Reducing Epitope Spread during Affinity Maturation of an Anti- GD2 Antibody

This information is current as Jian Hu, Xiaodong Huang, Chang-Chun Ling, David R. of September 27, 2021. Bundle and Nai-Kong V. Cheung J Immunol 2009; 183:5748-5755; Prepublished online 7 October 2009; doi: 10.4049/jimmunol.0901409

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2009 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

Reducing Epitope Spread during Affinity Maturation of an Anti-Ganglioside GD2 Antibody1

Jian Hu,* Xiaodong Huang,* Chang-Chun Ling,† David R. Bundle,‡ and Nai-Kong V. Cheung2*

Ab affinity maturation in vivo is always accompanied by negative selection to maintain Ag specificity. In contrast, in vitro affinity maturation can lead to epitope spread, resulting in loss of specificity. Anti-ganglioside-GD2 mAbs are clinically effective against neuroblastoma; pain and neuropathy are major side effects. We used structural relatives of GD2 to define epitope spread during in vitro affinity maturation of an anti-GD2 single-chain variable fragment (scFv) called 5F11-scFv. Clonal dominance identified by polyclonal sequencing was confirmed by analyzing individual clones. Affinity-matured mu- tations were introduced into scFv-streptavidin for functional studies. Without a negative selector, 19-fold affinity improve- ment (clone Q, where Q is the symbol for glutamine) was associated with strong cross-reactivity with GM2 and GD1b and

moderate cross-reactivity with GD3, resulting in positive immunohistochemical staining of all 13 non-neural normal human Downloaded from tissues, in contrast to none of 13 tissues with parental clone P. With GM2 as a negative selector, clone Y (where Y is the symbol for tyrosine) was generated with only weak cross-reactivity with GD1b, adrenal and thyroid glands, and no staining of other non-neural normal tissues. Even though there was only a 3-fold affinity improvement, clone Y showed significantly as (0.05 ؍ whereas clone Q was inferior (54% of clone P; p ,(0.04 ؍ higher tumor uptake over parental clone P (134%, p confirmed by tumor-to-normal tissue ratios across 16 organs (41% of clone P; p < 0.0001). Using the less efficient negative

selector GD3, a clone mixture (Q, V, and Y, where V is the symbol for valine) emerged. We conclude that epitope spread http://www.jimmunol.org/ during affinity maturation can be reduced by negative selection. Furthermore, efficiency of the negative selector depends on its cross-reactive affinity with the matured scFv. The Journal of Immunology, 2009, 183: 5748–5755.

ntibodies are accepted biologic tools and drugs for an the Ab created during in vitro affinity maturation could become array of human diseases. The affinity of an Ab can have cross-reactive with self-Ags. A a major impact on its therapeutic potential. When Ag provide a unique opportunity to study epitope density is low, high-affinity Ab is essential for successful targeting spreading because of their well-defined structural pathways. Di- (1). The affinity of Abs generated in vivo is typically no better than sialoganglioside GD2 (ganglioside names are abbreviated accord- by guest on September 27, 2021 0.1 nM (KD) (2, 3). To overcome this limitation, in vitro affinity ing to Svennerholm; Ref. 11) is a Ag highly expressed maturation is necessary, resulting in a 5,000-fold improvement in on tumors of neuroectodermal origin, including melanoma, neu- ϳ 3 affinity (final KD of 400 fM) (4). However, for therapeutic pur- roblastoma (NB), sarcoma, and small cell lung cancer. GD2 is an poses, affinity-matured Abs must maintain its high specificity. Loss attractive target for Ab-based (12). The anti-GD2 of specificity will diminish targeting efficiency and cause unfore- Abs 3F8 (mouse IgG3) and 14.18 have shown clinical potential seen toxicities. During normal in vivo affinity maturation of Ab- (13–15), and single-chain variable fragment (scFv) derived from producing B cells, binding to foreign Ags initiates their activation 5F11 (mouse IgM) has been genetically fused with streptavidin and differentiation. In contrast, binding to self-Ags leads to their (SA) and used successfully in multistep targeting to GD2-positive inactivation or deletion (5, 6), a process known as negative selec- human tumors (16). Specific anti-GD2 Abs bind to an epitope tion (7, 8). In pathologic conditions, e.g., autoimmune diseases, formed by the two sialic acids and N-acetylgalactosamine. Among lymphocytes that cross-react with self-Ags can become reacti- gangliosides there are at least four relatives with structural simi- vated. This may be due to the escape of lymphocytes from immune larities to GD2 (Fig. 1) (17). They are GM2, GD3, GD1b, and regulation or the emergence of new mutants from somatic hyper- GT2, characterized by one less sialic acid, one less N-acetylgalac- mutation (9, 10). One may expect that without negative selection, tosamine, one additional galactose, and one additional sialic acid, respectively. Being the nearest neighbors to GD2 in the synthesis pathway, they are potential cross-reactive Ags or epitopes during affinity maturation of anti-GD2 Abs. *Department of Pediatrics, Memorial Sloan-Kettering Cancer Center, New York, NY 10065; †Department of Chemistry, University of Calgary, Calgary, Alberta, This report is the first demonstration that affinity maturation can Canada; and ‡Department of Chemistry, University of Alberta, Edmonton, Al- yield high-affinity Abs with substantial epitope spread, resulting in berta, Canada poorer in vivo targeting. When the structural or epitope neighbors Received for publication May 5, 2009. Accepted for publication August 13, 2009. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 3 Abbreviations used in this paper: NB, neuroblastoma; CS, competitive selection; CSN, competitive selection in the presence of a negative selector; DOTA, 1,4,7,10- 1 This work was supported in part by National Institutes of Health Grant CA106450, tetraazacyclododecane-1,4,7,10-tetraacetic acid; GuHCl, guanidine chloride; IB50, Robert Steel Foundation, Hope Street Kids, William H. Goodwin, Alice Goodwin, concentration of competitor required to inhibit 50% binding; %ID/g, percentage of and the Commonwealth Foundation for Cancer Research, and the Experimental Re- injected dose of radiolabeled ligand per gram; IHC, immunohistochemistry; P, pa- search Center of Memorial Sloan-Kettering Cancer Center. rental (clone); SA, streptavidin; scFv, single chain variable fragment; NHS, 2 Address correspondence and reprint requests to Dr. Nai-Kong V. Cheung, Depart- N-hydroxysuccinimide. ment of Pediatrics, Memorial Sloan-Kettering Cancer Center, 1275 York Avenue, New York, NY 10065. E-mail address: [email protected] Copyright © 2009 by The American Association of Immunologists, Inc. 0022-1767/09/$2.00 www.jimmunol.org/cgi/doi/10.4049/jimmunol.0901409 The Journal of Immunology 5749

Construction of 5F11-scFv VH phage display library

The 5F11-scFv VH fragment was randomly mutated by PCR with a Gene- Morph II random mutagenesis kit (Stratagene) according to the manufac- turer’s manual. It was designed to introduce a single mutation into the

5F11-scFv VH gene by controlling the quantity of template and the number of PCR cycles. Library size was estimated by counting all variants with a single mutation, i.e., the number of possible deoxynucleosides in each po- ϫ ϭ sition multiplied by VH gene size, i.e., 4 396 1584. PCR products of mutated VH fragments were ligated into pHEN-1 containing the 5F11-scFv VL gene. Escherichia coli TG1 was used as the host. The 5F11-scFv phage library with a randomly mutated VH fragment contained a total of 1.44 ϫ 107 clones. By sequencing 20 random clones

from this library, the mutation rate for VH was estimated at 0.35 per clone. The ratio of the parental (P) clone to the mutated clone is 2942 ((1584/ 0.35) Ϫ 1584). Each mutant was estimated to have 3181 ((1.44 ϫ 107) ϫ (0.35/1584)) copies. Because a total of 1013 phagemids was used for se- lection, 2.2 ϫ 109 copies of each mutant were expected to be present. Even with stringent selection conditions, the probability of missing a promising candidate should be low. Affinity selection by competitive selection Downloaded from FIGURE 1. Structural pathways of gangliosides. In all affinity maturation experiments, 5F11-scFv-P-SA (P-SA, where P represents the parental clone) (16) or 3F8 (murine IgG3 specific for GD2) (20) was used as the competitor to eliminate low affinity clones. By Biacore affinity analysis, 3F8 could efficiently inhibit the binding of P-SA to GD2, are known, controlling specificity through negative selection can but not vice versa (data not shown). Selection without GM2-Sepharose as negative selector was done as avoid these undesirable clones. We demonstrated that the effi- follows. Approximately 1013 phagemids purified from a 5F11-scFv ciency of the negative selector (Ag used in negative selection) was phage display library were added into 14 ml of 2% BSA. After incu- http://www.jimmunol.org/ dependent on its binding affinity to these undesirable cross-reactive bation at room temperature for 1 h, 100 ␮l of GD2-Sepharose was clones. We further showed that the use of “polyclonal sequencing” added and incubated at room temperature for 2 h with shaking and then washed three times with PBS. During the first and second round selec- could facilitate single clone selection during affinity maturation. tion, P-SA was added to a final concentration of 100 ␮g/ml and 600 ␮g/ml and incubated for 24 and 72 h with shaking at 4°C, respectively. Materials and Methods During the third and fourth round selection, 3F8 was added to a final ␮ ␮ Conjugation of gangliosides to Sepharose matrix concentration of 600 g/ml and 750 g/ml, respectively, and incubated for 120 h with shaking at 4°C. After thorough washing with PBS for 10 GM3-Sepharose, GM2-Sepharose, GD3-Sepharose, and GD2-Sepharose times, phagemids binding to GD2 rescued from each round of selection were synthesized as previously described (18, 19). were amplified by transfection into E. coli TG1. When selection was

␮ by guest on September 27, 2021 Conjugation of GM OC H NH (GM3-Sepharose). N done with negative selector, 100 l of GM2-Sepharose was incubated 3 11 22 2 -hydroxysuccin- 13 imide (NHS)-activated Sepharose (Pharmacia) (3.4-ml dry volume) was with 10 phagemids in 14 ml of 2% BSA at room temperature for 1 h washed with 50 ml of ice-cold HCl (1 mM) and followed with 10 ml of to remove cross-reactive clones. This negative selection step was re- peated once for both first and second round selection. In separate ex- PBS (50 mM; pH 7.0). The wet gel and 4.04 mg of GM3OC11H22NH2 in 3.3 ml of PBS (50 mM; pH 7.0) reacted for 18 h at room temperature with periments, GD3-Sepharose, GM2-Sepharose, or GM3-Sepharose was shaking. The gel was filtered off and washed with 2 ml of PBS (50 mM; pH used as a negative selector under the same conditions. 7.0) and repeated four additional times. The combined filtrate was lyoph- ELISA for pooled phagemids ilized and unreacted GM3OC11H22NH2 was recovered by C18 Sep-Pak for the calculation of coupling efficiency. The recovered GM3OC11H22NH2 GD2 at 20 ng/well was coated onto a 96-well microtiter plate (Thermo was 1.86 mg and the coupling efficiency was ϳ54% (2.18 mg coupled; Electron). BSA (0.5%) in PBS was used as blocking reagent. Approxi- 2.72 ␮mol in 3.4 ml of dry gel). The functionalized gel was suspended in mately 2 ϫ 109 phagemids from each round of selection were mixed with 3.3 ml of PBS (50 mM; pH 7.0). Ethanolamine (0.2 ml) was added and the P-SA started at 650 ␮g/ml and added to GD2-coated wells for incubation mixture was gently shaken for 3 h. The gel was filtered off and successively at 37°C for 2 h. An HRP-conjugated anti-M13 mAb (GE Healthcare) was

washed with 3 ml of cold potassium biphthalate buffer (50 mM; pH 4.0), used to detect GD2 binding phagemids. IB50 (the concentration of com- and this step was repeated four additional times; the gel was then washed petitor required to inhibit 50% binding) was calculated using SigmaPlot 8.0 with 3 ml of PBS (50 mM; pH 8.0), and again this step was repeated four (Systat Software). additional times. After three washing cycles the gel was stored in 20% aqueous ethanol. The calculated trisaccharide incorporation density was Single clone selection by competitive ELISA or by washing with ␮ 0.80 mol of GM3OC11H22NH2/ml dry gel. 0.5 M guanidine hydrochloride (GuHCl) Conjugation of GM OC H NH (GM2-Sepharose). Tetrasaccharide 2 11 22 2 Rescued phagemids from each round of selection were transfected into E. GM OC H NH was conjugated to the NHS-activated Sepharose gel as 2 11 22 2 coli TG1. Three-milliliter aliquots were grown overnight without adding described above. GM OC H NH (5.02 mg) and 3.4 ml of gel (dry vol- 2 11 22 2 helper phage. The unseparated pool of plasmids (polyclonal plasmids) was ume) were coupled. The coupling efficiency was ϳ54% (2.70 ␮mol in 3.4 extracted for polyclonal sequencing. ml of dry gel). The calculated trisaccharide incorporation density was 0.80 After transfection of rescued phagemids, a sample was spread on a ␮mol of GM OC H NH /ml dry gel. 2 11 22 2 TYE plate containing 100 ␮l of ampicillin and 1% glucose to obtain Conjugation of GD3OC11H22NH2 (GD3-Sepharose). Tetrasaccharide single colonies. Single colonies were grown in 48-well plates (BD Bio- GD3OC11H22NH2 was conjugated to the NHS-activated Sepharose gel as sciences) with Luria-Bertani medium containing 100 ␮g/ml ampicillin. described above. GD3OC11H22NH2 (3.50 mg) and 3.4 ml of gel (dry vol- Isopropyl-␤-D-thiogalactoside was added to a final concentration of 1 ume) were coupled. The coupling efficiency was ϳ61% (1.98 ␮mol in 3.4 mM when OD600 reached 0.8–1.0 to express monoclonal scFv. Soluble ml of dry gel). The calculated trisaccharide incorporation density was 0.58 scFv supernatants were tested for high-affinity clones in ELISA using ␮ mol of GD3OC11H22NH2/ml dry gel. 3F8 as the competitor. The scFv supernatants were diluted 4-fold in ␮ Conjugation of GD2OC11H22NH2 (GD2-Sepharose). Pentasaccharide 0.5% BSA or 0.5% BSA containing a final concentration of 34.2 g/ml GD2OC11H22NH2 was conjugated to the NHS-activated Sepharose gel as 3F8 and then added to ELISA plates coated with 20 ng/well GD2. After described above. GD2OC11H22NH2 (3.92 mg) and 3.4 ml of gel (dry vol- incubation at 4°C for 3 days, bound scFv was detected with biotin- ume) were coupled. The coupling efficiency was ϳ70% (2.12 ␮mol in 3.4 conjugated mouse anti-c-Myc Ab and HRP/SA complex. Affinity of ml of dry gel). The calculated trisaccharide incorporation density was 0.62 scFv was estimated by its inhibition by 34.2 ␮g/ml 3F8; the percentage ␮ Ϫ ϩ mol of GD2OC11H22NH2/ml dry gel. of inhibition was expressed as (1 (OD450 of scFv 3F8 well)/(OD450 5750 REDUCING EPITOPE SPREAD DURING AFFINITY MATURATION

FIGURE 2. cDNA sequences of

5F11-scFv-VH parental clone, P, and its affinity-matured clones Q, V, and K (without negative selection), and clone Y (with negative selection). The under- lined sequences are the CDR 1, CDR2, and CDR3 regions, as labeled). Nucle- otide sequences for the amino acid mu- tations in CDR1 (from Glu (designated P for the parental clone) to Gln (Q), to Val (V), and to Lys (K)) and in CDR3 (from Phe (designated P for the paren- tal clone) to Tyr (Y)) are depicted in italics. Downloaded from http://www.jimmunol.org/

of scFv-only well)) ϫ 100. Any clone with at least 10% less inhibition at a retention time of 15.271 min with purity of 82, 63, and 65% for P-SA, by 3F8 than that of the parental clone (ϳ40%) was selected for gene Y-SA, and Q-SA, respectively. sequencing (Fig. 2). Alternatively, 0.5 M GuHCl was used as washing solution to select for Cross reactivity analysis by ELISA scFv clones with slow off rate. ScFv supernatants were diluted 4-fold in The a-series and b-series of gangliosides (GD3, GD2, GD1b, GT1b, GM3, 0.5% BSA and added to ELISA plates coated with 20 ng/well GD2. After GM2, GM1, and GD1a) were coated on ELISA plates at 20 ng/well. Gel- 2 h of incubation at 37°C, wells were washed with 0.5 M GuHCl three atin (0.01%) was used as the blocking reagent. P-SA, Y-SA, and Q-SA at times followed by PBS or washed with PBS alone. ScFv binding was the highest concentration of 3 ␮g/ml was diluted 1/3 in 0.5% BSA. HRP/ detected by mouse anti-c-Myc and HRP/goat anti-mouse Ab complex. In- by guest on September 27, 2021 Ϫ biotin was used as a detection agent. Wells without Ag were used as non- hibition of scFv binding by 0.5 M GuHCl was expressed as (1 (OD450 ϩ ϩ specific binding controls, and wells with Ag but no scFv-SA were used for of scFv 0.5M GuHCl washed well)/(OD450 of scFv PBS washed background subtraction. Cross-reactivity was expressed as the relative well)) ϫ 100. Any clone with at least 10% less inhibition by GuHCl than ϭ ϳ binding of other gangliosides to GD2, where cross reactivity that of the parental clone ( 20%) was selected for gene sequencing (OD Ϫ background)/(OD Ϫ background) ϫ 100%. (Fig. 2). 450(gangliosides) 450(GD2) Surface plasmon resonance analysis Conversion to scFv-SA format, scFv-SA expression, and purification The comparative affinity of 5F11-scFv-SAs was determined by BIA- CORE T100 (GE Healthcare). Gangliosides can be directly immobi- Because anti-GD2 scFv proteins were relatively unstable, we expressed the lized on sensor chip CM5 by hydrophobic interaction (22). The refer- newly identified clones as scFv-SA fusion proteins for functional studies. ence surface was immobilized with GM1. GM1 at 50 ␮g/ml in 20% ScFv fusion with SA (e.g., 5F11-scFv-SA or P-SA) was previously used to ethanol was passed through the reference surface with a flow rate of 5 test the functional properties of 5F11-scFv (16). A QuikChange II-E site- ␮l/min for 15 min and followed by five washes of 25 ␮lof20mM directed mutagenesis kit (Stratagene) was used to introduce identified mu- NaOH at a flow rate of 50 ␮l/min. To reduce mass transport as the tations into a plasmid containing the P-SA gene. E. coli XL-1 blue was limiting factor in Biacore measurements or multivalency during bind- used as host strain. The method of fermentation of P-SA in a BioFlow 3000 ing, the minimal amount of GD2 was used for immobilization on the fermentator (New Brunswick Scientific) was as previously described (16, Biacore sensor chip. Moreover, to ensure comparability of the reference 21), except that pH was controlled at 6.5 for both Y-SA and Q-SA (where channel (coated with GM1), the active channels were coated with equal Y and Q are the symbols for tyrosine and glutamine, respectively). The amount of GM1 mixed with GD2 at a ratio of 1:1 (final concentration purification of scFv-SA was based on the method described previously (16, of 50 ␮g/ml in 20% ethanol for GD2 and GM1, respectively) to ensure 21) with a minor modification. Briefly, scFv-SA was captured by 2-imi- an even distribution of the Ags. Immobilization was conducted for 15 nobiotin-Sepharose 4 fast flow (Affiland) and was eluted with 0.2 M so- min with a flow rate of 5 ␮l/min and followed by five washes of 25 ␮l dium acetate buffer (pH 4.6) containing 0.1 M NaCl. Eluates were directly of 20 mM NaOH at a flow rate of 50 ␮l/min. The GD2 coating con- ϫ neutralized with 0.5 M Tris buffer (pH 8.0), and then the product was centration was chosen to allow the analyte concentration (10 KD)to concentrated and changed to its storage buffer (30 mM Tris (pH 7.5) con- be ϳ100–200 resonance units. Affinity analysis was performed at 25°C taining 1 mM EDTA, 150 mM NaCl, and 5% sorbitol) by ultrafiltration anda30␮l/min flow rate in HBS-E buffer (0.01M HEPES (pH 7.4), 3 with a 100,000 m.w. cutoff membrane at 4°C. Protein concentration was mM EDTA, and 300 mM NaCl). Purified scFv-SA was diluted in ϭ calculated by A280/2.05. The A280 (1 mg/ml 2.05 arbitrary units) was HBS-E buffer at increasing concentrations (12.5, 25, 50, 100, and 200 calculated by Vector NTI Suit7 (Invitrogen). A TSK-GEL G3000SWxl nM). An association phase was run for 2 min followed by 3 min of size exclusion column (30 cm ϫ 7.8 mm; 5 ␮) (Tosoh Bioscience) was dissociation. At the end of each cycle, the surface was regenerated using ␮ used for HPLC analysis with 0.4M NaClO4 and 0.05 M NaH2PO4 (pH 6.0) 20 mM NaOH at a flow rate of 50 l/min over 1 min. BIACORE T-100 buffer as the mobile phase at a flow rate of 0.5 ml/min and analyzed at evaluation software (Version 2.0) was used for analysis. The biosensor 215 nM. curves obtained following injection of the samples over the active sur- Protein yield of P-SA was 200 mg/L; for Q-SA and Y-SA, it was 30–50 face were subtracted with the control curves obtained with the samples mg/L. After purification, the purified proteins were concentrated by ultra- injected over the reference surface before kinetics analysis. Based on ϳ filtration to 6 mg/ml in 30 mM Tris (pH 7.5) containing 1 mM EDTA, the association rate constant (kon, ka1) and the dissociation rate constant ϭ 150 mM NaCl, and 5% sorbitol. HPLC analysis exhibited one major peak (koff, kd1), the equilibrium constant (KD kd1/ka1) could be calculated. The Journal of Immunology 5751

FIGURE 3. Polyclonal sequencing to track the dominance of scFv populations. The color code for nucleotides is as follows: black, G; green, A; blue, C; and red, T. The size of each nucleotide peak reflects the abundance of the mutant scFv. A, Affinity maturation without negative selector; scFv with Glu (parental clone)3Gln (Q) (GAA3CAA in CDR1 (bold italics indicate nucleotide substitution) mutation became the dominant population starting from Downloaded from the third round (CS-3) through the fourth round of selection (CS-4). B, With GM2 as negative selector, scFv with Phe (parental clone)3Tyr (Y) (TTT3TAT in CDR3) became the dominant population from the third round (CSN-3) through the fourth round of selection (CSN-4). C–F, Affinity maturation was conducted with 20-fold fewer phagemids. C, With GM3 as negative selector, scFv with Glu (parental clone)3Gln (Q) (GAA3CAA) mutation became increasingly dominant from the third round (CSGM3-3) to the fourth round (CSGM3-4) to the sixth round of selection (CSGM3-6). D, With GM2 as negative selector, scFv with Phe (parental clone)3Tyr (Y) (TTT3TAT in CDR3) mutation became increasingly dominant from the fourth (GSGM2-4) to the fifth (CSGM2-5) to the sixth round of selection (CSGM2-6). E and F, With GD3 as the negative selector, scFv with mutations in CDR1 http://www.jimmunol.org/ (CAA (Q) and GTA (V) in E) and CDR3 (TAT (Y) in F) were all enriched. However, clone Q with Glu3Gln mutation (GAA3CAA in CDR1) rapidly became dominant from the fourth (GSGD3-4) to the fifth (CSGD3-5) to the sixth round of selection (CSGD3-6).

Immunohistochemistry (IHC) as the competing ligand for selection rounds no.1 and no.2 and 3F8 Stage 4 NB tumors and normal tissues were obtained at Memorial Sloan- for the subsequent rounds. The number of GD2-binding phagemids Kettering Cancer Center with institutional review board approval. Five- to recovered increased from the first through the fourth round (1.6 ϫ seven-micrometer sections of snap-frozen tissues were fixed in acetone for 108, 4.4 ϫ 108, 1.46 ϫ 109, and 9.23 ϫ 109 for CS-1, CS-2, CS-3, Ϫ 30 min at 20°C. Endogenous biotin-binding activity was blocked by and CS-4, respectively). With GM2-Sepharose as a negative se- by guest on September 27, 2021 sequential treatment with avidin and biotin (Vector avidin-biotin blocking lector, the number of GD2-binding phagemids recovered increased kit; Invitrogen) for 20 min each. Sections were incubated with 2 ␮g/ml ϫ 8 scFv-SA at room temperature for 1 h. Following washing, sections were only slightly from the first through the fourth round (2.1 10 , 8 8 8 incubated with HRP/biotin for 30 min at room temperature and subsequent 1.3 ϫ 10 , 3.1 ϫ 10 , and 7.1 ϫ 10 for CSN-1, CSN-2, CSN-3, incubation with 3,3Ј-diaminobenzidine for 5 min. H&E staining was also and CSN-4, respectively). performed. The IB50 of pooled phagemids to GD2 was used to estimate the In vivo biodistribution average phagemid affinity. As expected, after each round of selec- tion the enhancement of IB50 was consistent with an affinity gain. In vivo biodistribution was performed using a multistep targeting technique For CS, IB50 for pooled phagemids for each subsequent round was as previously described (16). Briefly, athymic nude mice with established Ͼ Ͼ ␮ human LAN-1 xenografts were fed biotin-free diet for 1 wk. Mice with 18, 589, 650, and 650 g/ml 3F8, respectively. As for CSN, ␮ measurable tumors (0.5- to 0.8-cm diameter at the time of Ab injection) IB50 of four rounds was 9, 20, 158, and 218 g/ml 3F8, respec- were injected i.v. with 900 ␮g of 5F11-SA. Twenty-four hours later, 450 tively. Using an IB of 6 ␮g per milliliter of the parental clone as ␮ 50 g of synthetic clearing agent (biotin-LC-NM-(Gal-NAc)16, where LC- the benchmark, CS-4 had a Ͼ108-fold affinity increase, whereas NM is amidocaproyl-N-methyl (16), was injected i.v. and followed 4 h later by 100 ␮Ci of 111In-DOTA-biotin i.v. (where DOTA is 1,4,7,10-tetraaza- CSN-4 had a 36-fold improvement. cyclododecane-1,4,7,10-tetraacetic acid). Mice were sacrificed 24 h after the injection of 111In-DOTA-biotin for biodistribution studies. The per- Polyclonal scFv sequencing to track clonal dominance centage of an injected dose (␮Ci) of radiolabeled ligand per gram of weight (%ID/g) of tissue was calculated for each mouse. The %ID/g of a tumor With each round of CS, certain clones became increasingly dominant measures the uptake intensity in the tumor and the ratio of %ID/g of a in the population. Polyclonal sequencing of these phagemid pools tumor vs the %ID/g of each normal tissue in each mouse represents tar- identified specific mutants (Fig. 3, A and B). These clones were later geting specificity in vivo. shown (see below) to have high affinity for GD2. By the fourth round, mutant clones (Table I) became fully dominant. Without a negative Results selector, CS yielded clone Q with a Glu to Gln (Q) mutation in the Affinity maturation with or without GM2 as a negative selector sixth amino acid of CDR1 (see Fig. 2, CS-4 graph in Fig. 3A, and ScFvs were selected on GD2-Sepharose in the presence of anti- Table I). In contrast, clone K, with a glutamic acid to lysine (K) GD2 competing Abs (competitors) either with negative selection mutation in CDR1, did not substantially expand from CS-3 to CS-4. (competitive selection in the presence of a negative selector To be certain that these polyclonal sequences were truly repre- (CSN)) or without negative selection (competitive selection (CS)). sentative of the clonal population, individual clones were isolated The first negative selector used was GM2-Sepharose. after selection with either 3F8 as the competitive inhibitor or 0.5 M 5F11-SA (P-SA) or 3F8 (both specific for GD2) were used as GuHCl as the washing buffer. We first chose clones with better competitors. CS in the presence of free Ag was not feasible be- affinity than that of the parental clone in the presence of a com- cause the glycolipid GD2 was not water soluble. We chose P-SA peting ligand (i.e., less inhibition by 3F8). At 38.4 ␮g/ml, 3F8 5752 REDUCING EPITOPE SPREAD DURING AFFINITY MATURATION

Table I. Affinity maturation of anti-GD2 5F11-scFv

Clone Selection Competing Anti-GD2 GM2 as Negative Location of Amino Acid Name Process Ligand Present Selector Mutation in VH change in VH P ϪϪ ϪϪϪ QCS ϩϪCDR1 Glu3Gln (Q) VCS ϩϪCDR1 Glu3Val (V) KCS ϩϪCDR1 Glu3Lys (K) Y CSN ϩϩCDR3 Phe3Tyr (Y)

inhibited 30–40% binding of 5F11-scFv (P-scFv). Of 1500 clones, values per Biacore analysis precluded reliable determination of the only 60 had Ͻ30% inhibition by 3F8. Upon sequencing these 60 cross-reactive affinity of Q-SA on GD3. clones, we consistently found mutations of the sixth amino acid, i.e., Glu, in the CDR1 H chain to Gln (Q), Lys (K), Val (V), and Gly (G) with frequencies of 81.6, 13.3, 3.3, and 1.7%, respectively (Fig. 2). In High-affinity clones matured without negative selection lost a subsequent experiment, 0.5 M GuHCl was used to eliminate low specificity for GD2 by ELISA affinity clones, which had reduced binding on ELISA. Washing thrice In vitro cross-reactivity was tested using both the a-series and b-series with 0.5 M GuHCl lowered ELISA binding of P-scFv by 20%. Fifty of gangliosides (Fig. 1), and results were summarized in Table II. Downloaded from of the 2400 clones with a Ͻ10% drop following 0.5M GuHCl wash Cross-reactivity with other gangliosides was defined as significant if were sequenced, and they all carried the GAA (Glu)3CAA (Gln) Q relative binding was Ͼ1% of binding to GD2. In contrast to P-SA, mutation. Thus, both polyclonal sequencing and monoclonal selection which had no cross-reactivity, Q-SA had strong cross-reactivity with pointed to a dominant candidate (i.e., clone Q) when selection was GM2 and GD1b and weak cross-reactivity with GD3 and GD1a, done without negative selection. whereas Y-SA had only weak cross-reactivity with GD1b. Cross-

In contrast, with GM2-Sepharose as a negative selector, the Y reactivity of Q-SA determined by ELISA was in agreement with the http://www.jimmunol.org/ clone with a TTT (Phe)3TAT (Tyr) mutation in CDR3 assumed affinity results obtained by Biacore (Table II). dominance after CSN-4 (Fig. 3B and Table I). In this case, no Q clone was detected. Moreover, the Y clone was not found in the sequencing data from CS-4, suggesting that they were competed High-affinity clone matured without negative selection lost its out by the high-affinity Q clone. tissue specificity by IHC P-SA, Y-SA, and Q-SA were tested for tissue specificity by IHC on High affinity of dominant clones confirmed by surface plasmon human NB tumors and normal human tissues. NB tumors are known resonance to express high levels of GD2. All 11 tumors were stained positive by

5F11-scFv was genetically fused to SA (e.g., 5F11-scFv-SA (P- P-SA, Y-SA, and Q-SA (Table III). Seventeen normal tissues were by guest on September 27, 2021 SA)) to make stable proteins to test their functional properties (16). also tested (Table III). Frontal lobe, pons, cerebellum, and spinal cord These purified scFv-SAs were analyzed by Biacore T100 for their all stained positive with both affinity-matured and parental clones as relative affinities to GD2 at concentrations of 12.5–200 nM. The expected, because GD2 is known to be present on neuronal tissues. affinities of Y-SA and Q-SA were superior by 3- and 19-fold, The remaining 13 normal tissues were all positive with Q-SA, in respectively, over P-SA (Table II). When tested against the struc- sharp contrast to complete negativity with P-SA. As for Y-SA, it tural neighbors of GD2, GM2 and GD1b, Q-SA showed higher showed only weak heterogeneous staining in thyroid and adrenal tis- affinity to GM2 than GD1b. The relatively low resonance unit sues, whereas the remaining 11 normal tissues were negative.

Table II. Affinity and cross-reactivity of scFv-SA on gangliosides

Clones P-SA Y-SA Q-SA

a Ϫ1 Ϫ1 ϫ 4 ϫ 5 ϫ 5 Affinity by SPR (GD2) Kon (M s ) 7.811 10 1.437 10 5.675 10 Ϫ1 ϫ Ϫ3 ϫ Ϫ4 ϫ Ϫ4 Koff (s ) 1.550 10 8.654 10 5.923 10 KD (nM) 19.8 6.02 1.04 Ϫ1 Ϫ1 ϫ 5 Affinity by SPR (GM2) Kon (M s ) ND ND 2.013 10 Ϫ1 ϫ Ϫ3 Koff (s ) ND ND 1.638 10 KD (nM) ND ND 8.14 Ϫ1 Ϫ1 ϫ 5 Affinity by SPR (GD1b) Kon (M s ) ND ND 1.595 10 Ϫ1 ϫ Ϫ3 Koff (s ) ND ND 1.807 10 KD (nM) ND ND 11.3 Cross-reactivity by ELISAb GD3/GD2 0.00% 0.31% 13.78% GD1b/GD2 0.36% 5.05% 25.74% GT1b/GD2 0.00% 0.14% 2.47% GM3/GD2 0.00% 0.14% 2.42% GM2/GD2 0.55% 0.71% 43.44% GM1/GD2 0.08% 0.00% 2.60% GD1a/GD2 0.00% 0.06% 4.45% Gelatin/GD2 0.32% 0.19% 1.09%

a Relative affinity was compared by surface plasmon resonance (Biacore T-100) using ganglioside (GD2, GM2, or GD1b) coated on CM5 chips. b Values of Ն1% were defined as cross-reactive; Ն15% was defined as strongly cross-reactive. The Journal of Immunology 5753

Table III. Tissue cross-reactivity by immunohistochemistrya Cross-reactive affinity of the negative selector determined the efficiency of negative selection b Organ P-SA Y-SA Q-SA We next tested the relationship between the efficiency of a negative Heart ϪϪϩ selector and its cross-reactive affinity with affinity-matured clones. ϪϪϩϩ ϭ Ileum GM2 had the highest cross-reactive affinity toward Q-SA (KD 8.14 kidney ϪϪϩ nM by Biacore; 43.44% cross-reactivity by ELISA). GD3 had mod- ϪϪϩ Liver erate cross-reactivity toward Q-SA (13.78% cross-reactivity by Lung ϪϪϩ Pancreas ϪϪϩϩ ELISA), whereas GM3 had minimal to no detectable cross-reactivity. Spleen ϪϪϩϩ We tested the individual efficiency of GM2-Sepharose, GD3-Sepha- Stomach ϪϪϩϩ rose, and GM3-Sepharose as negative selectors in affinity maturation ϪϪϩϩ Testes experiments labeled as CSGM2,CSGD3, and CSGM3, respectively Thyroid Ϫϩ/Ϫϩϩ Adrenal Ϫϩ/Ϫϩϩ (Fig. 3). In these experiments, we used 20-fold fewer starting phage- Sigmoid Colon ϪϪϩ/Ϫ mids. Polyclonal sequencing was used to follow the clonal evolution Skeletal Muscle ϪϪϩ during each of the consecutive selections. With GM2-Sepharose as a Pons ϩ ϩϩ ϩϩϩ negative selector (CSGM2), clone Y again became enriched by the ϩ Ϫ ϩϩ ϩϩϩ Cerebellum / sixth round of selection (CSGM2-6), and Q or K clones were never Spinal Cord ϩϩϩϩ Frontal Lobe ϩ ϩϩ ϩϩϩ detected (Fig. 3D). With GM3-Sepharose as a negative selector Stage 4 NB tumors (n ϭ 11) ϩϩ ϩϩ ϩϩ/ϩϩϩ (CSGM3), clone Q became dominant after the sixth round (CSGM3- Downloaded from a The minus sign (Ϫ) indicates negative. Positive was expressed as ϩ, ϩϩ, and 6), whereas clone K was soon competed out (Fig. 3C) as expected, ϩϩϩ according to intensity and homogeneity. Plus/minus (ϩ/Ϫ) heterogeneously because it cross-reacted strongly with GM3 (data not shown). With positive, i.e. some area was negative and some area was 1ϩ positive. The percentage GD3-Sepharose as negative selector (CSGD3), clone V was first de- positive and intensity of staining consistently ranked the clones as follows: Q-SA Ͼ Y-SA Ϸ P-SA. tected after the fourth round (CSGD3-4) (Fig. 3E), whereas clones Q b With clone Q, there was consistent diffuse staining of nuclei and cytoplasm in all and Y appeared after CSGD3-5 (Fig. 3, E and F). By CSGD3-6, tissues tested. ϳ40% population was represented by clone Q, ϳ30% by clone Y, ϳ20% by clone P, and ϳ10% by clone V (Fig. 3, E and F). The http://www.jimmunol.org/ reason why clone V appeared early on was because of its lower cross- High-affinity clone matured without negative selection lost its reactivity with GD3 compared with that of clone Q; it was finally tumor-targeting efficiency in vivo overtaken by clone Q, which had higher affinity to GD2. We conclude Athymic nude mice xenografted with human NB LAN-1 tumors that the lower affinity negative selectors (e.g., GD3) could not effi- were injected i.v. with either P-SA (n ϭ 10), Y-SA (n ϭ 5), or ciently remove all of the cross-reactive clones. The cross-reactive af- Q-SA (n ϭ 5). %ID/g of each scFv-SA for each organ, as well as finity of the negative selector appeared critical in determining the tumor to normal organ ratios, was compared in Fig. 4. Q-SA had efficiency of the negative selection step. In addition, when affinity Ϯ maturation was conducted using a 20-fold lower starting number of the lowest tumor uptake (2.93 0.50%ID/g) despite its 19-fold by guest on September 27, 2021 increase in affinity over the parental scFv-SA (P-SA, 3.99 Ϯ phagemids, clonal dominance was delayed by one to two rounds. 0.34% ID/g, p ϭ 0.05). It also had inferior tumor-to-normal organ ratios across the 16 organs tested (41 Ϯ 18% of clone P; p Ͻ 0.0001). In contrast, despite a mere 3-fold increase in affinity, Discussion Y-SA had significantly higher tumor uptake (5.29 Ϯ 0.54%ID/g; For healthy individuals, affinity improvement in vivo is part of the p ϭ 0.04). maturation of immune response and is always accompanied by

FIGURE 4. Efficiency of tumor targeting; %ID/g and tumor to organ ratio using clones P, Y and Q geneti- cally linked to SA in multistep target- ing. Athymic mice (n ϭ 5–10 per group) xenografted with NB LAN-1 were injected with a standard dose of 900 ␮g of scFv-SA, 450 ␮g of syn- thetic clearing agent, and 100␮Ci 111In DOTA-biotin as described pre- viously (16). Mice were sacrificed and their organ radioactivity was mea- sured to calculate the %ID/g in tumor and various organs. Tumor-to-organ ratio was calculated as follows: (%ID/g in tumor)/(%ID/g in organ). 5754 REDUCING EPITOPE SPREAD DURING AFFINITY MATURATION negative clonal selection. High-affinity clones mature whereas au- cepted that whereas B-cells with BCRs that recognize both self- toreactive clones are deleted. The Abs eventually achieve both Ags and foreign Ags would survive, those that recognize only the high affinity and Ag specificity. In contrast, most if not all in vitro foreign Ag will prevail because their BCR engagement is less affinity maturation strategies imitate adaptive immunity without chronic and specific T cell help is present. Self-reactive B cells that constraints. Very few reports incorporate steps to avoid cross-re- lack appropriate T cell help are rapidly censored or depleted. active clones. This is generally not due to the lack of foresight, In light of this B cell tolerance model, our findings can be in- but rather the difficulty in defining cross-reactive epitopes. Our terpreted as follows. If GD2 is the foreign epitope and GM2 is the study using gangliosides provides a working model to demon- self-epitope, affinity maturation during hypermutation will produce strate this breakdown in specificity during affinity maturation clones like Q and Y. The self-epitope should induce rapid B cell with subsequent epitope spreading. Knowing the epitope neigh- inactivation, i.e., negative selection. Thus, as immunity matures in borhood, strategies could therefore be tested to reduce this un- the presence of self-As, high-affinity clones (e.g., clone Y) emerge desirable epitope spread, thereby improving the quality of af- while retaining Ag specificity. However, if the censoring/deletion finity-matured anti-ganglioside Abs. pathway malfunctions (i.e., absence of negative selection), high- When an Ab clone evolves in affinity and spreads its epitope affinity clones (e.g., clone Q) will become dominant. The mere specificity, the cross-reacting peptides or oligosaccharides are usu- absence or depression of the self-epitope (e.g., GM2) during a ally structurally or conformationally related to the original Ag. specific time period or tissue space (e.g., during an infection) can Among gangliosides, a hierarchy of cross-reactivity can be derived be the reason for this malfunction. The ensuing cross-reactive Ab from their structural similarities. The four closest structural neigh- clones (e.g., clone Q) will be free to propagate and become patho- bors of GD2, namely GM2, GD3, GD1b, and GT2, differ from logic when the self-epitope GM2 re-emerges. Downloaded from GD2 by a single residue, representing four different structural di- We conclude that this report can provide insight into the affinity rections. Using GM2, GD3, and GM3, respectively, as negative maturation of carbohydrate (and ganglioside) specific Abs in vitro selectors we found that their cross-reactive affinity highly corre- and in vivo. More importantly, our findings may have broader lated with their ability to reduce clones with epitope spread. Al- implications for other epitopes of amino acid, nucleotide, or though one could increase the concentration of a negative selector origin, many of which are implicated in human diseases and some

to compensate for low affinity, the cost is prohibitive at high as therapeutic targets. We believe that epitope spread during af- http://www.jimmunol.org/ concentrations. finity maturation occurs both in vitro and in vivo. Thus, negative GM2 was the ideal negative selector because its high cross- selectors may potentially be necessary during high throughput reactivity was consistently detected among the high-affinity clones screens of peptide, aptamer, and nucleotide libraries for target- to GD2. We chose a novel ganglioside-oligosaccharide-Sepharose specific ligands. However, a major challenge remains, i.e., how to scaffold as a proof of principle for in vitro selection. This platform define the epitope neighborhood. Moreover, even with sophisti- should also have general applicability to most other . cated modeling, the ultimate proof of specificity of novel Abs and We introduced random single base pair mutations to the entire ligands will be in vivo testing in appropriate animal models before scFv-VH region, including both CDR and the framework regions being used in patients. (23). The efficiency of affinity maturation can be greatly improved by guest on September 27, 2021 by increasing the library size and by focusing mutations onto hot Acknowledgments spots (24). This focusing on CDR regions is particularly important We thank Dr. Irene Cheung (Memorial Sloan-Kettering Cancer Center, if simultaneous mutations in the scFv are desired. Although “poly- New York, NY) for critically reviewing the manuscript and Dr. P. Zhang clonal sequencing” is highly efficient for tracking clonal domi- for expertise in ganglioside conjugation to Sepharose. nance for single mutations, the presence of simultaneous mutations in a bigger library may make the analysis more complex. Disclosures Epitope spread has clinical implications for ganglioside-specific The authors have no financial conflict of interest. Abs. Anti-GD2 Abs are known to induce significantly painful side effects (25–27), with occasional reports of cranial or peripheral References neuropathy. Anti-GD1a Abs preferentially immunostained motor 1. Zuckier, L. S., E. Z. Berkowitz, R. J. Sattenberg, Q. H. Zhao, H. F. Deng, and fibers thought to cause motor neuropathies such as acute motor M. D. Scharff. 2000. Influence of affinity and antigen density on antibody local- axonal neuropathy (28). GD1b Abs preferentially immunostained ization in a modifiable tumor targeting model. Cancer Res. 60: 7008–7013. 2. Batista, F. D., and M. S. Neuberger. 1998. Affinity dependence of the B cell the large dorsal root ganglion neurons, potentially responsible for response to antigen: a threshold, a ceiling, and the importance of off-rate. Immu- sensory ataxic neuropathies (28). High-affinity anti-GD2 Abs with nity 8: 751–759. 3. Rathanaswami, P., S. Roalstad, L. Roskos, Q. J. Su, S. Lackie, and J. Babcook. promiscuous specificity would potentially cause significant motor 2005. Demonstration of an in vivo generated sub-picomolar affinity fully human and sensory neuropathies if they were to be given to patients. In monoclonal antibody to interleukin-8. Biochem. Biophys. Res. Commun. 334: contrast, with GM2 negative selection during affinity maturation 1004–1013. 4. Steidl, S., O. Ratsch, B. Brocks, M. Durr, and E. Thomassen-Wolf. 2008. In vitro the high-affinity clones isolated were much more restricted in their affinity maturation of human GM-CSF antibodies by targeted CDR-diversifica- cross-reactivity and would likely to be safe for clinical use. tion. Mol. Immunol. 46: 135–144. Our in vitro findings also raise the possibility that autoimmune 5. Carsetti, R., G. Kohler, and M. C. Lamers. 1995. Transitional B cells are the target of negative selection in the B cell compartment. J. Exp. Med. 181: Abs can develop when there is no negative selection during so- 2129–2140. matic hypermutation. B cell tolerance, whether central or periph- 6. Strasser, A., H. Puthalakath, L. A. O’Reilly, and P. Bouillet. 2008. What do we know about the mechanisms of elimination of autoreactive T and B cells and what eral, is a complex process for preventing the development of au- challenges remain. Immunol. Cell Biol. 86: 57–66. toimmunity during an immune response to an epitope on a foreign 7. Nossal, G. J. 1994. Negative selection of lymphocytes. Cell 76: 229–239. pathogen. In germinal center follicles of peripheral lymphoid tis- 8. Palmer, E. 2003. Negative selection–clearing out the bad apples from the T-cell repertoire. Nat. Rev. 3: 383–391. sues, self-reactive BCRs are continually generated through somatic 9. Vanderlugt, C. L., and S. D. Miller. 2002. Epitope spreading in immune-mediated hypermutation (29, 30). With affinity maturation, autoimmunity, if diseases: implications for immunotherapy. Nat. Rev. 2: 85–95. not prevented, can be severe, especially because follicular B-cells 10. Deshmukh, U. S., H. Bagavant, J. Lewis, F. Gaskin, and S. M. Fu. 2005. Epitope spreading within lupus-associated ribonucleoprotein antigens. Clin. Immunol. differentiate into long-lived memory cells and plasma cells that in 117: 112–120. turn will produce autoantibodies indefinitely. It is generally ac- 11. Svennerholm, L. 1964. The gangliosides. J. Lipid Res. 5: 145–155. The Journal of Immunology 5755

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