a function of antibody maturity? 6

8. Experimental Techniques Antibody dissociation constants were determined using techniques in which the experimental observable is proportional to the fraction of ligand bound Antibody was titrated into a solution of Prodan in PBS Prodan fluorescence experiments done using a Perkin Elmer LS50B fluorimeter Prodan fluorescence anisotropy measured with a Panvera Beacon 2000 using an excitation filter centered at 360 nm and a transmission filter centered at 490 nm 7

9. Experimental Techniques Multifrequency phase fluorometry was used to measure the fluorescence lifetimes and lifetime distributions of the Trp residues in the antibodies and Prodan The phase and modulation data were fit to a combination of discrete lifetimes and Gaussian distributions of lifetimes 8 Discrete:Gaussian Distribution: Intensity decay: Fractional contribution to fluorescence intensity:

10. The emission spectrum of Prodan blue-shifts with decreasing solvent polarity The emission spectrum of Prodan in acetone resembles that of Prodan bound to the antibodies studied Both free (low anisotropy) and bound (high anisotropy) Prodan fluoresce in the range of the emission filter used in the anisotropy experiments, therefore, the anisotropy in this wavelength range can be used to determine the fraction of Prodan bound Prodan Emission and Anisotropy 9

11. Emission Spectra of Prodan Bound to the Mature 38C2 Antibody Excitation is at 361 nm Prodan emission shifts to 445 nm upon binding to the 38C2 antibody The emission intensity at 445 nm increases with increasing antibody concentration At 490 nm, both free and bound Prodan contribute to the fluorescence signal 10

12. Prodan Binds Specifically in the Antibody Binding Site Excitation is at 361 nm Prodan emission shifts to 445 nm upon binding to the 38C2 antibody The emission intensity at 445 nm increases with increasing antibody concentration At 490 nm, both free and bound Prodan contribute to the fluorescence signal Addition of hapten to a solution of Prodan bound to an antibody resulted in the displacement of Prodan 11

13. The fluorescence anisotropy of Prodan is related to the fraction of Prodan bound to antibody A plot of the anisotropy vs. the concentration of free antibody binding sites, [S], allows the dissociation constant, K d, of the antibodies to be determined according to the following relationship: The concentration of free antibody was approximated by the total antibody concentration, which is valid as the total Prodan concentration used (20 nM-100 nM) was at least ten-fold lower than the observed K d values Determination of Antibody Dissociation Constants 12

14. Determination of Antibody Dissociation Constants From Anisotropy Data 13

15. Summary of Antibody Binding Affinity Data 14 Antibody Heavy Chain CDR3 Sequence % Trp residues in binding site K d ’ /K dq (84G3) Trp quenching by hapten K d /K d (84G3) Prodan anisotropy 84G3 Unknown 42% (10/24) 11 38C2 CKIYKYSFSYW 42% (10/24) 14 Tertiary CIRGGTAYNRYDGAYW 38% (10/26) 315 Secondary (2c26.1) CATAHYVNPGRFTKTLDYW 38% (10/26) 5417 Primary CTRWGYAYW 43% (12/28) Non-specific binding 9

16. Multifrequency Phase Fluorometry 15 The best fit to the phase and modulation data for Prodan bound to 38C2 gives two lifetime components One is a discrete lifetime of <1 ns (below the resolution of the instrument) and one is a distribution centered at 4.0 ns with a full-width at half- maximum of 0.8 ns

17. Steady-State and Multifrequency Phase Fluorometry 16 System  L  (ns) fw L (ns)f L * 22 Ex Max. (nm) Em Max. (nm) Stokes Shift (cm -1 ) Prodan1.81 (0.06) discrete0.421.443755287700 +84G35.03 (0.12) 0.960.781.633754735500 +38C24.16 (0.03) 0.770.801.243604525700 +Tertiary4.03 (0.05) 1.000.461.393794614700 +Secondary2.90 (0.02) 1.860.381.303804946000 Primary4.72 (0.03) 0.910.401.663894614000 IgG1.66 (0.07) discrete0.401.833755317800 The remainder of the fluorescence intensity is due to a discrete lifetime component with a lifetime <1 ns, below the resolution of our instrument

18. Both the blue-shifted emission and increased anisotropy of Prodan can be used as a probe of binding affinity of antibodies. Though raised against a diketone hapten, the antibodies studied exhibit K d values ranging from 0.122 – 2.1  M. The 50-fold range of Prodan binding constants determined for the antibodies studied are consistent with the binding behavior of the antibodies for the hapten as measured by tryptophan quenching (more mature antibodies bind more strongly). The emission spectrum of Prodan bound to antibodies is blue-shifted relative to that observed for Prodan in solution or in the presence of a non-specific antibody, indicating the hydrophobic nature of the antibody binding sites. The lifetime data exhibit a marked increase (factor of three) in the lifetime of Prodan when bound to an antibody. Lifetime distribution widths decrease with increasing antibody maturity, suggesting that affinity maturation produces antibodies with increasingly rigid binding sites. Conclusions 17

19. NSF C-RUI collaborators –Prof. Richard Goldsby, creation of antibody family –Prof. David Hansen, synthesis of hapten –Prof. David Ratner and Nalini Sha-Mahoney, genetic characterization of antibodies –Phil Chiu ‘02, Trp quenching data Camille and Henry Dreyfus Scholar/Fellow Program for Undergraduate Institutions Faculty Research Awards Progam, Amherst College 18 Acknowledgements

20. Emission Spectra of Prodan Bound to the Tertiary Antibody Excitation is at 361 nm As seen with the 38C2 antibody, the prodan emission shifts to 445 nm upon binding to the tertiary antibody, and the intensity at 445 nm increases with increasing antibody concentration Again, both bound and free prodan contribute to the fluorescence signal at 510 nm 14

21. Quenching of Intrinsic Tryptophan Fluorescence on Hapten Binding

22. Emission Spectra of Prodan Bound to the Mature 38C2 Antibody Excitation is at 361 nm Emission intensity at 445 nm is due to bound prodan, and is observed to increase with increasing antibody concentration

23. Emission Spectra of Prodan Bound to the Mature 38C2 Antibody Excitation is at 361 nm Emission intensity at 445 nm is due to bound prodan, and is observed to increase with increasing antibody concentration

24. Summary of Antibody Binding Affinity Data 18 Antibody Heavy Chain CDR3 Sequence % Trp residues in binding site K d /K d 38C2 Trp quenching by hapten K d from prodan anisotropy (fluorescence) 38C2 CKIYKYSFSYW 42% (10/24) 1 0.51 ± 0.06  M (0.75 ± 0.06  M) Tertiary CIRGGTAYNRYDGAYW 38% (10/26) 3 0.8 ± 0.3  M (0.73 ± 0.04  M) Secondary (A2c26.1) CATAHYVNPGRFTKTLDYW 38% (10/26) 54 1.8 ± 0.7  M Secondary (A2c22.1) CTRGNYGYVGAYW 38% (10/26) 180 * N/A Primary CTRWGYAYW 43% (12/28) Non-specific binding 1.1 ± 1.0  M * Ligand used was acetyl acetone, not the hapten

25. Determination of Antibody Dissociation Constants From Anisotropy Data 17

26. Determination of Antibody Binding Constants From Tryptophan Fluorescence Quenching 11

27. Quenching of the intrinsic tryptophan fluorescence of the antibodies is related to the fraction of antibody binding sites filled (F b ) according to the following relationship: A plot of F 0 -F vs. the total ligand concentration (L t ) can be used to determine the dissociation constants (K d ) of the antibodies according to the following relationship: Determination of Antibody Dissociation Constants 8 F b : Fraction of binding sites filled F 0 : Trp fluorescence in the absence of ligand F: Trp fluorescence in the presence of ligand F mq : Trp fluorescence with maximum quenching S t : Total concentration of antibody binding sites (all other parameters defined above)

28. Excitation at 295 nm for 38C2, 284 nm for IgG The fluorescence intensity due to intrinsic tryptophan residues in the antibodies decreases with increasing hapten concentration The fluorescence quenching observed for the mature 38C2 antibody (specific binding) is greater than that observed for IgG (non-specific binding) Quenching of Intrinsic Tryptophan Fluorescence on Hapten Binding 38C2 Increasing hapten 9 IgG Increasing hapten

29. Quenching of Intrinsic Tryptophan Fluorescence on Hapten Binding 10 Increasing hapten Tertiary Increasing hapten Secondary (A2c26.1) Secondary (A2c22.1) Primary exc =295 nm in all cases

30. Ab concentrations  3  M Primary antibody shows non-specific binding similar to IgG (data not shown) Other antibodies show binding behavior more similar to 38C2 Because 38C2 forms a covalent bond with the hapten, the value reported is an apparent K d, K dq Non-specific binding occurs at high hapten concentrations Use of fluorescent ligand would allow for lower ligand concentrations Determination of Antibody Dissociation Constants From Tryptophan Fluorescence Quenching 11