Structural basis for cancer by the PNAS PLUS first-in-class checkpoint inhibitor

Udupi A. Ramagopala,1,2, Weifeng Liua,b,1, Sarah C. Garrett-Thomsona,1, Jeffrey B. Bonannoa, Qingrong Yanb,3, Mohan Srinivasanc, Susan C. Wongc, Alasdair Bellc,4, Shilpa Mankikarc, Vangipuram S. Ranganc, Shrikant Deshpandec, Alan J. Kormanc,5, and Steven C. Almoa,5

aDepartment of Biochemistry, Albert Einstein College of Medicine, Bronx, NY 10461; bDepartment of Microbiology and Immunology, Albert Einstein College of Medicine, Bronx, NY 10461; and cBiologics Discovery California, Bristol–Myers Squibb, Redwood City, CA 94063

Edited by James P. Allison, MD Anderson Cancer Center, University of Texas, Houston, TX, and approved April 6, 2017 (received for review November 1, 2016) Rational modulation of the immune response with biologics repre- expressed on activated T effector cells as a result of ligand binding. sents one of the most promising and active areas for the realization Extrinsic effects of CTLA-4 are the consequence of the ability of of new therapeutic strategies. In particular, the use of function CTLA-4 expressed on Tregs to remove B7 ligands from the surface blocking monoclonal targeting checkpoint inhibitors such of dendritic cells or presenting cells, resulting in significantly as CTLA-4 and PD-1 have proven to be highly effective for the reduced suppression (16). This regulatory mechanism of trans- systemic activation of the human to treat a wide endocytosis may also be operative in activated T effector cells (17, range of cancers. Ipilimumab is a fully human targeting 18). CTLA-4–B7 interactions also have intrinsic effects on Treg, CTLA-4 that received FDA approval for the treatment of metastatic dampening their proliferation or activation (19). in 2011. Ipilimumab is the first-in-class immunotherapeutic Antibodies to CTLA-4 also operate through multiple mecha- for blockade of CTLA-4 and significantly benefits overall survival of nisms. Recent studies in murine models suggest that antibodies patients with metastatic melanoma. Understanding the chemical and targeting CTLA-4 delete intratumoral Treg cells through an Fcγ physical determinants recognized by these mAbs provides direct receptor (FcγR)-dependent process (20, 21). Although Treg de- insight into the mechanisms of pathway blockade, the organization – pletion does not require ligand blocking, ample evidence indicates of the antigen antibody complexes at the cell surface, and opportu- that inhibition of ligand binding to CTLA-4 is an important factor nities to further engineer affinity and selectivity. Here, we report the INFLAMMATION contributing to the antitumor activity of anti–CTLA-4 antibodies in IMMUNOLOGY AND 3.0 Å resolution X-ray crystal structure of the complex formed by murine models as well as in humans. These data include the ipilimumab with its human CTLA-4 target. This structure reveals that β demonstration in murine models that targeting of the effector T-cell ipilimumab contacts the front -sheet of CTLA-4 and intersects with – the CTLA-4:Β7 recognition surface, indicating that direct steric overlap compartment contributes to the antitumor activity of anti CTLA-4, whereas exclusive targeting of the Treg cell compartment failed between ipilimumab and the B7 ligands is a major mechanistic con- — tributor to ipilimumab function. The crystallographically observed to elicit tumor protection thus highlighting the importance of binding interface was confirmed by a comprehensive cell-based bind- ing assay against a library of CTLA-4 mutants and by direct biochem- Significance ical approaches. This structure also highlights determinants responsible for the selectivity exhibited by ipilimumab toward CTLA-4 relative to Biologics represent a major class of therapeutics for the treat- the homologous and functionally related CD28. ment of malignancies, autoimmune diseases, and infectious diseases. Ipilimumab is the first-in-class immunotherapeutic for immunotherapy | X-ray crystallography | CTLA-4 | ipilimumab | cancer blockade of CTLA-4 and significantly benefits overall survival of patients with metastatic melanoma. The X-ray crystal structure ctivation of the immune system to target and eliminate ma- of the ipilimumab:CTLA-4 complex defines the atomic interac- Alignancies is recognized as one of the most promising directions tions responsible for affinity and selectivity and demonstrates forcancertherapy(1–4). Two broad strategies for immunotherapy that the therapeutic action of ipilimumab is due to direct steric may be envisaged: inhibition of negative regulators of immune re- competition with the B7 ligands for binding to CTLA-4. sponsiveness (collectively known as checkpoint blockade) (2, 5–8) Author contributions: U.A.R., W.L., S.C.G.-T., J.B.B., Q.Y., M.S., S.C.W., A.B., S.M., V.S.R., and activation of costimulatory pathways (8). A powerful example is S.D., A.J.K., and S.C.A. designed research; U.A.R., W.L., S.C.G.-T., Q.Y., M.S., S.C.W., A.B., provided by antibodies targeting cytotoxic T lymphocyte-associated S.M., V.S.R., and S.D. performed research; U.A.R., W.L., S.C.G.-T., J.B.B., Q.Y., M.S., S.C.W., antigen 4 (CTLA-4), a T-cell surface molecule, which like the ho- A.B., S.M., V.S.R., and S.D. analyzed data; and U.A.R., W.L., S.C.G.-T., M.S., A.J.K., and S.C.A. mologous CD28 (∼30% sequence identity) binds the B7-1 and B7-2 wrote the paper. ligands (9). While CD28 is constitutively expressed and is required, Conflict of interest statement: S.C.A., S.C.G.-T., U.A.R., W.L., and Q.Y. declare no compet- ing financial interests. A.B. is a former employee of Bristol–Myers & Squibb. A.J.K., M.S., in conjunction with TCR engagement, for T-cell activation, CTLA-4 S.C.W., S.M., V.S.R., and S.D. are employees and stockholders of Bristol–Myers & Squibb. is a negative regulator of T-cell function expressed after T-cell ac- This article is a PNAS Direct Submission. tivation to terminate the response. Ipilimumab, a fully human an- Freely available online through the PNAS open access option. tibody targeting CTLA-4 (marketed as Yervoy), demonstrated Data deposition: The crystallography, atomic coordinates, and structure factors have been improved overall survival in two phase-III clinical trials of metastatic deposited in the Protein Data Bank, www.rcsb.org/pdb/home/home.do (PDB ID code melanoma (10, 11) and received FDA approval for the treatment of 5TRU). metastatic melanoma in 2011. Ipilimumab is the first-in-class im- 1U.A.R., W.L., and S.C.G.-T. contributed equally to this work. munotherapeutic for blockade of CTLA-4 and significantly benefits 2Present address: Biological Sciences Division, Poornaprajna Institute of Scientific Re- overall survival of patients with metastatic melanoma. Notably, search, Bangalore 562110, India. combination therapies involving ipilimumab and other immuno- 3Present address: Janssen Pharmaceuticals Inc., Titusville, NJ 08560. modulators/checkpoint antibodies, such as those targeting PD-1 or 4Present address: F-Star Biotechnology Ltd., Cambridge, CB22 3AT, United Kingdom. – – tremelimumab (anti CTLA-4) and anti PD-L1, can result in en- 5To whom correspondence may be addressed. Email: [email protected] or alan. hanced activity (12–15). [email protected]. Multiple mechanisms have been described for CTLA-4 function. This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. These include negative signals from intrinsic effects of CTLA-4 1073/pnas.1617941114/-/DCSupplemental.

www.pnas.org/cgi/doi/10.1073/pnas.1617941114 PNAS Early Edition | 1of10 Downloaded by guest on September 26, 2021 both modalities for antitumor activity (6). Blockade of CTLA-4 also similarity, both CTLA-4 and CD28 use the stereochemical fea- promotes Treg suppression in vitro (6, 19). tures of a shared proline-rich motif (MYPPPY), present in the Human clinical studies with tremelimumab, which, like ipili- loops joining the F and G β strands, to bind the B7-1 and B7-2 mumab, blocks the interactions of CTLA-4 with its ligands, ligands (9). An essential property of any CTLA-4 therapeutic demonstrate that this antibody also has antitumor activity in ad- antibody is the ability to specifically engage CTLA-4, while dition to inducing adverse events (22). Moreover, long-term sur- exhibiting little or no cross-reactivity with CD28. In the case of vival of melanoma patients treated with either ipilimumab (23) or , recognition of CD28 and inhibition of tremelimumab have been reported (24). Notably, tremelimumab ligand binding could inhibit T-cell activation, which would op- harbors the IgG2 isotype and thus cannot engage FcγRs, sup- pose the desired therapeutic activity. To define the nature of the porting a mechanism of action that relies largely on competitive inhibitory mechanism and the specificity exhibited by ipilimumab inhibition with the B7 ligands (ref. 20 and references therein). In for CTLA-4, we report the crystal structure of the complex contrast, ipilimumab is an IgG1 that can engage human FcγR; formed by a Fab fragment of ipilimumab and human CTLA-4, as consistent with this behavior, ipilimumab was shown to mediate well as complementary biochemical studies that confirm the antibody-dependent cell-mediated cytotoxicity (ADCC)-facilitated epitope recognized by ipilimumab. This work unambiguously depletion of Treg cells in vitro (25). However, only small numbers defines the ipilimumab recognition surface on CTLA-4, which of patients have been analyzed for depletion of Treg at the tumor partly overlaps the B7 ligand binding surfaces, indicating that site by ipilimumab (26, 27). Of note, FcγR polymorphisms have no direct steric competition contributes to the function of ipilimu- impact on the survival of ipilimumab-treated patients (28). Im- mab. This work also highlights the determinants responsible for portantly, all aspects of ipilimumab-mediated CTLA-4 blockade the highly selective binding to CTLA-4 and provides the foun- require specific and high-affinity recognition of CTLA-4. dation for structure-guided engineering of ipilimumab variants CTLA-4 and CD28 are type-I integral membrane proteins with new in vitro activities (e.g., altered affinities for CTLA-4 composed of a single Ig variable domain (IgV), a transmembrane and Fc receptors) for the realization of enhanced in vivo segment, and a cytoplasmic tail bearing various signaling motifs. therapeutic functions. The IgV ectodomains share ∼30% sequence identity and exhibit a two-layered beta sheet involving the A′GFCC′C′′ strands of Results and Discussion the front sheet and the ABED strands of the back sheet. Both Overall Structure of the Human CTLA-4:Ipilimumab Complex. The molecules exist as covalent homodimers due to a disulfide bond structure of the complex between monomeric human CTLA-4 formed between cysteines in the stalk segments connecting the (residues 1–118) and the Fab fragment derived from ipilimumab IgV and transmembrane domains. A wide range of structural and was determined and refined to a resolution of 3.0 Å, with Rwork biochemical data demonstrate that, despite this modest sequence and Rfree of 20.3% and 26.8%, respectively (Fig. 1A, Fig. S1A,and

Fig. 1. Structure of the CTLA-4:ipilimumab complex. (A) The concave CTLA-4 front face, formed by the CC′ (beige) and FG (MYPPPY loop; dark purple) strands, is buried between CDRs of ipilimumab. CDRs, FG, and CC′ strands are colored: LCDR3 (olive) and LCDR1 (teal) make the hydrogen bonding interaction with the G strand. HCDR2 (pink) and HCDR1 (light gray) pack against F and C strands and participate in both hydrogen bond and hydrophobic interactions. HCDR3 (magenta) inserts into the center of the front face of CTLA-4 and exclusively participates in hydrophobic interaction. (B) Rainbow representation (N to C termini transition from blue to red) of CTLA-4 molecules with front and back strands labeled white and black, respectively. The G-strand β bulge conserved among the antigen receptors is showninred.(C) CTLA-4 rotated 90° around the vertical axis relative to B, highlighting the concave front surface, with approximate location of CDRs represented by arrows in respective color (as used in A). The coordinates of the ipilimumab:CTLA-4 complex have been deposited in PDB as ID code 5TRU.

2of10 | www.pnas.org/cgi/doi/10.1073/pnas.1617941114 Ramagopal et al. Downloaded by guest on September 26, 2021 Table 1). The two independent copies of the complex in the 53Tyr, 57Asn, and 59Tyr), HCDR3 (101Trp and 102Leu), LCDR1 PNAS PLUS asymmetric unit exhibit similar overall organization and superpose (31Ser and 33Tyr), and LCDR3 (93Gly, 94Ser, 95Ser, and 97Trp) with a Cα–root-mean-square deviation (Cα–rmsd) of 0.74 Å, cal- (Fig. 2A). The G strand from CTLA-4 is positioned along the cleft culated over all experimentally defined Cα atoms of CTLA-4 formed between the VL (LCDR1 and LCDR3) and VH (HCDR1 A B (2–116) and the VH (2–118) and VL (2–108) domains of the ipi- and HCDR2) domains (Fig. 2 and ), whereas the adjacent F limumab Fab fragment (Fig. S1;theCα–rmsd is 1.24 Å when the strand packs against HCDR1 and HCDR2 (Fig. 2A). Together these constant domains of the Fab are included). The Cα–rmsd between contacts account for almost all of the hydrogen bonding interactions the two molecules of CTLA-4 in the asymmetric unit is 0.65 Å. within the complex. 101Trp and 102Leu from HCDR3 contact the The ipilimumab Fab exhibited typical CDR structural parameters, center of the concave hydrophobic patch on the front face of CTLA-4 39 46 93 with elbow angles for the two independent molecules in the formed by Leu, Val, and Ile, which extends the binding in- asymmetric unit of 168° and 170°. Due to the similarity of the two terface toward the CC′ loop located opposite the FG loop (Fig. 2A). complexes, the discussion below refers to one of the CTLA-4:Fab Overall, ∼13 hydrogen bonds and more than 90 contacts less complexes in the asymmetric unit (Fig. 1 and Fig. S1B), denoted L than 4.0 Å contribute to the CTLA-4:ipilimumab Fab interface for light chain, H for heavy chain, and C for CTLA-4. (Table S1). The total surface area buried at the binding interface is 2 2 The binding interface is formed by residues from the C, C′,F, ∼1,880 Å , with 990 Å contributed by the ipilimumab Fab and 2 and G strands (Fig. 1 B and C) of the front β-sheet of CTLA-4 and 890 Å by CTLA-4, which is at the high end of observed values – 2 – light chain complementarity determining regions 1 and 3 (LCDR1 (i.e., 1,175 1,755 Å )forantigen antibody complexes (31). These and LCDR3) and heavy chain CDRs 1, 2, and 3 (HCDR1, extensive interactions are consistent with the high affinity in- HCDR2, and HCDR3) (Fig. 1). The only potential interaction teraction between CTLA-4 and the ipilimumab Fab [equilibrium dissociation constant (Kd) of 10.6 nM]. Both the heavy chain involving LCDR2 is the 4.2 Å approach between the side-chain 2 2 hydroxyl of 50Tyr and the main-chain carbonyl of 44Ser on CTLA-4 (605 Å ) and light chain (385 Å ) make significant contributions [numbering is consistent with previous CTLA-4 structural reports; to the binding interface. The F and G strands, which include the 99MYPPPY104 loop, bury 498 Å2 of surface area, representing e.g., Protein Data Bank (PDB) ID codes 1I85 and 1I8L (29, 30)]. 2 93 95 97 ∼60% of the total buried surface area (890 Å ) contributed by CTLA-4 residues from the F and G strands ( Ile, Lys, Glu, 100 103 2 106Leu, and 108Ile), the FG loop (99MYPPPY104)aswellas33Glu, CTLA-4. The YPPP segment buries only 56 Å of surface 35 39 46 ′ area upon binding ipilimumab, with the major contributors from Arg, and Leu from the C strand and Val from the C strand 99 104 52 the CTLA-4 MYPPPY loop being Tyr-104 and Met-99, which form an extended interface with residues from HCDR2 ( Ser, 2 bury 115.0 and 85.3 Å of surface area, respectively. Thus, although INFLAMMATION the FG loop is involved in the recognition of ipilimumab, this loop IMMUNOLOGY AND Table 1. Crystallographic data and refinement statistics is not completely buried, as observed in the CTLA-4:B7-1 and CTLA-4:B7-2 complexes (29, 30). PDB ID 5TRU An important consideration is that the species crystallized in Source 24–ID–E, APS these studies is monomeric CTLA-4, whereas the physiologically Wavelength, Å 0.979 relevant cell surface receptor is a disulfide-linked homodimer in- Resolution limits, Å 34.9–3.0 volving Cys-122, which is outside the well-ordered globular domain

Space group C2221 and not included in the construct used in this crystallographic Unit cell, Å, a, b, c 95.84, 197.50, 148.12 analysis. Structural and modeling analyses demonstrate that ipili- No. of observations 181,876 mumab could readily accommodate the observed interactions No. of unique reflections 28,514 within the bona fide CTLA-4 dimer. In particular, the unique mode Completeness, % 99.7 (99.9) of CTLA-4 dimerization, involving “side-to-side” contact between Mean I/σI 14.6 (2.0) CLTA-4 monomers, places the FG loops distal to the dimer in-

Rmerge on I, %* 14.3 (82.0) terface, such that the relevant epitope in each monomer is highly † Rpim,% 5.2 (31.5) solvent accessible and appropriately positioned to recapitulate the Redundancy 6.4 (6.6) interactions observed between the CTLA-4 monomer and the intact 2 BWilson,Å 63 ipilimumab mAb (Fig. S2).

CC1/2 0.965 (0.881) Refinement statistics Cell-Based Evaluation of the CTLA-4:Ipilimumab Recognition Interface. Resolution limits, Å 34.9–3.0 To confirm the crystallographically observed binding interface, we No. of reflections, work/free 26945/1447 generated a library of 118 dimeric CTLA-4 mutants, which were Protein atoms 8036 transiently expressed in HEK293 cells and challenged with soluble ‡ ipilimumab. This approach provides a gold standard for evaluating Rcryst,% 0.203 (0.349) extracellular interactions, as the members of the mutant library Rfree, %, 5% of data 0.268 (0.390) rmsd Bonds, Å/angles, ° 0.009/1.46 undergo all requisite co- and posttranslational modifications (e.g., Mean B, Å2§ 71 correct disulfide bond formation and glycosylation) and are pre- Mean B, Å2, for chains L, H, l, h, C, 59, 65, 80, 68, 84, 84 sented in the context of the mammalian cell surface environment and c, respectively§ (32). In the context of cell surface expression, mutation of residues Fo, Fc correlation 0.93 (EDS) S20, R35, R40, Q76, D88, K95, E97, Y104, L106, and I108 of Ramachandran plot statistics, % 94.9 (favored), 4.5 (allowed), human CTLA-4 resulted in significant loss of ipilimumab binding A B A — and 0.6 (outlier) (Fig. 3 and and Fig. S3 ). Of the residues identified R35, K95, E97, Y104, L106, and I108—are observed to make direct Parentheses indicate statistics for the high–resolution data bin for X-ray contacts with ipilimumab in the crystal structure. Residues R40 and refinementP P data. P P and D88 form a salt bridge that links two loops in the membrane *Rmerge = hkl i jIhkl,i – j/ hkl i Ihkl,i. † P P P P proximal region of CTLA-4; disruption of this interaction likely R = {1/[N – 1]}1/2 × jI – j/ I , where N is the pim hkl hkl i hkl,i hkl hkl i hkl,i causes local or global structural perturbations, resulting in loss of redundancy.P P ‡ ipilimumab binding. Similarly, mutation of S20 or Q76 to aspartic Rcryst = hkl jFobshkl – Fcalchklj/ hkl Fobshkl, where Fobs and Fcalc are ob- served and calculated structure factors, respectively. acid also resulted in loss of ipilimumab binding, despite residing §Average B factors were calculated with program BAVERAGE in the CCP4 on the opposite side of CTLA-4 relative to the ligand-binding site. suite. O- and N-linked glycosylation algorithms predict that S20 and N75

Ramagopal et al. PNAS Early Edition | 3of10 Downloaded by guest on September 26, 2021 (adjacent to Q76) are potentially glycosylated, suggesting that to B7-2 (34). These observations suggest a mechanism that cou- mutations at residues 20 and 76 may affect installation or in- ples ligand binding with formation of the CTLA-4 dimer interface. teraction with the carbohydrate moieties and potentially overall The biological consequences of this behavior will require further structural stability (Fig. S3B) (33). The likelihood that mutations evaluation. at these four residues (S20, R40, N75, and D88) cause global structural perturbation/destabilization of CTLA-4 is supported by Biochemical Confirmation. To further evaluate the contributions to the observation that they also result in the loss of binding to B7-1, the binding interface, a selected subset of CTLA-4 mutants were B7-2, and ICOS-L, despite the observation that these residues purified and their interactions with ipilimumab Fab were evaluated reside outside of the experimentally determined binding interfaces by size exclusion chromatography (SEC) and native gel analysis. In (Fig. 3 A and B and Fig. S3C). particular, 95Lys, 99Met, and 104Tyr, which all contact the Fab, and These mapping studies also highlight features of the binding 105Tyr, which lies outside the crystallographically observed binding surface recognized by B7-1, B7-2, and ICOS-L. A number of interface, were mutated to alanine in dimeric human CTLA-4 con- CTLA-4 mutations (R35A, R35D, K95A, K95D, E97A, E97R, taining the native interchain disulfide. Interactions between the Y104A, Y104D, I108A, and I108D) that strongly impair B7-1 wild-type and mutant dimeric CTLA-4 molecules and the Fab were and B7-2 binding are consistent with contacts observed in the examined by SEC (Figs. 5A and 6A). The dimeric K95A and Y104A CTLA-4:B7-1 and CTLA-4:B7-2 crystal structures (29, 30) (Fig. mutants did not exhibit any interaction with the Fab, whereas the S3C). In addition, mutations at residues E33, E48, and Y100, dimeric M99A mutant exhibited significantly decreased association also present at the CTLA-4:B7-2 binding interface, result in compared with the wild type (Fig. 6A). The Y105A mutation did modest reductions in binding between CTLA-4 and B7-2. Again, not significantly affect the interaction between dimeric CTLA-4 and mutations at residues R35, K95, E97, Y104, and I108 severely the Fab, consistent with the placement of 105Youtsidetheobserved diminished ipilimumab binding, consistent with the direct steric ipilimumab recognition interface (Fig. 6A). The behavior of the blockade of CTLA-4 ligand binding by ipilimumab. This conclusion wild-type dimeric CTLA-4 is consistent with the function blocking is further supported by results from a competition experiment activity of intact ipilimumab. demonstrating that ipilimumab, but not a control mAb, directly The interactions between the Fab and the wild-type dimeric competes with hB7-1 for bindingtobeadscoatedwithhCTLA-4 CTLA-4 and dimeric mutants were further evaluated by native (Fig. 4 A–C). In addition, mutation of a number of CTLA-4 resi- nonreducing PAGE analysis. The ipilimumab Fab does not enter dues distal to the ligand-binding interfaces (e.g., V10, L12, S14, the gel under native PAGE conditions due to the overall positive T69, T71, N78, Q82, G83, R85, A86, Y115, D118, and E120) charge of the Fab (pI of 8.8) at pH 8.0. Formation of the dimeric exhibited impaired binding to B7-1, B7-2, and ICOS-L (Fig. S3D). CTLA-4:Fab complex results in a species capable of entering the Importantly, all of the CTLA-4 mutants analyzed for binding gel, whereas unbound dimeric CTLA-4 results in a distinct band showed similar mCherry expression (Fig. S3E), making it unlikely (Fig. 5B). As shown by the native PAGE results, the wild-type that these mutations caused severe structural instability or protein dimeric CTLA-4 interacted with Fab and formed new species misfolding. It is more likely that mutations of residues on CTLA-4 consistent with the formation of the complex in the gel (Fig. 5B). distal to the observed ligand binding site caused more subtle local Dimeric K95A, M99A, and Y104A CTLA-4 mutants did not structural perturbations. Three of these residues (Y115, D118, form any new species with ipilimumab Fab under native PAGE and E120) contribute to the CTLA-4 dimer interface, with the conditions (Fig. 6B). Dimeric Y105A CTLA-4 productively remaining residues arranged in a patch that extends toward the interacted with the Fab, as demonstrated by the appearance ligand binding surface (29, 30). The mechanistic underpinnings for of a new band in the native PAGE (Fig. 6B). Importantly, as the binding properties of these mutants is not clear but may be evidenced by fluorescence-monitored thermal denaturation, all related to the previous report that CTLA-4 lacking the interchain of these dimeric CTLA-4 mutants are fully folded under the disulfide exists as an interconverting population of noncovalent di- conditions used in these experiments (SI Materials and Methods mer and monomer, which fully resolves to monomer when bound and Fig. S4). These results are fully consistent with the observed

Fig. 2. Critical interactions between ipilimumab and CTLA-4. (A) Surface representation of ipilimumab (heavy chain in light gray; light chain in dark gray)

showing the cleft formed between the VH and VL domains. Residues on the FG (purple) and CC′ (beige) strands of CTLA-4 facing toward the Fab are shown in stick representation. For clarity, side chains on the FG and CC′ strands facing away from the Fab are not shown. The G strand extends along the cleft with hydrophobic residues (104Tyr–108Ile) intercalating into the cleft between the two domains. The F and C strands stack against HCDR2 and HCDR1. 101Trp and 102Leu from the tip of the HCDR3 loop contacts the center of the CTLA-4 front face and interacts with a hydrophobic patch formed by 39Leu, 46Val, and 93Ile. (B) Stick representation of the 99MYPPPY104 loop followed by G-strand residues (purple). The main-chain carbonyl and amide groups of the edge G-strand residues not involved in interstrand interactions with the F strand participate in hydrogen bonding interactions with LCDR3 (olive) and LCDR1 (teal) residues. The 99MYPPPY104 loop (FG loop) packs against a cluster of aromatic residues, and 59Y(pink) from HCDR2 contacts the center of the FG loop and also par- ticipates in a hydrogen bonding interaction with the carbonyl oxygen of 99M.

4of10 | www.pnas.org/cgi/doi/10.1073/pnas.1617941114 Ramagopal et al. Downloaded by guest on September 26, 2021 crystallographic interface and support the physiological rele- PNAS PLUS vance of the CTLA-4: ipilimumab Fab complex. Interactions of B7 ligands with wild-type CTLA-4 and mutants were confirmed by native nonreducing PAGE analysis as well as by SEC (Figs. S5, S6, and S7).

Epitope Verification and Specificity Determinants of Ipilimumab. The challenge of intact ipilimumab with proteolytically generated (i.e., AspN, GluC, and Trypsin) peptide fragments derived from the disulfide-linked human CTLA-4–Fc , coupled with MS, identified a discontinuous epitope involving three different linear peptides (Fig. S8). P1 stretches from 26YASPGKATEVRVTVLRQA42,P2from43DSQVTEVCAA- TYMMGNELTFLDD65,andP3from96VELMYPPPYYLGIG109. From Table S1, it can be seen that residues from HCDR2 and HCDR3 interact with P1 and P2, respectively, whereas HCDR2, HCDR3, and LCDR3 interact with P3. Most of the interaction energy between CTLA-4:B7-1 and CTLA-4:B7-2 is derived from contacts with 99MYPPPY104 (9), which is contained within the P3 segment recognized by ipilimumab, consistent with the function blocking activity of ipilimumab. To understand the specificity of ipilimumab toward CTLA-4, linear peptides from both native and reduced human CD28–Fc fusion pro- tein were generated, and their interactions with ipilimumab-coupled beads were examined. After elution from antibody-decorated beads, the mass and sequences of the bound peptides were characterized by MALDI-TOF MS and nano-liquid chromatography (nano-LC)–

MS/MS, respectively. Of particularnoteistheCD28trypticpep- INFLAMMATION tide, 96IEVMYPPPYLDNEK108, which did not bind ipilimumab IMMUNOLOGY AND (Fig. S8). In contrast, the corresponding peptide from CTLA-4, 98LMYPPPYYLGIGN110, which is part of peptide P3 (Fig. S8), bound well to ipilimumab. Even though the sequence 99MYPPPY104 was present in both linear peptides from CTLA-4 and CD28, the observation that only the CTLA-4–derived peptide binds implies that residues outside of this region play important roles in recognition of the 99MYPPPY104-containing epitope, which is consistent with the crystallographic data as discussed above (also see Table S1).

Mechanism of CTLA-4 Recognition and Blockade by Ipilimumab. The ability of CTLA-4 and CD28 to recognize both the B7-1 and B7-2 ligands is, in part, the consequence of the conserved FG loop (99MYPPPY104) shared by both receptors (Fig. 7). The in- troduction of mutations in this loop resulted in greater than 90% loss of affinity to the B7 ligands, identifying this segment as the core of the ligand binding surface on both CTLA-4 and CD28 (9). Direct structural analyses of the CTLA-4:B7-1 and CTLA-4:B7-2 complexes (29, 30) showed that the 99MYPPPY104 loop contributes ∼80% of the interfacial contacts with the B7 ligands. The three consecutive proline residues in the FG loop assume an unusual cis–trans–cis conformation, and with the exception of 99Met, all of the main-chain carbonyl atoms are directed away from the ligand binding surface (Fig. 8A). Given its chemical composition and conformation, this loop is devoid of free amide nitrogens or solvent- accessible carbonyls (except for the terminal residues 99Mand104Y) and thus lacks determinants typically associated with directionality and specificity (i.e., hydrogen bond donors and acceptors). This unique and highly strained main-chain conformation provides sig- nificant geometric complementarity and hydrophobicity to support recognition of the concave surfaces presented by the front sheets of Fig. 3. Mapping the ipilimumab-binding epitope on cell surface-expressed human CTLA-4. (A) Wild-type and mutant CTLA-4 constructs (118 total) were transiently expressed as C-terminal mCherry fusions in HEK293 suspension cells. Two days posttransfection, CTLA-4–expressing cells were queried with experiments for those CTLA-4 mutants that resulted in ≤50% binding to a either the ipilimumab or cells transiently expressing particular query. (B) Residues at which mutations caused a significant loss of hB7-1-GFP, hB7-2-GFP, or hICOS-L-GFP and analyzed by flow cytometry to ipilimumab binding are mapped onto the CTLA-4 crystal structure. The res- determine the percentage of mCherry-positive cells (CTLA-4–expressing) idues highlighted in red make direct contact with ipilimumab, the two blue bound (anti-human Alexa 488 signal reports on ipilimumab binding; GFP residues R40 and D88 form a salt bridge, and the two green residues signal reports on B7-1, B7-2, or ICOS-L binding). The chart highlights (colored (S20 and Q76) affected ipilimumab binding even though they are distal to background) the average percent bound and SD from three independent the ipilimumab recognition surface.

Ramagopal et al. PNAS Early Edition | 5of10 Downloaded by guest on September 26, 2021 B7 complexes, the tip of the FG loop (99MYPPPY104)fromCTLA-4 contacts the front face of the B7 ligands (Fig. 8 C and D and Fig. S9 A and B), with the CC′ loop of CTLA-4 positioned away from the B7 surface. However, in the CTLA-4:ipilimumab complex, the –101PPP103− tip of the FG loop is only partially engaged, allowing for the association of the entire CTLA-4 front face with ipilimumab (Fig. 8E and Fig. S9C). In this way, the ipilimumab interaction surface not only sterically occludes the conserved 99MYPPPY104 surface from availability to B7 ligands but also ex- tends its interaction toward the opposite side of the CTLA-4 IgV domain (i.e., CC′ loop) (Fig. 8F). The recognition of this extended CTLA-4 surface by ipilimumab (relative to the B7 ligands) is the consequence of the highly twisted architecture common to the front sheet of IgV domains of the antigen receptors, which results in the protrusion of the long FG and CC′ loops away from the plane of the front sheet, creating a concave surface on the CTLA-4 front sheet (Fig. 1C)(35). The differences in total buried surface area upon binding CTLA-4 (∼1,885 Å2 for ipilimumab vs. ∼1,250 Å2 for the B7 ligands), the greater number of hydrogen bonds (∼13 for ipilimumab vs. ∼5–7 for the B7 ligands), and the larger number of van der Waals contacts are consistent with the Kds exhibited by ipilimumab

Fig. 4. Ipilimumab directly competes with hB7-1 for binding to hCTLA-4 in a bead-based FACS assay. (A) Schematic representation of the assay. (B) Pro- tein A beads saturated with hCTLA-4 hIgG1 were titrated with hB7-1 mIgG2a protein. FACS analysis was used to determine the GeoMean of the FL1 channel (488) for ALL BEADS gated. Data represent the average of three independent experiments with error bars showing the SD. (C) Protein A beads loaded with hCTLA-4 hIgG1 were saturated with hB7-1 mIgG2a (5 nM) and incubated with increasing concentrations of control mAb or ipilimumab. Data show the GeoMean of Fl1 normalized to the 5-nM titration point from that same experiment. All data show an average of three independent ex- periments with error bars showing SD.

the B7 ligands. The structures of the unliganded murine and human CTLA-4 molecules demonstrate that this FG loop adopts essen- tially the same detailed conformation in both forms (35), indicating that CTLA-4 presents a preformed B7-recognition surface. Al- though a structure of CD28 bound to a B-7 ligand is not available, the conserved sequences and conformation of the FG loop in the structure of a CD28:Fab complex (36), as well a series of muta- genesis experiments (9), supports a mode of interaction with B7 ligands similar to that observed for CTLA-4. Ipilimumab exploits the unique features of the FG loop and sandwiches the front sheet of CTLA-4 between LCDR1 and LCDR3 on one side and HCDR1 and HCDR2 on the other (Fig. 2A). The region between LCDR3 and HCDR2 is rich in aro- matic residues and stacks against the surface presented by the 99 104 B Fig. 5. Biochemical confirmation of the ipilimumab:CTLA-4 binding in- MYPPPY loop (Fig. 2 ). Residues in the tips of LCDR3 and terface, and formation of the wild-type dimeric hCTLA-4:Fab complex LCDR1 are positioned proximal to the edge G-strand side of the demonstrated by gel filtration and native PAGE gel. (A) Gel filtration of front sheet of CTLA-4 and participate in a series of hydrogen dimeric hCTLA-4 and Fab complex. The chromatograms for Fab, dimeric bonding interactions with main-chain atoms of the G strand (Fig. hCTLA-4, and the dimeric hCTLA-4:Fab mixture are indicated as black, blue, 2B). HCDR1 and HCDR2 stack almost perpendicular to the C, F, and red curves, respectively. It should be noted that the ipilimumab Fab and G stands of CTLA-4 and provide both hydrophobic and (molecular mass, ∼50 kDa) reproducibly exhibits aberrantly long retention ∼ hydrogen bonding interactions with the solvent-exposed residues times relative to the smaller dimeric CTLA-4 (molecular mass, 25 kDa). (B) Native PAGE analysis of the dimeric hCTLA-4 and Fab complex. The on the F and C strands as well as those located toward the termini concentration of wild-type dimeric hCTLA-4 was held constant at 10 μM with of the FG loop (Table S1). The HCDR3 loop contacts the center of increasing concentration of Fab. Wild-type dimeric hCTLA-4 and Fab were theconcavesurfaceofCTLA-4onits front sheet, with interactions mixed in molar ratios of 4:1, 2:1, 1:1, 1:2, and 1:4 from lane 1 to lane 5. Lanes extending toward the CC′ loop (Figs. 1A and 2A). In the CTLA-4: 6 and 7 show Fab and dimeric hCTLA-4 alone, respectively.

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Fig. 6. Gel filtration and native PAGE analysis of dimeric mutants and Fab. (A) The chromatograms for Fab, dimeric hCTLA-4 mutants, and the dimeric hCTLA- 4:Fab mixture are indicated as black, blue, and red curves, respectively. (Top Left) K95A. (Top Right) Y104A. (Bottom Left) M99A. (Bottom Right) Y105A. All peaks of the corresponding proteins are indicated by arrows. The formation of the complex is indicated by a red arrow. (B) Distinct migration bands for dimeric hCTLA-4 mutants, Fab, and the hCTLA-4 mutant:Fab complex are diagnostic for interactions of dimeric hCTLA-4 mutants and the Fab. The con- centrations of dimeric hCTLA-4 mutants were held constant at 10 μM. (Top Left) Dimeric mutant K95A and Fab were mixed in molar ratios of 4:1, 2:1, 1:1, 1:2, and 1:4, from lane 1 to lane 5. Lanes 6 and 7 show Fab and dimeric mutant K95A alone, respectively. (Top Right) Dimeric mutant Y104A and Fab were mixed in molar ratios of 4:1, 2:1, 1:1, 1:2, and 1:4, from lane 1 to lane 5. Lanes 6 and 7 show Fab and dimeric mutant Y104A alone, respectively. (Bottom Left) Dimeric mutant M99A and Fab were mixed in molar ratios of 2:1, 1:1, and 1:2, from lane 1 to lane 3. Lanes 4 and 5 show Fab and dimeric mutant M99A alone, respectively. (Bottom Right) Dimeric mutant Y105A and Fab were mixed in molar ratios of 2:1, 1:1, and 1:2, from lane 1 to lane 3. Lanes 4 and 5 show Fab and dimeric mutant Y105A alone, respectively.

and B7 ligands for CTLA-4 (∼10 nM and ∼0.1–1 μMrange,re- main fold. Overall, the structure of the Fab-bound human spectively) and the observation that ipilimumab competes effec- CTLA-4 monomer is similar to that in the apo–CTLA-4 homo- tively with the B7 ligands for binding CTLA-4. It is this direct dimer structure (35), the CTLA-4:B7-1 complex (30), the CTLA- competition between ipilimumab and the B-7 ligands that, in 4:B7-2 complex (29), and the CTLA-4:Lipocalin (37) complex, part, underlies the therapeutic efficacy of ipilimumab. with Cα–rmsds that range between 0.85 and 1.1 Å. Similarly, the Cα–rmsds calculated with ipilimumab-bound human CTLA-4 Ipilimumab Specificity. Although the B7 ligands interact with both against human CD28 (36) and murine CTLA-4 (38) structures CTLA-4 and CD28, the ability of ipilimumab to discriminate are 1.51 Å and 1.27 Å, respectively. between these two functionally distinct receptors is critical for its Nearly all features of the FG loop (99MYPPPY104) are con- therapeutic efficacy. CTLA-4 and CD28 share ∼30% sequence served among the structurally characterized CD28:CTLA-4 family identity, including conservation of the critical core residues and members, including the sequences and the specific side-chain disulfide linkages that are essential for maintaining the IgV do- conformations; the sole exception is found in the apo–CTLA-4

Ramagopal et al. PNAS Early Edition | 7of10 Downloaded by guest on September 26, 2021 Fig. 7. Sequence alignment of CTLA-4 and CD28. CTLA-4 residues interacting with ipilimumab are marked with blue triangles. The filled circles indicate residues from B7-1 (crimson) and B7-2 (purple) that make contacts less than 4.0 Å. Insertion in CD28, before the β bulge on the G strand, is shown with an arrow. Notice the sequence differences in the G-strand residues just after the 99MYPPPY104 loop. The three discontinuous stretches of CTLA4 (P1, 26YASPGKATEVRVTVLRQA42; P2, 43DSQVTEVCAATYMMGNELTFLDD65; and P3, 96VELMYPPPYYLGIG109) identified as part of the ipilimumab epitope, by nano- LC–MS/MS, are shown by brown lines above the sequence of CTLA-4.

homodimeric structure, where 100Tyr adopts a different rotamer and 93Phe, respectively, and may contribute to the ability of ipili- conformation to accommodate crystal contacts (35). The confor- mumab to discriminate between CTLA-4 and CD28 (Fig. 8). mation of this loop in the Fab-bound CD28 structure is also similar As members of the antigen receptor group of the IgSF (35), both CTLA-4 and CD28 possess β bulges in C′ and G strands, to that found in CTLA-4, including side-chain rotamers, with an 48 110 overall Cα–rmsd of ∼0.3 Å (Fig. 8 A and B). Based on these ob- which in CTLA-4 are centered on Glu and Asn, respectively (in CD28, these bulges are centered on 46Glu and 110Asn, re- servations, these segments are not likely to be major determinants spectively) (Fig. 1B) (39). The –Gly–X–Gly– sequence of G-strand for the discrimination between CTLA-4 and CD28. Although many β bulge observed in CTLA-4 is present in all VL domains and other CTLA-4 residues that interact with ipilimumab are conserved more than 98% of VH domains of mABs (39). Notably, in CD28, in CD28, there are a few important differences in sequence and the first glycine in this sequence is replaced by serine. In CD28, conformation. 39Leu and 93Ile in CTLA-4, which interact with the single residue insertion 108Glu (between the equivalent of 101Trp and 102Leu from HCDR3, are replaced in CD28 by 38His 108Gly and 109Ile in CTLA-4), just before the G-strand β bulge,

Fig. 8. Mechanism of ipilimumab action. (A) Identical residues between CTLA-4 and CD28 are colored blue, and insertions in the CTLA-4 sequence are colored red; both are mapped onto the CTLA-4 structure (yellow). (B) Identical residues between CTLA-4 and CD28 are colored blue, and insertions in the CD28 sequence are colored red; both are mapped onto the CD28 structure (yellow). Note that the MYPPPY-loop surfaces contours are similar. (C) Mode of B7-2 (light blue) interaction with the MYPPPY-loop surface. (D) Mode of B7-1 (pink) interaction with the MYPPPY-loop surface. (E) Mode of CTLA–4and ipilimumab interactions. (F) Superposition of the CTLA-4:B7-2 and CTLA-4:ipilimumab structures, based on the CTLA-4 component in each complex, is pre- sented. Superposition of the CTLA-4:B7-1 and CTLA-4:ipilimumab complexes is shown in Fig. S9. These superpositions indicate that ipilimumab and the B7 ligands compete for overlapping binding surfaces on CTLA-4.

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Fig. 9. Determinants of selectivity. (A) The 99MYPPPY104 loop conformations in CTLA-4 (purple) and CD28 (yellow) are very similar. Note that only the carbonyl moiety from 99M and the amide nitrogen from 104Y are facing toward the viewer and are available for interaction with the ligands. (B) Insertion in the edge G strand following the conserved β bulge causes the G strand of CD28 to protrude away from the F strand, resulting in the loss of many interstrand interactions with the F strand. (C) Possible clashes between the LCDR1 and the extended protrusion of the G strand due to insertion at 109K on CD28 (yellow). The G strand from CTLA-4 is colored purple. INFLAMMATION

exaggerates the protrusion of the G strand (Figs. 1B,7,and8B). In Kd for EGFR, which is expressed at significantly higher levels on IMMUNOLOGY AND addition, on the G strand, immediately following the conserved the malignant cells (41). Similar studies, which examined scFvs 99MYPPPY104 loop, all residues within the span of 105–109 are against EGFR (derived from the C10 anti-EGFR antibody) (44) different in CD28 (Figs. 7 and 9). In particular, 107Asn preceding the and ErbB2 (derived from the 4D5 trastuzumab antibody) (45) with insertion is involved in two hydrogen bond interactions with the Kds spanning ∼2–3 orders of magnitude, have reinforced this F-strand backbone, which further perturbs the canonical interstrand concept. Together, these studies demonstrate that apparent Kds interaction between the F and G strands (Fig. 9 B and C), pushing not only control selectivity between target and nontarget cells but the two strands farther apart than is typical. In CTLA-4, this region is can also impact CAR T-cell function (e.g., cytokine production) – – involved in continuous main chain main chain and main chain side and suggest that on and off rates (i.e., kon and koff) will have im- chain interactions with LCDR1 and LCDR3 (Fig. 2B). Superposition portant mechanistic contributions to the ultimate therapeutic of CD28 on the CTLA-4:ipilimumab complex predicts that the dis- function. These examples and considerations underscore the need tortion of the G strand results in not only loss of hydrogen bonds but to achieve optimal, not maximal, affinities (and kinetics) to attain C also a substantial steric clash with LCDR1 (Fig. 9 ). The detailed the desired biological/therapeutic activity. In the case of ipilimu- structural differences in the G strands of CTLA-4 and CD28 are mab, affinity-attenuated variants could be generated, which would likely the major determinants responsible for the specificity ex- exhibit selectivity for activated Treg cells present at tumor sites due hibited by ipilimumab. In addition, in CTLA-4 there is an insertion to their higher expression levels of CTLA-4, compared with other 47 ′ β of residue T just before the C -strand bulge (between the activated T cells (20, 21). Additionally, it might be possible to equivalent of residues 45Vand46EinCD28;Figs.7and8A), which ′ engineer ipilimumab variants that are more responsive to the re- probably contributes to the differences in the CC loop conforma- duced pH often associated with the tumor microenvironment (46– tion. However, this insertion is distal to the recognition interface 48). Thus, the structure of the CTLA-4:ipilimumab complex pro- and is not predicted to significantly impact the binding or selectivity. vides the foundation for the rationale design of affinity-modulated An important consideration for normal physiology and ther- ipilimumab variants with distinct T-cell subset-targeting profiles apeutic efficacy is that an optimal biological outcome is not al- and more selective and efficacious therapeutic properties. ways associated with the strongest achievable binding (i.e., very high affinity between receptor and ligand). This concept is il- Materials and Methods lustrated by numerous physiological processes, such as the need Detailed methods can be found in SI Materials and Methods. for reversible binding interactions in oxygen delivery, replication, Monomeric and dimeric CTLA-4 and the monomeric IgV domain of hB7-2 transcription, and translation, and these same principles are di- were refolded as previously described from inclusion bodies (29, 49). Ipili- rectly relevant to the development of therapeutic strategies mumab Fab fragments were prepared by papain digestion. Crystals of the (reviewed in ref. 40). Notable examples are provided by recent CTLA-4:ipilimumab Fab complex were obtained by sitting drop vapor dif- reports of chimeric antigen receptor (CAR) T cells engineered fusion and the structure determined by molecular replacement. The crys- to present scFv modules spanning a range of affinities for the tallographically determined binding interface was confirmed by a high- target antigen (41, 42). These studies demonstrate that scFvs throughput FACS analysis using a library of CTLA-4 mutants expressed on with reduced affinities had increased selectivity for solid tumors, the surface of HEK293 cells as well as by direct biochemical characterization relative to normal cells, due to the requirement for higher sur- of ipilimumab binding to wild-type CTLA-4 and mutant CTLA-4 variants. face density of target to support productive engage- ment, a state often afforded by the targeted tumor cells. For ACKNOWLEDGMENTS. We thank the staff of the 24-ID-E beamline, Advanced Photon Source, Argonne National Laboratory for assistance with X-ray diffrac- example, CAR T cells bearing scFvs derived from nimotuxumab, tion data collection. This work was supported by National Institutes of Health but not cetuximab (43), effectively discriminated between ma- Grants HG008325, GM094662, and GM094665 (to S.C.A.); and the Albert Einstein lignant cells and nonmalignant cells based on its ∼10-fold poorer Cancer Center (P30CA013330).

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