An Engineered Lipocalin Specific for CTLA-4 Reveals a Combining Site with Structural and Conformational Features Similar to Antibodies

An engineered lipocalin specific for CTLA-4 reveals a combining site with structural and conformational features similar to antibodies

D. Scho¨ nfelda, G. Matschinerb, L. Chatwella, S. Trentmannb, H. Gilleb,M.Hu¨ lsmeyerb, N. Brownc, P. M. Kayec, S. Schlehuberb, A. M. Hohlbaumb, and A. Skerraa,1

aMunich Center for Integrated Protein Science and Lehrstuhl fu¨r Biologische Chemie, Technische Universita¨t Mu¨ nchen, 85350 Freising-Weihenstephan, Germany; bPieris AG, 85354 Freising, Germany; and cCentre for Immunology and Infection, Department of Biology and Hull York Medical School, University of York, Wentworth Way, York YO10 5YW, United Kingdom

Edited by Gregory A. Petsko, Brandeis University, Waltham, MA, and approved March 25, 2009 (received for review December 31, 2008) Biomolecular reagents that enable the specific molecular recog- vitamins, hormones, and secondary metabolites or even catalytic nition of proteins play a crucial role in basic research as well as functions in some cases. medicine. Up to now, antibodies (immunoglobulins) have been In the human body, Ϸ10 different lipocalins are abundant widely used for this purpose. Their predominant feature is the (3), including the neutrophil gelatinase-associated lipocalin vast repertoire of antigen-binding sites that arise from a set of (NGAL or Lcn2, Lipocalin 2) (4). Lcn2 plays a role in innate 6 hypervariable loops. However, antibodies suffer from practical immunity by scavenging iron-charged microbial siderophores, disadvantages because of their complicated architecture, large in particular FeIII⅐enterobactin, and thus inhibiting bacterial size, and multiple functions. The lipocalins, on the other hand, infection (5). Whereas Lcn2 binds its ligand with high affinity have evolved as a protein family that primarily serves for the [KD ϭ 0.4 nM (4)] and specificity, other lipocalins are more binding of small molecules. Here, we show that an engineered promiscuous and exhibit moderate KD values in the low- lipocalin, derived from human Lcn2, can specifically bind the T micromolar range. A striking example is ␣ -acid glycoprotein cell coreceptor CTLA-4 as a prescribed protein target with 1 (AGP) (6), which can bind hundreds of different low- BIOCHEMISTRY subnanomolar affinity. Crystallographic analysis reveals that its molecular-weight compounds, including many pharmacologi- reshaped cup-like binding site, which is formed by 4 variable loops, provides perfect structural complementarity with this cally active substances, and thus influences their plasma ‘‘antigen.’’ Furthermore, comparison with the crystal structure circulation and distribution. However, lipocalins lack mecha- of the uncomplexed engineered lipocalin indicates a pro- nisms of somatic variation such as those known for Igs (7). nounced induced-fit mechanism, a phenomenon so far consid- Hence, we set out to investigate whether the binding site of a ered typical for antibodies. By recognizing the same epitope on lipocalin can be reshaped by in vitro mutagenesis and selection CTLA-4 that interacts with the counterreceptors B7.1/B7.2 on to specifically recognize a prescribed protein ‘‘antigen’’ with antigen-presenting cells the engineered Lcn2 exhibits strong, biomedical relevance. cross-species antagonistic activity, as evidenced by biological The cytotoxic T lymphocyte-associated antigen 4 (CTLA-4, effects comparable with a CTLA-4-specific antibody. With its CD152) is a critical T cell regulatory molecule (8, 9) that has proven stimulatory activity on T cells in vivo, the CTLA-4 block- attracted attention as a target for immunotherapy of cancer (10, ing lipocalin offers potential for immunotherapy of cancer and 11) as well as HIV (12) and other infectious diseases (13, 14). A infectious disease. Beyond that, lipocalins with engineered few days after antigen-dependent stimulation, CTLA-4 becomes antigen-binding sites, so-called Anticalins, provide a class of up-regulated and effectively competes with binding of the co- small (Ϸ180 residues), structurally simple, and robust binding stimulatory molecule CD28 to CD80 (B7.1) and CD86 (B7.2) on proteins with applications in the life sciences in general. antigen-presenting cells (APCs), thereby attenuating the T cell. Blockade of the physiological interaction between CTLA-4 and antigen ͉ immunoglobulin ͉ protein crystallography ͉ protein engineering these counterreceptors abolishes its negative immunoregulatory function. Accordingly, application of antibodies that bind to hereas members of the Ig superfamily, either as soluble human CTLA-4 and prevent its interaction with B7.1 and B7.2 Wantibodies or as membrane receptors, are primarily re- appears effective in cancer treatment (9), especially in combi- sponsible for the molecular recognition of protein antigens in nation with tumor vaccination (15). Because an Fc effector higher organisms, the lipocalins comprise a class of proteins that region is most likely not required for the functional blockade, exclusively bind small molecules (1). Lipocalins are found in engineered lipocalins with antagonistic activity could provide most phyla of life, from bacteria to man, and generally serve for alternative therapeutic agents. the transport, storage, or sequestration of endogenous or exog- enous compounds of low molecular weight. They are secretory proteins—usually compact monomers with 150 to 180 residues— Author contributions: S.S., A.M.H., and A.S. designed research; D.S., G.M., L.C., S.T., H.G., and in vertebrates, they mostly occur in the body fluids, including M.H., N.B., and P.M.K. performed research; D.S., G.M., L.C., S.T., H.G., M.H., N.B., P.M.K., S.S., blood and secretions. A.M.H., and A.S. analyzed data; and D.S., G.M., and A.S. wrote the paper. The lipocalins share a common architecture with a strongly Conflict of interest statement: G.M., S.T., H.G., M.H., S.S., and A.H. are employees of Pieris ␤ ␣ AG, a biotech company that commercializes the Anticalin technology and A.S. is cofounder conserved 8-stranded antiparallel -barrel and an -helix at- and shareholder of Pieris AG. N.B. and P.M.K. received funding from Pieris AG. D.S. was tached to its side (2). At one end, the ␤-barrel is closed by short funded by a collaboration between Pieris AG and the Technische Universita¨t Mu¨ nchen. loops and densely packed side chains, forming a hydrophobic This article is a PNAS Direct Submission. ␤ core. At the other end, 4 loops connect the -strands in a Data deposition: The atomic coordinates have been deposited in the Protein Data Bank, pairwise fashion and shape the entrance to the ligand pocket. www.pdb.org (PDB ID codes 3BX7 and 3BX8). Among the natural lipocalins, these loops are remarkably vari- 1To whom correspondence should be addressed. E-mail: [email protected]. able in terms of length, conformation, and amino acid sequence, This article contains supporting information online at www.pnas.org/cgi/content/full/ which reflects the variety of observed binding specificities for 0813399106/DCSupplemental.

www.pnas.org͞cgi͞doi͞10.1073͞pnas.0813399106 PNAS Early Edition ͉ 1of6 Downloaded by guest on September 27, 2021 .1 25. 50. 75. 100 . Lcn2 QDSTSDLIPAPPLSKVPLQQNFQDNQFQGKWYVVGLAGNAILREDKDPQKMYATIYELKEDKSYNVTSVLFRKKKCDYWIRTFVPGCQPGEFTLGNIKSY Library ...... h...... x.x.x.xx.xx...... x.xx...x.x.a.....s...... #001 ...... h...... R.L.D.QH.MN...... I.PH...E.T.a.....s...... #002 ...... h...... R.L.D.QH.MN...... I.PH...E.T.a.....s...... #003 ...... h...... R.L.D.QH.MN...... ISSH...E.T.a.....s...... ====A=====------#1------===B======C===--#2---===D======E=----#3

101 . 125 . 150 . 178 . Kd [nM] Lcn2 PGLTSYLVRVVSTNYNQHAMVFFKKVSQNREYFKITLYGRTKELTSELKENFIRFSKSLGLPENHIVFPVPIDQCIDG - Library xxx...... x.xx.x.x.a..i...... a...... - #001 GDK...... L.ED.A.F.a..i...... a...... 79.8 ± 0.1 #002 GDK...... Y...... LAED.A.F.a..i...... a...... 23.8 ± 0.02 #003 GDK...... D...Y...... LAED.A.F.a..i...... a...... 9.0 ± 0.02 -----===F======G===---#4---===H===

Fig. 1. Amino acid sequences of CTLA-4-specific engineered lipocalins in comparison with the human wild-type Lcn2 scaffold. Randomized positions are indicated by x, whereas 3 fixed conservative amino acid exchanges (Q28H, L137I, T145A) allowing the introduction of unique cloning sites for the central gene cassette are indicated as lowercase letters. Additional side chains were exchanged to remove the exposed free Cys residue of Lcn2 (C87S) (3) as well as the 3 positively charged side chains that play a role for recognition of the natural ligand FeIII⅐enterobactin (R81A, K125X, K134A). Dots represent amino acids identical with the wild-type Lcn2 sequence (SWISS-PROT entry P80188). The 8 structurally conserved strands A–H of the ␤-barrel (2) and the 4 loops #1–4 that form the binding site are labeled. C76 and C175 are connected by a disulfide bond. Affinity values (KD) measured via kinetic binding analysis of the soluble lipocalin variants by using human CTLA-4-Fc immobilized on a surface plasmon resonance chip are given at the end of each amino acid sequence.

Results recommendations for testing of monoclonal antibody products We have constructed a mutant genetic library of Lcn2 by for human use). Notably, the engineered lipocalin shows no randomizing 20-aa positions in the structurally variable loop cross-reactivity with the structurally related T cell coreceptor region (Fig. 1). This lipocalin library with Ϸ2 ϫ 1010 different CD28 (18) because no binding activity was detectable in corre- combinatorial variants [see supporting information (SI) Text] sponding Biacore measurements (Fig. S1) for the entire series of was subjected to several cycles of bacterial phage display variants obtained in the course of the affinity maturation. selection (16) against human CTLA-4-Fc, a soluble recombi- Furthermore, the engineered lipocalins exert potent antago- nant fusion protein of the extracellular domain of this receptor. nistic activity against CTLA-4 by effectively preventing binding Enriched clones were individually expressed in the periplasm of the soluble counterreceptors B7.1 and B7.2 in ELISA and by of Escherichia coli (3), followed by ELISA screening of the strongly enhancing CTLA-4-attenuated T cell activation in a soluble proteins, which led to the identification of several Lcn2 mixed lymphocyte reaction (MLR) cell culture assay (Fig. 2B). variants with differing epitope specificities on the prescribed Finally, a PEGylated Lcn2 variant (PRS-010#004a) was dem- target. onstrated to have similar in vivo efficacy as a CTLA-4-specific One lipocalin candidate (PRS-010#001) was found to com- monoclonal antibody in promoting resistance to the intracellular petitively inhibit the interaction between CTLA-4 and its re- pathogen Leishmania donovani (Fig. 2C), an infection previously combinant ligands B7.1 and B7.2 in ELISA as well as Biacore shown to be under negative regulatory control by CTLA-4 (19). binding experiments, revealing a dissociation constant (KD)of80 The Lcn2 variant PRS-010#003 was successfully crystallized, nM (Fig. S1). Compared with human wild-type Lcn2, PRS- and its X-ray structure was elucidated both as apo-protein and 010#001 exhibits 19 side-chain replacements in the mutagenized as complex with the bacterially produced extracellular domain segments (Fig. 1), thus demonstrating high tolerance of this (residues 3–126) of human CTLA-4 (Table S2). In the crystal lipocalin for structural alterations in its loop region. The affinity structure of the complex, 1 molecule of the engineered lipocalin of the initial lipocalin variant for CTLA-4 was further raised in is bound to 1 subunit of the CTLA-4 target. The extracellular 2 cycles of in vitro evolution via error-prone gene amplification receptor domain shows the known Ig V-type fold, whereby its C and selection under conditions of increasing stringency. terminus is connected—via the native disulfide bridge arising First, PRS-010#002 with improved affinity was selected from Cys-122—to a symmetry-related subunit (Fig. 3 and Fig. (Fig. 1). In a subsequent round, the Lcn2 variant PRS-010#003 S3). The interface between the 2 CTLA-4 monomers exhibits was obtained, which shows a significantly better target affinity essentially the same mainly hydrophobic contact area as in of 9.00 Ϯ 0.02 nM in conjunction with high thermostability previous (lower-resolution) crystallographic analyses of this (melting temperature, Tm ϭ 65.5 °C). Further affinity matu- protein (20, 21). ration resulted in the variants PRS-010#004 and PRS- In the complex, each CTLA-4 monomer rests with its wedge- 010#005, which carry additional mutations in the loop region like tip—formed at one end of the ␤-sandwich—in the binding and exhibit KD values of 2.7 nM and even of 240 pM (Table S1). site of the engineered lipocalin. The arrangement resembles that The latter engineered lipocalin has an affinity for the protein of an egg in a cup (Fig. 4), whereby contacts with all 4 reshaped antigen that is Ϸ10-fold better (in a Biacore analysis) than that loops at the open end of the ␤-barrel are involved. Because the of a humanized CTLA-4 specific antibody which has been crystallized CTLA-4 dimer represents a similar quaternary under clinical investigation (11, 17). structure as it is anticipated for the native receptor on the T cell This series of engineered lipocalins also shows functional surface (20, 21), this mode of interaction should allow the binding activity toward CTLA-4 as a native transmembrane simultaneous attachment of 2 copies of the engineered lipocalin protein, which was demonstrated both by flow cytofluorimetry without steric hindrance. of transfected CHO cells (Fig. 2) and by histochemical staining Similarly to the complex, the apo-structure of PRS-010#003 of acetone-fixed cryosections of spleen and tonsil tissues con- reveals the characteristic lipocalin fold and is thus closely related taining activated T cells (Fig. S2). The protein target is recog- to the crystal structure of the parental human Lcn2 (4). Super- nized in a highly specific manner, because unspecific tissue position of 58 conserved C␣ positions in the ␤-barrel (2) with the cross-reactivity was not detected during immunohistochemical wild-type lipocalin results in a remarkably small root mean- analysis of 32 nonmalignant human tissues (according to FDA square deviation of 0.34 Å. In contrast, all 4 loops at the open

2of6 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.0813399106 Scho¨nfeld et al. Downloaded by guest on September 27, 2021 A end of the ␤-barrel, which carry most of the substituted side 200 chains in the engineered protein, show significant conformational deviations from Lcn2 (Fig. 4). #003 In the complex of PRS-010#003 with the extracellular 150 #004 BNI3 domain of CTLA-4, the 4 loops provide an almost perfectly complementary shape for the target protein, covering its 100 twisted ␤-sandwich from one side. The ‘‘epitope,’’ comprising the FG loop (also known as CDR3-like segment) with its 50 center around positions 100–104 as well as the BC loop and strand CЈ of CTLA-4, points directly into the binding site of the

Mean Fluorescence Intensity 0 engineered lipocalin (Fig. S4). Remarkably, this epitope co- 10-1 100 101 102 103 104 incides with the surface region that is involved in the physio- Concentration [nM] logical interaction between CTLA-4 and the V-type domains of B7.1 and B7.2 (20, 21) (Fig. 3 A and B), hence explaining B 1600 the experimentally observed antagonistic activity of the engi- 1400 neered lipocalin. 2 1200 Overall, a protein surface of 2,378 Å becomes buried upon 2 1000 complex formation between PRS-010#003 (1,155 Å ) and 2 800 CTLA-4 (1,223 Å ). The resulting interface has more than

600 double the size described for the CTLA-4/B7.1 complex (1,255 2 IFN γ [pg/ml] Å ) (21), which reflects the up to 1,000-fold stronger affinity of 400 the lipocalin variants for CTLA-4 compared with the B7.1 200 receptor (KD ϭ 0.4 ␮M) (22). A large number of protein–protein 100 contacts via hydrogen bonds and hydrophobic as well as aromatic 1 µM 2 µM 1 µM 2 µM 1 µM 2 µM 1 µM 2 µM 4 µM 4 µM None 0.5 µM 0.5 µM 0.5 µM 0.5 µM interactions are involved, whereby 10 of the 20 exchanged 0.25 µM 0.25 µM 0.25 µM 0.25 µM 0.125 µM 0.125 µM CTLA4-Ig BNI3 isoMAb Lcn2 #004 residues participate in specific contacts with the CTLA-4 target (Fig. 3B and Fig. S5). BIOCHEMISTRY p<0.001 p<0.001 The bound FG loop of CTLA-4 contains the 99MYPPPYY105 C 110 100 motif, whose 3 consecutive Pro residues adopt a cis-trans-cis 90 configuration with important implications for B7.1 binding (21). 80 70 The PPP segment is directly centered at the lipocalin cavity (Fig. 60 S4; for a detailed description, see SI Text), leading to contacts 50 ␤ 40 with several core residues in the -barrel (Fig. 4A). Notably, in 30 light of the proven antagonistic activity of the engineered 20 10 lipocalin, the observed structural mode of interaction with

Parasite load [% control] 0 CTLA-4, which involves similar interfaces with the ectodomains IgG 4F10 Lcn2 #004a of B7.1 and B7.2 as described in previous structural studies (20, Fig. 2. Demonstration of antagonistic activity of CTLA-4-specific engineered 21), lends further support to the assumed signaling mechanism lipocalins. (A) Competitive binding with a soluble B7.1-Fc fusion protein to the between APCs and T cells. native receptor expressed on CHO cells. Biotinylated recombinant human B7.1- The extracellular domain of CTLA-4 shows close structural muIg (Ancell) was mixed with different concentrations of the engineered lipoca- relationship to CD28, the stimulatory coreceptor on T cells (18). lin or a CTLA-4-specific antibody (BNI3; BD Bioscience) and incubated with CHO In particular, the FG loop with the central sequence motif cells stably transfected with human CTLA-4 cDNA. After 2 hours, cells were MYPPPY is fully conserved. In addition, CD28 and CTLA-4 washed and bound B7.1 was quantified by using streptavidin–phycoerythrin share the same binding site on their counterreceptors B7.1/B7.2. conjugate via flow cytofluorimetry. EC50 values were 14.5 nM (PRS-010#004), 74.0 nM (PRS-010#003), and 5.6 nM (BNI3). Note that the EC50 values determined in this However, superposition of the CD28 crystal structure on the one experiment were consistently higher than in surface plasmon resonance mea- of CTLA-4 in complex with the lipocalin gives rise to several surements with purified proteins (see Fig. 1). The apparently stronger binding of steric clashes (Fig. S6), thus explaining the lack of measurable the bivalent monoclonal antibody was due to its avidity effect. No competing cross-reactivity. binding activity could be detected with wild-type Lcn2. (B) Immunostimulatory Comparison of the CTLA-4 complex with the apo-structure activity determined in an MLR assay. PHA T cell blasts and irradiated allogeneic CD80ϩ and CD86ϩ JY B cells were incubated in the presence of the indicated of PRS-010#003 reveals significant changes in the conforma- concentrations of the engineered lipocalin (PRS-010#004), wild-type Lcn2 (here as tion of its loops and adjacent parts of the ␤-barrel due to fusion with an albumin-binding domain) (33), a monoclonal antibody (BNI3) or an binding of the target protein (Fig. 4 A and B and Fig. S7). isotype control (anti-hCD14 mAb; BD Bioscience). Human CTLA-4-Ig (5 ␮g/mL; Interestingly, loop #3, which is partly disordered in the Chimerigen) was added in another control to fully block CD28-dependent co- apo-protein, adopts a well-defined conformation upon com- ␥ stimulation. After 3 days, supernatants were analyzed for IFN production by plex formation, thus revealing an induced fit of this flexible ELISA. Error bars show the standard deviation of triplicate assays. Cytokine measurements in the absence of JY cells showed no nonspecific T cell activation. segment. This structural phenomenon is well-known for anti- (C) BALB/c mice were infected with L. donovani parasites and subsequently bodies (23). Compared with the apo-lipocalin, the whole upper treated with either (i) PEGylated PRS-010#004a (30 mg/kg i.p.) or control recom- part of the ␤-barrel widens in the complex with CTLA-4, binant wild-type Lcn2 (15 mg/kg; as fusion with an albumin-binding domain), because strands G and H together with loop #4 are bent daily for 2 weeks from day ϩ1 of infection or (ii) with a CTLA-4-specific hamster outward. Consequently, there is also an induced fit for loop #4, monoclonal antibody (4 mg/kg; 4F10; BD Bioscience) or unrelated hamster IgG, albeit more subtle than for loop #3. In contrast, loop #1, which given once at day ϩ1. Spleen parasite load was determined at day 28 of infection. Data are presented as percentage of parasite burden in treated compared with carries the largest number (altogether 7) of side-chain substi- untreated mice and is pooled from 2 independent experiments (n ϭ 9–11 mice tutions, does not show a significant conformational change per group). Neither wild-type Lcn2 nor hamster IgG had a significant effect on upon complex formation, whereas the C␣ positions 72–75 of parasite burden. loop #2 move slightly by up to 1.8 Å.

Scho¨nfeld et al. PNAS Early Edition ͉ 3of6 Downloaded by guest on September 27, 2021 Fig. 3. Crystal structure of the engineered lipocalin PRS-010#003 in complex with the extracellular domain of human CTLA-4. The recombinant homodimer of CTLA-4 with the 2 bound lipocalin molecules is shown in shades of blue and violet, respectively (Middle), whereby both halves are related by 2-fold crystallographic symmetry. A disulﬁde bridge (orange) links the C termini of both receptor ectodomains. The 20 randomized side chains are highlighted in red for the right lipocalin molecule whereby its contact surface with the CTLA-4 target is shown. In the left CTLA-4 monomer all residues that differ from the murine protein are highlighted in green. The complex between 1 monomer of human CTLA-4 and B7.1 (PDB ID code 1I8L) (21) is shown with similar orientation of the receptor ectodomain (Left; blue and light green, respectively). The counterreceptor B7.1 and the engineered lipocalin bind to the same region of CTLA-4, however, in a different manner. For comparison, the ectodomain of the neuronal membrane protein MOG, which shares a common fold with CTLA-4, is shown in complex with the Fv region of the cognate antibody 8-18C5 (Right; MOG colored cyan, VH in orange and VL in yellow; disulﬁde bridges are colored black, with the one of MOG hidden in this perspective; PDB ID code 1PKQ) (26).

Discussion smaller (1,664 Å2) than the one between PRS-010#003 and The engineered lipocalin exhibits a mechanism of epitope rec- CTLA-4. ognition that strongly resembles the well-known interactions The finding that the engineered lipocalin and a typical between antibodies and their antigens (24). In this respect, 2 antibody can tightly bind the same kind of structural epitope characteristic phenomena are seen: first, high structural plastic- demonstrates that the extraordinary functional properties of ity of the binding site, which becomes apparent from the antibody CDRs for antigen recognition are less unique than significant alterations in loop backbone conformation compared previously assumed (7). In fact, the question arises why the with the wild-type Lcn2 resulting from the amino acid exchanges immune system chose Igs for this task rather than lipocalins; in the engineered apo-lipocalin; second, a pronounced induced possibly, this may be due to the large diversity of binding sites fit for one of the loops (#3, from disordered to ordered) as well that can be generated in a rather simple biological mechanism as significant conformational rearrangements for 2 others (#2 via combinatorial pairing of Ig light and heavy chains. and #4) upon complex formation with CTLA-4. Notably, this Interestingly, the 2 CTLA-4 antagonistic antibodies that are currently under clinical investigation (27, 28) seem to recog- mechanism was not observed for binding of wild-type Lcn2 to its nize a slightly different epitope, because they do not show natural ligand FeIII⅐enterobactin (4). cross-reactivity between primates and mice. In contrast, PRS- The mode of interaction with the protein ‘‘antigen,’’, which 010#003 binds murine CTLA-4 with just 4-fold-lower affinity involves all 4 structurally variable loops of the engineered than the human target, thus facilitating the use of correspond- lipocalin, is comparable with the role of the 6 hypervariable ing animal models for in vivo studies as shown here for loops (CDRs) in the antibody combining site. There, diversity Leishmania infectivity. This species cross-reactivity of the arises from the recombination of an inherited set of gene engineered lipocalin is in line with the close structural rela- segments, followed by somatic hypermutation (7). Although tionship of CTLA-4 from both organisms (20, 21, 29) and the lipocalins cannot take advantage of such a genetic mechanism, known fact that both human and mouse B7 receptors can bind in vitro mutagenesis and selection has led here to an altered to either human or mouse CTLA-4 (30, 31). Eighty-six percent target specificity, yielding a molecular interface with an of the interface between CTLA-4 and B7.1 is also buried in the area even larger than the buried surface usually observed complex with the engineered lipocalin. Of the altogether 41 aa 2 for antibody/antigen complexes (2,380 versus 1,550 Å on that differ between the extracellular domains of human and average) (25). murine CTLA-4 (20), only 4 residues occur in this region. The surprising resemblance regarding the mode of target In conclusion, combinatorial engineering of a lipocalin that binding between the engineered lipocalin and a typical Ig is naturally binds a small molecule (FeIII⅐enterobactin, with a total illustrated by a structural comparison (Fig. 3) with the Fv region buried surface of just Ϸ800 Å2) has led to a dramatic alteration of the murine monoclonal antibody 8–18C5 (26). Its Fab frag- in the mode of ligand binding and a much extended interface ment was crystallized in complex with the ectodomain of rat with the prescribed protein target. Notably, this is an otherwise myelin oligodendrocyte glycoprotein (MOG), an Ig-type recep- unknown example of a lipocalin from a higher organism that tor whose extracellular domain has a fold homologous to the one specifically recognizes another protein with high affinity, exhib- of CTLA-4. In this antibody/antigen complex, the FG loop of iting an association constant similar or even better than for a MOG is similarly placed at the center of the combining site, in monoclonal antibody (17). Compared with humanized antibod- this case surrounded by CDRs H3 and L3 as well as H1. ies that are currently under advanced clinical investigation, in However, because of the generally shorter CDRs of this anti- particular for cancer therapy (32), such a CTLA-4-specific body—especially if compared with the extended loop #2 of the human lipocalin should provide several advantages with respect engineered lipocalin—its interface with the antigen is much to improved tissue penetration, lack of Ig Fc-mediated receptor

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Fig. 4. Structural variations in the binding site of the engineered lipocalin as a consequence of mutagenizing its loop region and of an induced ﬁt upon target complexation. Crystal structures of (A) PRS-010#003 in complex with CTLA-4, (B) uncomplexed PRS-010#003 (chain B from the asymmetric unit), and (C) Lcn2 in complex with its natural ligand FeIII⅐enterobactin (PDB ID code 1L6M (4), chain A; ligand shown in a space-ﬁlling representation), together with their mutual superposition in 2 different orientations (D and E). The variable loop regions are colored, whereby the C␣ positions of the 20 randomized amino acids are indicated as spheres. The structurally conserved ␤-barrel (2) is colored in shades of gray, whereas the loops at its closed end and the ␣-helix attached to its side are shown in light gray for all 3 structures. Loop #3 is partially disordered in the crystal structure of the apo-lipocalin as illustrated by a dotted line. The 3 Pro residues at the center of the CTLA-4 epitope are highlighted as stick representation.

interactions, and tunable blood clearance. In general, the class of For structural studies, the dimeric extracellular domain of human CTLA-4 binding proteins based on the lipocalin scaffold, dubbed anti- and the Lcn2 variant PRS-010#003 were separately produced in E. coli. calins, offers the benefits of small size and composition of a Puriﬁed protein preparations were mixed, and the resulting complex was single polypeptide chain, efficient microbial production, and isolated via gel ﬁltration and crystallized in sitting drops with 1.4 M sodium malonate (pH 8.4) at 20 °C. The PRS-010#003 apo-protein was crystallized easy preparation of fusion proteins, thus opening many appli- with 1.45 M ammonium sulfate, 0.1 M sodium cacodylate (pH 5.45), and cations in biological research and medicine. 0.1% (vol/vol) Anapoe X-405 at 20 °C. Native synchrotron datasets were collected at the Berliner Elektronenspeicherring-Gesellschaft fu¨r Synchro- Experimental Procedures tronstrahlung. The crystal structure of the apo-protein was solved by A random library comprising Ϸ2 ϫ 1010 mutants of Lcn2 was constructed via molecular replacement using the coordinates of human wild-type Lcn2. PCR assembly using degenerate oligodeoxynucleotides and applied for The structure of the lipocalin/CTLA-4 complex was similarly solved by using selection by phage display and panning against the immobilized extracel- also the coordinates of the extracellular region of human CTLA-4. Details lular domain of human CTLA-4 fused to an IgG1-Fc region. Enriched Lcn2 of all methods are described in SI Text. variants were expressed as soluble proteins in E. coli and screened in ELISA for binding to human CTLA-4-Fc in comparison with control proteins. ACKNOWLEDGMENTS. We thank H. Schwartz for setup of initial cell-based Binding of biotinylated B7.1-Fc to CTLA-4-transfected CHO-K1 cells upon assays, E. Braungart and P. So¨hlemann for help in the screening and initial competition with Lcn2 variants was measured in a FACSCalibur instrument characterization of lipocalin variants, M. Aloe for technical assistance, G. Katzmann and H.-J. Christian for protein production, Joost van Nerven (Bio- using a streptavidin–phycoerythrin conjugate. CTLA-4-attenuated T cell ceros) for performing the MLR assay, and U. Mu¨ller and J. Schulze for support activation was tested in an MLR assay using human PBMCs. In vivo activity during synchrotron measurements at beam line 14.1 of the Berliner Elek- of a PEGylated Lcn2 variant was compared with that of an anti-CTLA-4 tronenspeicherring-Gesellschaft fu¨r Synchrotronstrahlung (BESSY) and Free monoclonal antibody in a mouse model of Leishmania donovani infections. University Berlin at BESSY, Germany.

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