Antibody light chain variable domains and their biophysically improved versions for human immunotherapy

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1 Paper Type mab 6:1, ; January/February 2014; 2014 Lande Biocience Report Antibody light chain variable domain and their biophyically improved verion for human immunotherapy Dae Young Kim 1, Rebecca To 1, Hiba Kandalaft 1, Wen Ding 1, Henk van Faaen 1, Yan Luo 1,, Joeph D Schrag 2, Nadereh St-Amant 3,, Mary Hefford 3, Tomoko Hirama 1,, John F Kelly 1, Roger MacKenzie 1,4, and Jamhid Tanha 1,4,5, * 1 Human Health Therapeutic; National Reearch Council Canada; Ottawa, ON Canada; 2 Human Health Therapeutic; National Reearch Council Canada; Montréal, QC Canada; 3 Centre for Vaccine Evaluation; Biologic and Genetic Therapie Directorate; Health Canada; Ottawa, ON Canada; 4 School of Environmental Science; Ontario Agricultural College; Univerity of Guelph; Guelph, ON Canada; 5 Department of Biochemitry, Microbiology, and Immunology; Univerity of Ottawa; Ottawa, ON Canada Current affiliation: Caprotec Bioanalytic GmbH; Berlin, Germany; Canadian Nuclear Safety Commiion; Ottawa, ON Canada; Koteahicho 1-chome; Tokorozawa, Japan Keyword:, ingle-domain antibody, diulfide linkage, thermal tability, proteae reitance Abbreviation: CDR, complementarity-determining region; CID, colliion induced diociation; DDA, data dependent analyi; ETD, electron tranfer diociation; Fab, fragment antigen-binding; FR, framework region; HBS-EP buffer, 10 mm HEPES, ph 7.4, 150 mm NaCl, 3 mm EDTA and 0.005% P20 urfactant; GI, gatrointetinal; K D, equilibrium diociation contant; MALS, multiangle light cattering; Mapp, apparent molecular ma; M for, formula molecular ma; M MALS, molecular ma determined by MALS; MS, ma pectrometry; RU, reonance unit; dab, ingle-domain antibody; SEC, ize-excluion chromatography; cfv, ingle chain Fv fragment of an antibody; SPR, urface plamon reonance; T m, melting temperature; TRE, thermal refolding efficiency;, antibody heavy chain variable domain; H, camelid heavy chain antibody variable domain;, antibody light chain variable domain; VNAR, hark IgNAR (Ig New Antigen Receptor) variable domain We et out to gain deeper inight into the potential of antibody light chain variable domain ( ) a immunotherapeutic. To thi end, we generated a naïve human phage diplay library and, by uing a method previouly hown to elect for non-aggregating antibody heavy chain variable domain (VH), we iolated a diverity of domain by panning the library againt B cell uper-antigen protein L. Eight domain repreenting different germline origin were hown to be non-aggregating at concentration a high a 450 μm, indicating VL repertoire are a rich ource of nonaggregating domain. In addition, the demontrated high expreion yield in E. coli, protein L binding and high reveribility of thermal unfolding. A ide-by-ide comparion with a et of non-aggregating human revealed that the had imilar overall profile with repect to melting temperature (T m ), reveribility of thermal unfolding and reitance to gatrointetinal proteae. Succeful engineering of a non-canonical diulfide linkage in the core of did not compromie the non-aggregation tate or protein L binding propertie. Furthermore, the introduced diulfide bond ignificantly increaed their T m, by C, and pepin reitance, although it omewhat reduced expreion yield and ubtly changed the tructure of. Human and engineered verion may make uitable therapeutic due to their deirable biophyical feature. The diulfide linkage-engineered may be the preferred therapeutic format becaue of their higher tability, epecially for oral therapy application that neceitate high reitance to the tomach acidic ph and pepin. Introduction A antibody-baed therapeutic, full-length monoclonal antibodie have little competition o far. 1-3 In fact, mot approved monoclonal antibodie and thoe in regulatory review are canonical IgG antibodie ( mab/about/). The diadvantage of thee molecule, uch a cotly and time-conuming production in mammalian cell line, large (~150 kda) and complex molecular tructure, poor tiue penetration and inability to acce cryptic epitope, and the fact that the Fc portion of the antibody i not needed in many intance or may even be harmful, have reulted in the creation of a niche that can be occupied by antibody fragment. 2,4,5 Thee maller antibody fragment, including ingle-domain antibodie (dab), have unique feature that may make them the preferred therapeutic format for many application. Currently, there are *Correpondence to: Jamhid Tanha; jamhid.tanha@nrc-cnrc.gc.ca Submitted: 09/11/2013; Revied: 10/15/2013; Accepted: 10/16/ mab 219

2 numerou antibody fragment in clinical development, with ome being dab. 2,4 dab, e.g., human, human, camelid H, have become a viable option in the antibody-baed therapeutic tool box that alo include IgG, antigen-binding fragment (Fab), ingle chain Fv fragment (cfv), and their many derivative. Appealing feature of dab include their high affinity (nm - pm equilibrium diociation contant (K D ) range) for cognate antigen, 6-29 mall ize (~15 kda) and imple tructure, ingle domain nature, modularity, low immunogenicity, high-level expreion in microorganim uch a bacteria, high thermal, chemical and proteae tabilitie, high olubility and aggregation reitance, ability to acce cryptic epitope, and eae of genetic manipulation and diplay library contruction. 5,30,31 H are more convenient to obtain due to their better biophyical propertie and the exitence and acceibility of in vivo naïve and immune H repertoire ource, but human and have the perceived advantage of being le immunogenic in human therapy. A number of report have implied human may be uperior therapeutic candidate compared with human becaue of their lower tendency to aggregate, which may tranlate to lower immunogenicity and in turn higher therapeutic efficacy for. In vivo, human contruct are the reult of genetic recombination between germline gene egment and J L. The firt two complementarity-determining region (CDR1 and CDR2) and a part of the CDR3 up to reidue 95 are encoded by egment gene; the ret of the CDR3 and the entire framework region (FR) 4 are encoded by J L gene egment. 35 Human are claified a either κ or λ ubtype, with even gene egment ubgroup ( 1 7) within the κ cla and 11 V λ gene egment ubgroup (V λ 1 11) within the λ cla ( org/). 36,37 In general, domain exhibit higher olubility and tability than V λ domain, poibly due to a higher packing denity in their upper core and a more hydrophilic C-terminu, and among the ubgroup, 3 ubgroup member exhibit the bet propertie in term of olubility and thermodynamic tability. 33,38 A ignificant proportion of human, predominantly of cla, bind to the B cell uper-antigen protein L domain are imilar to in term of overall tructure. They are compoed of two β-heet that are formed by everal anti-parallel β-trand and pack face-to-face to form β-andwich tructure. 42 Alo, imilar to, they poe a pair of cyteine reidue at patially equivalent poition (Kabat poition 23 and 88) 43 that form a highly conerved diulfide linkage. Thi linkage, which pin together the two β-heet in the core of domain, play a critical role in maintaining the tructural integrity of. 44,45 Previouly, it wa hown that engineering an additional diulfide linkage in the core of a et of human improved their aggregation reitance and thermotability. 46,47 Given the overall tructural imilarity between and, it i hypotheized that the ame engineering approach, with imilar tability improvement, hould be applicable to. Here, to further explore the merit of human a therapeutic modalitie, we et out to perform an extenive biophyical characterization of a et of tet. We contructed a naïve human phage diplay library, and from it iolated a diverity of domain with protein L binding property by a phage election method that wa previouly hown to be highly elective for nonaggregating human domain. 48 We then characterized a repreentative ample of for propertie uch a expreion yield, non-aggregation, thermal tability, reverible thermal unfolding, tructural integrity, and proteae reitance. Next, we determined if a non-canonical diulfide linkage engineering approach previouly hown to improve the thermotability and proteae reitance of camelid H and human 46,47,49-52 would do the ame for the preent. Thu, we engineered human with an additional, non-canonical diulfide linkage between Cy48 and Cy64 in β-trand C and D. We then performed pair-wie biophyical comparion between wild-type and their correponding Cy mutant domain. Reult Identification and equence analyi of human Eentially the ame election method employed to iolate non-aggregating from a human phage diplay library wa applied to a human library for iolating oluble, monomeric. 48 A human library with a ize of tranformant wa contructed. Twenty-four clone (plaque) from the library titer plate were iolated and their gene were amplified by PCR and then equenced. The equence were divere in term of germ-line origin, although 75% of the were of V λ origin (data not hown). Three round of panning againt protein L reulted in enrichment for large plaque. Thirty-four of the large plaque were equenced and 32 unique equence were identified (Fig. 1). Except for HVLP389, which i from the λ cla (ubgroup V λ 1, V germline 1b), the remaining 31 belonged to the cla. Of the 31 κ cla, 24 fell within the 3 ubgroup and 7 fell within the 1 ubgroup. Sixteen of the 24 3 equence utilized the L6 V germline equence, while the remaining equence utilized A27, L2, and L16 V germline equence. The 1 ubgroup originated from the O2/O12 or A30 V germline equence. Noticeable mutation occurred at poition 96. The germline amino acid at thi poition are aromatic and hydrophobic amino acid Trp, Phe, Tyr, Leu or Ile for κ cla, and Tyr, Val or Ala for λ cla. In the elected pool of κ cla, however, only 5 out of 31 had their germline amino acid at poition 96: HVLP325, HVLP349, HVLP388, HVLP3109, and HVLP393; 21 had charged amino acid, of which 20 were poitively charged, 2 had Pro, 1 had Gln, 1 had Ser, and one had Thr at poition 96. Moreover, 18 of the κ cla had their lat three germline reidue ( ) replaced with amino acid Thr, Val, and Leu, which are only found in λ cla. Expreion yield and aggregation tatu of human Eight of the elected repreenting different V germline origin were expreed in E. coli TG1 in 1 L culture and purified: HVLP324, HVLP325, HVLP335, HVLP342, HVLP351, HVLP364, HVLP389, and HVLP3103 (Fig. 2A; Table 1). All were expreed in good yield ranging from 6.2 mg for HVLP325 to ~75 mg for HVLP335 and HVLP364. The aggregation tendency of the human wa aeed by Superdex mab Volume 6 Iue 1

3 Figure 1. Amino acid equence of elected from a human phage diplay library by panning againt protein L. The dot in the equence entrie indicate amino acid identity with HVLP333. Dahe are included for equence alignment. See V BASE ( php?&mmn_poition=1:1) for equence numbering and CDR and FR deignation. L6, A27, L2, L16, O2/O12, A30, and 1b are V germline gene egment deignation. J germline gene egment deignation are in bracket. NF, not found. ize-excluion chromatography (SEC). 47 At a concentration of 0.6 mg/ml (43 μm) all were eentially free of aggregate and gave ingle, ymmetrical peak (Fig. 3A). HVLP351, HVLP342, HVLP335 and HVLP3103, were till monomer when teted at their highet concentration available, i.e., 0.89 mg/ml (64 μm), 1.0 mg/ml (72 μm), 4.9 mg/ml (352 μm), and 5.9 mg/ml (430 μm), repectively, although light tailing wa oberved for the HVLP335 monomeric peak at 5.9 mg/ml, uggeting interaction with the column matrix. The apparent molecular mae (M app ) of, calculated from their elution volume (Fig. S1), ranged from 6.9 kda to 24.3 kda, with a mean M app ± SEM of 13.7 ± 2.2 kda and a M for ± SEM of 13.8 ± 0.04 kda. Variation in M app for non-aggregating with imilar formula molecular mae (M for ) ha been reported previouly. 32 Such variation wa alo oberved in the cae of highly non-aggregating and may have been the reult of weak tranient interaction with the column material or monomer/dimer equilibria. 53 The non-aggregating tatu of wa confirmed by a urface plamon reonance (SPR) aay baed on the Ni 2+ -Hi 6 tag interaction that involved flowing Hi-tagged over Ni 2+ -immobilized enorchip urface. 46 Similar to previou reult obtained with monomeric, for the two diociation phae window teted, all the C-terminally Hi 6 -tagged gave k off that are very imilar to thoe of the monomeric H control, but dratically fater than thoe for the dimeric control with two C-terminal Hi 6 tag, confirming the monomeric tatu of the (Fig. 3B). Thi concluion wa accurately endored by the multiangle light cattering (MALS) experiment, which howed that the had M MALS (molecular mae determined by MALS) that were very imilar to their M for (Table 1). Protein L binding of human A anticipated, all elected bound to protein L in SPR analyi (Fig. S2A; Table 1). The K D of binding to protein L were in the μm range, with HVLP324 and HVLP342, which belong to 1 ubgroup, howing additional maller K D of 0.07 μm and 0.04 μm, repectively, when Biacore analye were performed at a low concentration range (1 10 nm) (Table 1). The etimated toichiometry of binding, :protein L, were 7 for 1 ubgroup member, 3 for all 3 ubgroup member except for HVLP351 which wa 2, and 1 for HVLP389, a V λ 1 member. T m of human To ae the thermotability of the, T m of were determined baed on ellipticity data auming a two-tate ytem, which i in agreement with the oberved denaturation curve correponding to a harp tranition into denaturation (Fig. 4A). mab 221

4 Figure 2. Structure of a et of human choen for detailed biophyical analye. (A) Amino acid equence of eight human and their Cy mutant verion. The amino acid poition (48 and 64) where mutation to cyteine reidue were made are marked. (B) Homology tructure of wild-type (left) and Cy mutant (right) of HVLP324. The native and engineered non-canonical diulfide linkage are hown a light grey and dark grey phere, repectively. Protein homology tructure were obtained uing the Geno3D automatic modeling tool ( The figure were drawn with PyMOL ( and Diulfide by Deign (verion 1.20) freeware. 80 T m, taken at midpoint of the igmoidal denaturation curve of molar ellipticity change (Δθ) v. temperature ( C), were in the range of C (median T m = 61.9 C) (Table 1). The lowet T m wa that of HVLP389, which i the only of λ family among the eight, and the highet T m wa that of HVLP325, which howed light aggregation (Fig. 3A; Table 1). The T m of eight non-aggregating (HVHP428, HVHP413, HVHP414, HVHP421, HVHP429, HVHP44, HVHP420, and HVHP419) iolated previouly in the ame manner a 48 were alo determined for comparion. The T m range wa C and the median T m wa 57.6 C; the T m difference between and were not ignificant (Mann-Whitney tet, two-tailed; P = ) (Fig. 4B and D). Thermal refolding efficiency of human The ability of the human to refold following thermal denaturation wa aeed by determining their thermal refolding efficiencie (TRE), which i calculated a the ratio of the K D for the binding of the native to protein L (K D n) to the K D for the binding of the heat-denatured/cooled (refolded) to protein L (K D ref). 48,54 K D were determined by SPR (Fig. 5; Fig. S3; Table 1). Figure 5A and B compare the protein L binding enorgram profile for HVLP335 and HVLP325, repectively, in native and refolded tate at 20 μm concentration. It can be een that for HVLP335, with near perfect refolding (95%) (Table 1), the enorgram for the native and refolded pecie are almot uperimpoable. In contrat, for HVLP325 with 222 mab Volume 6 Iue 1

5 Table 1. Biophyical characteritic of Subgroup M for (kda) HVLP HVLP324S HVLP HVLP325S HVLP HVLP335S HVLP HVLP342S HVLP HVLP351S HVLP HVLP364S h HVLP HVLP3103S HVLP V λ 1 HVLP389S M MALS (kda) a ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± 0.68 Expreion yield (mg) b 2.5, 7 0.5, 1.1 K D (µm) :protein L c T m ( C) ΔT m ( C) TRE (%) d 4 µm 20 µm GI proteae reitance (%) e Trypin Chymotrypin Pepin 0.2, 7: f , 7: ND 0.06 g ND g f 0.5, : , : ND g ND g : : ND g ND g , , , 7: f , 7: ND 0.05 g ND g f 1.2, : , : ND g ND g , : ND g ND g 72.3 ND g ND g , : : ND g ND g , : : ND g ND g a Mean ± SEM; b Expreion yield value are per liter of bacterial culture. Two expreion yield value correpond to two independent expreion trial; c Stoichiometry of -protein L binding; d Thermal refolding efficiency at 4 and 20 µm concentration, repectively; e Percentage proteolytic reitance value are at proteae concentration of 10 µg/ml (ee alo Fig. 6E); f Smaller K D value correpond to the binding of HVLP324, HVLP324S, HVLP342, and HVLP342S to the high affinity ite on protein L; g ND, not determined; h HVLP364S additionally gave a ignificant econd peak (Fig. 3A) with a maller eluion volume and a M MALS of ± 0.47 kda. 65% TRE (Table 1), a ignificant drop in binding i oberved for refolded pecie compared with the native pecie when enorgram at the ame concentration are compared. At 4 μm concentration, TRE are in the range of %, except for HVLP351, which i lightly lower at 87%, indicating a near perfect refolding for (median = 92%) (Fig. 5C). At 20 μm concentration, the TRE ignificantly decreae, with having a TRE range of 65 95% and a TRE median dropping to 84.5% (Wilcoxon matched-pair igned rank tet, two-tailed; P = ) (Fig. 5C). However, for four (HVLP335, HVLP364, HVLP389, HVLP3103), TRE value till remain above 90%, and for three of the four, TRE do not change when concentration are increaed from 4 μm to 20 μm. For comparion, the TRE of the eight non-aggregating at 4 μm and 20 μm concentration are alo included (Fig. 5C; Fig. S4). Two of the eight (HVHP428 and HVHP413) howed low TRE of 48% and 7% at 4 μm, with the remaining 6 (HVHP414, HVHP421, HVHP429, HVHM44, HVHP420, HVHP419) having TRE of 90 98% (median = 92%). A with, the TRE of at 20 μm concentration decreaed ignificantly (median = 78.5%, Wilcoxon matched-pair igned rank tet, two-tailed; P = ), with HVHP428 and HVHP413 failing to refold (TRE = 2% and 1%, repectively). Statitical analyi of TRE revealed that were comparable to in term of reveribility of thermal unfolding [Mann-Whitney tet, two-tailed; P = (4 μm), P = (20 μm)]. Moreover, mab 223

6 Figure 3. Size-excluion chromatography and SPR analye of wild-type and mutant. (A) Superdex 75 ize-excluion chromatogram of with monomeric peak marked with arrowhead. For HVLP364S, the dimeric peak ituated to the left of the monomeric peak i viible. (B) SPR analyi of binding to a Ni 2+ -NTA enorchip. A llama H Monomer (A ) and a Dimer 81 were ued a control. Meaurement were taken at two diociation phae window ( and ) and the mean value obtained from two independent trial performed in duplicate were recorded in the table. SPR experiment were performed with SEC-purified. the drop in TRE value a a function of increaed concentration of and are ignificant and expected a previouly reported. 55 No ignificant correlation between TRE and T m wa found. Proteae reitance of human In addition to thermotability, e.g., T m, proteae reitance i alo a meaure of protein tability. To determine proteae reitance, were treated with major gatrointetinal (GI) proteae, trypin, chymotrypin, and pepin, at variou proteae concentration (Fig. 6; Table 1). A expected, a gradual decreae in proteae reitance of wa oberved a a function of proteae concentration for all three proteae (Fig. 6A, B, and C). At the highet trypin concentration (20 μg/ml), the proteae reitance of wa in the range of 0 75% with a median reitance of 16% (Fig. 6A). The, however, demontrate higher reitance to chymotrypin (Fig. 6B). For example, the demontrated a median reitance of 75% at a chymotrypin concentration of 10 μg/ml compared with 33% for trypin at the ame concentration; at a higher chymotrypin concentration of 20 μg/ ml, had a proteae reitance range and median of % and 67.5%, repectively. howed the leat reitance to pepin (Fig. 6C). At 1 μg/ml pepin concentration, two of the (HVLP364 and HVLP389) were digeted almot completely with 8% and 2% pepin reitance, repectively, and at 10 μg/ ml pepin concentration, the reitance of all decreaed to below 34% (median reitance = 1.5%) (Table 1). At 20 μg/ ml pepin concentration, the pepin reitance range and median were 0 23% and 0%, repectively. We compared to the eight non-aggregating in term of reitance to the three proteae at 10 μg/ml proteae concentration (Fig. 6D). We found that there wa no ignificant difference between and with repect to reitance to trypin (Mann-Whitney tet, two-tailed; P = ). were ignificantly more reitant to chymotrypin than (Mann- Whitney tet, two-tailed; P = ), while the oppoite wa true with repect to reitance to pepin (Mann-Whitney tet, two-tailed; P = ). Theoretical number of proteae cleavage ite were determined by a proteae digetion prediction webware ( peptide_cutter). It wa found that had ignificantly higher number of proteae cleavage ite than for all three proteae [trypin ite median: 8.5 and 10.5 for and, repectively, (Mann-Whitney tet, two-tailed; P = ); chymotrypin ite median: 10 and 13.5 for and, repectively, (Mann-Whitney tet, two-tailed; P = ); pepin ite median: 30 and 39.5 for and, repectively [Mann-Whitney tet, two-tailed; P = )]. Diulfide linkage engineering of human To improve the tability of human, we created 8 mutant (HVLP324S, HVLP325S, HVLP335S, HVLP342S, HVLP351S, HVLP364S, HVLP389S, and HVLP3103S) with a pair of Cy ubtitution at amino acid poition 48 and 64 (Fig. 2A). All were expreed well in E. coli, albeit with lower yield compared with wild-type, with expreion yield ranging from 1.1 mg/l of bacterial culture in haker flak for HVLP324S to ~11 mg/l for HVLP342S (Table 1). To determine if the engineered Cy pair formed the deired diulfide linkage in the mutant, ma pectrometry (MS) wa performed by analyzing tryptic diget of mutant. The MS analye revealed that the diulfide linkage wa formed a intended in all mutant (Fig. S5; Table 2). The diulfidelinked peptide ion appeared prominent in the urvey of LC-MS chromatogram with tryptic peptide of the mutant. The expected diulfide-linked peptide equence correponding to each mutant were confirmed by manual de novo equencing. When there wa only one diulfide linkage between two peptide, the exact diulfide linkage poition wa confirmed by an almot complete diulfide-linked y fragment ion erie from one peptide with the other peptide attached a a modification via a diulfide bond that remain intact under colliion-induced diociation 224 mab Volume 6 Iue 1

7 Figure 4. Thermotability analyi of antibody domain. Thermal unfolding curve of (A) wild-type, (B), and (C) mutant. T m were calculated and incorporated into Figure 4D and Table 1. The non-aggregating are decribed in ref. 48. (D) Graph comparing the T m of wild-type to thoe of and mutant. (CID), 47,49,56 e.g., the diulfide linked peptide ATLSCR (P1) and GSGTLFTLTISSLEPEDSAVYFCQQR (P2) of HVLP325S (Table 2; MS 2 data not hown). When there were three peptide linked by two diulfide bond, the y fragment ion containing the linkage cloe to the N-terminal of two peptide wa difficult to oberve. Neverthele, an almot complete y ion erie of each peptide wa oberved. For example, a prominent ion at m/z (6+) from HVLP324S tryptic diulfide-linked peptide LLCFAASTLQSGVPSR (P1), FSCSGSGTDFTLTISNLQPEDFATYYCQQSYSTPR (P2) and VTITCR (P3) wa oberved from the urvey of LC-MS chromatogram (Fig. S5B (top panel); Table 2). Informative y fragment ion were oberved from P2 with P3 a a modification via a diulfide bond, and an almot complete y ion erie of P1 wa oberved a well (Fig. S5B, top panel). To further confirm the above diulfide bond formation in the mutant HVLP324S, the electron tranfer diociation (ETD)-MS 2 pectrum of the peptide ion [M + 5H] 5+ at m/z (5+) from the ame diulfide-linked peptide of HVLP324S wa acquired (Fig. S5B, middle panel). The mot abundant charge-reduced ETD fragment ion [M + 5H] 4+ at m/z (4+) wa elected for CID to obtain the ETD-CID-MS 3 pectrum of the m/z (5+) ion (Fig. S5B, bottom panel). The intact P1, P2, and P3 ion at m/z (1+), (1+), and (2+), repectively, were all oberved at relatively high abundance upon diociation of the diulfide linkage of the three linked peptide by ETD. Tryptic peptide linked by the engineered diulfide bond were poitively identified for all mutant. Thee fragment are recorded in Table 2. Aggregation tatu of diulfide linkage-engineered human Next, we aimed to determine if the engineered diulfide linkage compromied the non-aggregation tatu of. To thi end, we aeed the mutant by Superdex 75 SEC. Except for HVLP364S, which eemingly formed dimeric aggregate at ~17%, the remaining even mutant were monomeric (Fig. 3A), indicating that imilar to wild-type, mutant with the extra diulfide linkage were aggregation-reitant. Moreover, the light aggregation oberved in HVLP325 diappeared in the mutant verion, HVLP325S. The M app of mutant, calculated from their elution volume, were imilar to thoe of wild-type verion, ranging from 4.9 kda to 23.5 kda with a mean M app ± SEM of 11.4 ± 2.3 kda compared with 13.7 ± 2.2 kda for the wild-type. The SEC reult were further confirmed by the MALS data, which howed that the were indeed monomeric a their experimental M MALS were very cloe to their theoretical M for (Table 1). Furthermore, the minor HVLP364S peak identified a correponding to a dimer in mab 225

8 Figure 5. Thermal refolding efficiency determination of human and by SPR. (A), (B) Repreentative SPR enorgram for the binding of native and heat-denatured/cooled (refolded) human to immobilized protein L. Data are from TRE experiment performed at 20 µm concentration. concentration ued to contruct each enorgram et wa 12.5, 18.8, 25, 37.5, 50, 75, and 100 nm for HVLP325 and 12.5, 20, 25, 30, 38, 50, and 75 nm for HVLP335. See Figure S3 and S4 for SPR data for all and. The K D n and K D ref determined from Native and Refolded enorgram pair were ued to determine TRE in (C). (C) TRE of wild-type and obtained under 4 µm and 20 µm domain concentration condition in refolding experiment. Line connect TRE for the ame clone. SEC had indeed the M MALS for a dimeric : ± 0.47 kda (v. the expected M for of kda). Probing conformational change in diulfide linkage-engineered human by protein L binding To probe any poible tructural change brought about by the non-canonical diulfide linkage in mutant, the binding of mutant to protein L wa quantified by SPR. We found that all mutant, with the exception of HVLP324S, HVLP342S, and HVLP351S, bound to protein L with almot the ame K D (and k on and k off ) a their wild-type counterpart (Fig. S2; Table 1), indicating that, for the majority of, there were no tructural change due to the engineered diulfide linkage, or if there were any, they were too ubtle to be ened by protein L. HVLP324S, HVLP342S and HVLP351S howed 2- to 3-fold affinity increae toward protein L low affinity binding ite compared with wild-type counterpart. In contrat, in all cae the toichiometry of :protein L binding remained unchanged between the wild-type and correponding mutant. In the cae of HVLP364S, the K D eemed to be in the low micromolar range, but a reliable K D, and conequently a reliable toichiometry, could not be determined by SPR due to aggregate contamination in the ample. A homology tructure of HVLP324S ugget that the non-canonical diulfide linkage and protein L binding ite occupy ditinct location in mutant (Fig. 7). In contrat, for a with a imilar diulfide linkage mutation, the non-canonical diulfide linkage i very intimate with and imbedded within the protein A binding ite (Fig. 7). Thermal tability of diulfide linkage-engineered human To determine the effect of the non-canonical diulfide linkage on the thermal tability of, the T m of mutant were determined and compared with thoe for wild-type. The T m were in the range of C (median T m = 72.9 C) compared with C (median T m = 61.9 C) for wild-type (Fig. 4C and D; Table 1). The thermotability improvement were ignificant, reflecting a T m increae (ΔT m ) range and median of C and 12.4 C, repectively (Fig. 4D; Table 1) (Wilcoxon matched-pair igned rank tet, two-tailed; P = ). Although the reult indicated that the engineered diulfide linkage tabilized the regardle of their germline ubtype (κ or λ), the thermotability improvement were more pronounced for the κ3 and λ1 ubgroup member compared with the κ1 ubgroup member. GI proteae reitance of diulfide linkage-engineered human Previouly, H were hown to have acquired proteae reitance with the addition of a imilar non-canonical diulfide linkage. 49 We therefore invetigated the effect of the non-canonical diulfide linkage on the reitance of to trypin, chymotrypin, and pepin. Mutant were digeted with varying concentration of proteae under the ame digetion condition a for wild-type (Fig. 6). We oberved that there wa no ignificant overall difference between wild-type and mutant with repect to reitance to trypin or chymotrypin (Fig. 6A, 6B, and 6E; Wilcoxon matched-pair igned rank tet, two-tailed; P = , , , and for trypin at 1 μg/ml, 5 μg/ml, 10 μg/ml, and 20 μg/ml, repectively; P = , , , and for chymotrypin at 1 μg/ml, 5 μg/ml, 10 μg/ml, and 20 μg/ml, repectively). Mutant, however, howed improved reitance to pepin. The pepin reitance median of wild-type were 73% and 1.5% at enzyme concentration of 1 μg/ml and 10 μg/ml, repectively, and decreaed to 0% at the concentration of 20 μg/ ml (Fig. 6C). In contrat, mutant had pepin reitance median of 100%, 54%, 39.5%, and 25.9% at enzyme concentration of 1, 10, 20, and 50 μg/ml, repectively. The pepin reitance improvement were ignificant at 1 and 10 μg/ml enzyme concentration (Fig. 6E; Wilcoxon matched-pair igned rank tet, two-tailed; P = and , repectively), but not o at 20 and 50 μg/ml enzyme concentration (p = for both concentration condition). Moreover, the correlation between proteae reitance and T m wa explored (Fig. 6F). It wa found that in general, with higher T m had higher reitance to pepin 226 mab Volume 6 Iue 1

9 (Pearon correlation, ; P = , r 2 = , at 1 μg/ml enzyme concentration; Pearon correlation, ; P = , r 2 = , at 10 μg/ ml enzyme concentration; Pearon correlation, ; P = , r 2 = , at 20 μg/ml enzyme concentration; Pearon correlation, ; P = , r 2 = , at 50 μg/ ml enzyme concentration). No ignificant correlation wa oberved in the cae of trypin or chymotrypin (data not hown). Dicuion Numerou publication in the pat two decade have firmly etablihed the uitability of dab a therapeutic and diagnotic agent. Human and dab, in particular, have been purued a therapeutic due to their expected lower immunogenicity in patient compared with other clae of dab uch a camelid H and hark V NAR. 4,5,32 A number of tudie have highlighted a bona fide affinity reagent, and a few, in particular, have pointed to an inherent property of a being more aggregation reitant than. 23,32-34,57-61 Thu, from the aggregation point of view, human may be preferable over human a immunotherapeutic. In thi tudy, we et out to obtain a deeper undertanding of with repect to a number of biophyical propertie, including their aggregation tendencie. Human domain are known for their general tendency to aggregate. Previouly, a phage election method wa ued to Figure 6. GI proteae reitance profile for human and. (A), (B), (C) Trypin, chymotrypin, and pepin reitance (re.) of human (open circle) and their correponding Cy mutant (cloed circle). (D) Graph comparing to in term of reitance to trypin, chymotrypin, and pepin at 10 µg/ml proteae concentration. Horizontal line in graph (A) (D) repreent median. (E) Graph howing trypin, chymotrypin, and pepin reitance of human (open circle) and their correponding Cy mutant (cloed circle) in pair-wie manner at 10 µg/ml proteae concentration (ee alo Table 1). Line connect proteae reitance value for each to that for it correponding mutant verion. The p value in graph (D) and (E) were obtained by the Mann-Whitney tet (two-tailed) and Wilcoxon matched-pair igned rank tet (two-tailed), repectively, uing GraphPad Prim (GraphPad Software). (F) A correlation graph of pepin reitance v. T m (Pearon correlation, ; p = , r 2 = ). Data are from digetion experiment performed at 10 µg/ml pepin concentration. Open circle, wild-type ; cloed circle, mutant. obtain excluively non-aggregating human domain from a naïve human phage diplay library that wa propagated a plaque. 48 The library, with a ize of tranformant, wa panned againt the B cell uper-antigen protein A, and equencing of more than 110 clone with complete open reading frame yielded a total of 15 non-aggregating. By applying mab 227

10 Figure 7. Homology tructure of HVLP324S and huvham302s, comparing the poitioning of protein L binding ite to protein A binding ite relative to the engineered non-canonical diulfide linkage (blue phere) for the and, repectively. huvham302s i a previouly decribed human that lot it protein A binding activity by 3.5-fold upon the introduction of a non-canonical diulfide linkage at poition 49 and Red phere repreent the native, canonical diulfide linkage, pink- and green-colored region CDR and FR, repectively. Amino acid reidue forming protein L binding ite 1 and 2 (top, left panel) and protein A binding ite (top, right panel) are hown. The binding ite reidue were identified baed on the publihed crytal tructure of the complex between a human antibody Fab fragment (2A2, κ1 ubtype, 3 family) and a ingle Peptotreptococcu magnu protein L domain 64 ( ) and between Fab 2A2 and domain D of Staphylococcu aureu protein A 82 ( ). Middle and bottom panel how ide and top view of the protein L and protein A binding ite in dot and tick preentation. Protein homology tructure were obtained a decribed in the Figure 2 legend. The figure were drawn with PyMOL ( and manipulated uing Adobe Photohop CS2 oftware. eentially the ame election approach, we obtained a total of 32 unique equence out of 34 creened clone from a 200-fold maller-ized library. Given that the election method ha been hown to elect only for non-aggregating domain, thi high yield iolation of implie that human are more frequently aggregation reitant than human, which i conitent with previou finding. 32,33 Thi i further upported by the fact that our ubet of randomly elected protein L binding repreenting different germline origin were indeed non-aggregating a hown by ize-excluion chromatography, hexa-hitidine capture SPR and multiangle light cattering experiment. Thi alo confirm the power of the aforementioned election approach for the iolation of non-aggregating protein. However, an intrinic ability of protein L to creen out tructurally compromied, aggregating domain in favor of non-aggregating protein L binder during panning experiment i a poibility that may have contributed to the trong elective power of the approach. It i not urpriing that from a library coniting motly of V λ cla domain, the vat majority of the protein L binding iolated (31 out of 32 unique equence) were of type ( 3 and 1 ubgroup) with only one being of a V λ type (HVLP389). Previou tudie have hown that protein L predominantly bind to the type domain, pecifically to thoe in the ubgroup 1, 3, and 4, with a paucity of binding to λ cla. 40,41,62-66 Given the elective nature of our approach for table (non-aggregating) domain, the predominance of 3 ubgroup type (24/31) followed next by 1 ubgroup type (7/31), and the abence of any 4 ubgroup type, in the pool of elected binder may be a reflection of the relative tability of. Previouly, it wa hown that of the four domain repreenting the conenu equence of human ubgroup 1 4, the 3 wa the mot thermodynamically table followed by the 1, with the 4 appearing to be the leat table of the three. 33 Alo, unlike the firt two, which were monomeric, the 4 formed dimer, an indication of it aggregation tendency. 33 In fact, a bleak election for the V λ cla in thi tudy may not have been jut the reult of their general lack of binding to protein L, but alo the reult of their lower tability compared with cla. 33 Conitent with thi i the fact that our lone λ cla binder belong to the V λ 1 ubgroup, a ubgroup whoe one repreentative wa hown to be more thermodynamically table than two other repreenting ubgroup V λ 2 and V λ 3. It i alo poible, however, that the relative proportion of 3 and 1 binder may imply reflect their relative proportion in the original, unelected library. Mutation with repect to germline equence were oberved for the pool of elected, but, in the abence of mutational tudie, it i very difficult to aign olubility role to mutation poition. Significant mutation at poition 96, e.g., mutation from a hydrophobic germline amino acid to a poitively-charged amino acid in the vat majority of, including 6 of the 8 non-aggregating, repreentative, ugget a olubility role for poition 96. Conitent with thi, previou tudie with immunoglobulin κ1 light chain have uggeted that while an aromatic or hydrophobic reidue at poition 96 enhance dimerization, a charged amino acid (Arg) at the ame poition reult in table light chain monomer. 67 It wa explained that Arg-Arg charge repulion at poition 96 of monomer would interfere with dimer formation. 67 Further mutational tudie are requireto 228 mab Volume 6 Iue 1

11 Table 2. Diulfide linkage determination of by MS analye V Tryptic peptide a M for ΔM c L (Da) (Da) (Da) HVLP324S VTITCR...LLCFAASTLQSGVPSR... FSCSGSGTDFTLTISNLQPEDFATYYCQQSYSTPR b b b HVLP325S ATLSCR...GSGTLFTLTISSLEPEDSAVYFCQQR LLCFDTSNR...FSCR b b 0.03 b M exp HVLP335S HVLP342S HVLP351S HVLP364S LLCYGTSNR...FSCSGSGTHFTLTINR b b b ATLSCR...LEPGDFAVYYCQQYGSSPR VTITCR...LCYGASSLQGGVPSR...FSCSGSGTEFTLTI SGLQPEDFATYYCLQHHTYPR b b 0.05 b ATLSCR...LLCYDASNR... FSCSGSGTDFTLTISSLEPE DFAVYYCQQR b b 0.26 b LLCYGASSR...FSCSGSGTDFTLTISR b b b ATFSCR...LEPEDFAVYYCQQYDTSPR HVLP389S HVLP3103S LLCYGNDK...FSCSK b b 0.06 b VTISCSGSSYNIGENSVSWYQQLPGTAPK SGTSAT LGITGLQTGDEADYYCGTWDSNLR ATLSCR LLCYGASTR FSCSGSGTDFTLTISSLQV EDVAVYYCQQYYTTPK b b 0.04 b a Major tryptic peptide containing diulfide linkage are hown, with connecting cyteine reidue ingle or double underlined (native or engineered Cy (C), repectively) and boldfaced (ee Fig. S5 for experimental detail). The triple dot between peptide denote equence dicontinuity, which wa caued by the lo of equence after trypin digetion; the dicontinuing peptide, however, are held together by diulfide linkage(); b The very cloe match between M for (formula molecular ma) and M exp (experimental molecular ma) indicate the preence of the Cy48-Cy64 diulfide linkage. c ΔM = M for - M exp. In addition, the diulfide linkage were confirmed by de novo equencing the CID or ETD pectra of the diulfide linked peptide (e.g., ee Fig. S5B). determine if the preence of a poitively-charged amino acid at poition 96 lead to that are more aggregation reitant, and if o, whether a negatively-charged amino acid would have the ame effect. Other tudie with immunoglobulin domain howed that ubtitution with negatively-charged amino acid, Ap ubtitution in particular, were more effective than poitively-charged ubtitution in increaing the aggregation reitance of. 68 We alo oberved that at FR4 poition 105, 106, and 107, intead of the typical Ap/Glu, Ile, and Ly, the majority of the κ cla had Thr, Val, and Leu, amino acid, repectively, which are characteritic of λ cla. Whether the ubtitution have a role in improving the biophyical propertie of the κ cla remain to be een. The importance of FR4 reidue in improving the aggregation reitance of immunoglobulin variable domain ( H and ) ha been uggeted. 69,70 For example, H, which are known for their high aggregation reitance, have a highly conerved 105Q mutation compared with the aggregation prone. Similarly, with the 105Q mutation have been hown to have improved aggregation reitance compared with a correponding wild-type. Alo, the role of J egment, which code for the FR4 amino acid, in increaing the thermal tability and non-aggregation of H ha been hown by other. 69 SPR binding experiment on the eight repreentative nonaggregating confirmed their protein L binding activity. Of interet were the two 1 type binder, which unlike the 3 and V λ type that bound to protein L with imilar micromolar affinitie (1 3 μm), bound to protein L with low (0.2 μm and 0.6 μm) and high (0.04 μm and 0.07 μm) affinitie. High and low affinity binding of human Fab and light chain of 1- ubgroup type to 2 ditinct ite on ingle Ig binding domain of protein L have been reported previouly. 63,64 Here a differential :protein L toichiometry wa alo oberved, with the λ type having a 1:1 binding toichiometry, the 3 having a 3:1 binding toichiometry in the majority of cae, and 1 having a 7:1 binding toichiometry. Previou SPR binding tudie with 5 Ig-binding domain of a protein L howed that while all 5 domain bound to a human light chain of 1 ubgroup, only 3 bound to a human light chain of 3 ubgroup, upporting the higher oberved toichiometry for 1 binder compared with 3 binder and the 3:1 toichiometry found for the 3 binder. A binding toichiometry of 7:1 in the cae 1 ubgroup binder i plauible, given that our protein L conited of 4 Ig-binding domain with each Ig-binding domain having up to 2 binding ite that can imultaneouly engage with 2 ite on 1 ubgroup binder. 63,64,71 A higher affinity and toichiometry (higher avidity) in the cae of 1 type antibodie hould tranlate to their more enitive detection by protein L. The low affinity and lack of avidity a a reult of a 1:1 toichiometry, a hown here for the V λ type HVLP389, may be the reaon for the reported weak interaction between human Ig λ-light chain and protein L. 41,65,66 Thu, failing to detect a V λ -protein L interaction hould not be interpreted a the lack of protein L binding activity on the part of a V λ type antibody. It hould, however, be mentioned that the toichiometry value are etimate. If the urface i not fully active the theoretical R max for 1:1 binding cannot be mab 229

12 attained, reulting in underetimation of the binding toichiometry. Iothermal titration calorimetry may provide a more accurate mean of determining the binding toichiometrie. Conitent with being highly non-aggregating at concentration a high a 450 μm, the howed high reveribility of thermal unfolding at relatively high concentration. In thi repect, and with repect to T m, the performed a well a our et of non-aggregating. The oberved lack of correlation between T m and aggregation reitance, expreed in term of TRE, i conitent with previou finding that howed that aggregation reitant may not necearily have high T m or thermodynamic tability. 72 Conitent with thi finding i the fact that the with the highet T m from among the eight in thi tudy (HVLP325) wa alo the only one that howed ome degree of aggregation. The were different from with repect to GI proteae reitance pattern. That i, while were more reitant to chymotrypin, the were more o to pepin, with both and being comparable in term of reitance to trypin. The oberved proteae reitance data for only chymotrypin could be explained in term of the number of potential cleavage ite, i.e., had a ignificantly lower number of potential chymotrypin cleavage ite than. Even when only the more proteae acceible CDR equence were conidered in the calculation of theoretical number of cleavage ite, the obtained number did not fully explain the oberved difference between proteae reitance profile of and (data not hown). Previouly for H, increae in pepin reitance were correlated with increae in T m, but thi correlation cannot explain the better pepin reitance of here becaue both and had very imilar T m. 49 A dicued previouly, proteae enitivity i a function of a number of variable including the theoretical number of proteolytic ite, the location of proteolytic ite, and protein compactne and thermodynamic tability. 49,73,74 In an effort to further improve the biophyical propertie of, we introduced a pair of cyteine reidue at amino acid poition 48 and 64, hypotheizing that thi would lead to the formation of a diulfide linkage in the dab core. Previouly, it wa hown that the ubtitution for a pair of cyteine at patially equivalent poition in H and led to the formation of diulfide linkage in all domain teted, with ubequent improvement in thermotability and proteae reitance. 46,49-52 We too find here that all the with the added Cy pair have the intended diulfide linkage, a well a improved thermotability (T m ) and proteae reitance. We find that the increae in T m (ΔT m ) are relatively high ( C, median ΔT m : 12.4 C) compared with thoe obtained for a previouly reported et of H with imilar engineered diulfide linkage (ΔT m : 4 12 C, median ΔT m : 7 C). 49 Thi may, at leat partly, be due to the fact that T m were obtained under different aay condition and intrument etting. It cannot be excluded, however, that a noncanonical diulfide linkage may have been a better fit to the overall fold of, leading to their overall higher ΔT m gain. 49 Thi may alo explain the differential T m gain oberved among the mutant that were characterized under identical condition. We alo find that the thermotability gain due to the engineered diulfide linkage i more pronounced for the κ3 and λ1 member, compared with the κ1 member; however, general concluion hould await further experiment involving a tatitically appropriate ample ize. In term of proteae reitance, the non-canonical diulfide linkage led to increae in pepin reitance of, without compromiing their trypin or chymotrypin reitance. Thi i imilar to the reult obtained with a et of H with imilar non-canonical diulfide linkage. 49 A with the H, poitive correlation between pepin reitance and T m were alo oberved. The higher pepin reitance of the mutant may be due to the fact that they may have a more compact and thermodynamically table tructure equating to higher reitance to acid-induced unfolding under pepin digetion condition (ph = 2; pepin i more effective on denatured protein) compared with the wild-type without the non-canonical diulfide linkage. 49 Higher T m of mutant, uggeting their higher thermodynamic tabilitie, and the fact that H with imilar engineered diulfide linkage became reitant to unfolding at ph 2 and 37 C upport thi peculation. 49 Importantly, the introduction of the non-canonical diulfide linkage into doe not appear to compromie their aggregation reitance. Thi wa hown to be the cae for H and with imilar non-canonical diulfide linkage a well. 46,49 In the cae of, thi led to improvement in the aggregation reitance of mutant compared with the wildtype counterpart without the non-canonical diulfide linkage. The biophyical improvement gained through the introduction of the non-canonical diulfide linkage do come at the expene of expreion yield a mutant demontrated ignificantly lower expreion yield than wild-type in E. coli. Thi wa alo reported in the cae of H, which like the here, were expreed in E. coli. 49 Thu, the relatively lower expreion yield of Cy mutant domain compared with wild-type one may have to do with the limited capacity of E. coli in folding protein with higher diulfide linkage uch a the Cy mutant in thi tudy. Thi hould be reolved by expreing the mutant in eukaryotic microorganim, e.g., yeat or mammalian cell, with the capacity to fold complex protein uch a thoe with multiple diulfide linkage. The biophyical improvement alo come at the expene of undeirable conformational change for mutant, which were alo reported in the cae of H and. 46,49 The oberved differential proteae reitance profile between wild-type and correponding mutant, a well a change to protein L binding for ome of the mutant, upport thi concluion. However, for the majority of, the conformational change, a determined by binding meaurement of wild-type and Cy mutant againt protein L, are too ubtle to be ened by protein L, which bind to in a conformation-dependent manner. 41 Thi i in harp contrat to the reult obtained with our Cy mutant, 46 where tructural change a a reult of the introduction of non-canonical diulfide linkage were more eaily probed with protein A and led to up to 10-fold reduction in protein A binding of mutant. Such dicrepancy could be due to the fact that, while for the protein L binding ite i too far from the engineered diulfide linkage to be affected by it, for the protein A binding ite 230 mab Volume 6 Iue 1