Chapter 2 Isolation and Validation of Anti-B7-H4 scFvs from an Ovarian Cancer scFv Yeast-Display Library Denarda Dangaj and Nathalie Scholler Abstract B7-H4 (VTCN1, B7x, B7s) is an inhibitory modulator of T-cell response implicated in antigen tolerization. As such, B7-H4 is an immune checkpoint of potential therapeutic interest. To generate anti-B7-H4 targeting reagents, we isolated antibodies by differential cell screening of a yeast-display library of recombinant antibodies (scFvs) derived from ovarian cancer patients and we screened for functional scFvs capable to interfere with B7-H4-mediated inhibition of antitumor responses. We found one antibody binding to B7-H4 that could restore antitumor T cell responses. This chapter gives an overview of the methods we developed to isolate a functional anti-B7-H4 antibody fragment. Key words pAGA2 vector, p416 BCCP vector, Magnetic sorting, Flow sorting, Cell panning, Homologous recombination, Immune checkpoint, Functional assays, Tumor-associated macrophages, CD3-stimulated T cells, T2 APCs, Humanized mouse model of ovarian cancer

1

Introduction B7-H4, also called B7x/B7s, is a B7 superfamily member recently identified as an inhibitory modulator of T-cell response [1–3]. When present at the surface of antigen presenting cells, B7-H4 negatively regulates T cell activation, possibly through interaction with a ligand that remains to be identified [4]. B7-H4 mRNA is widely expressed but the restricted pattern of protein expression in normal tissues suggests posttranscriptional regulation. B7-H4 expression in tumor tissues is observed in various types of human cancers such as breast [5], ovarian [6], pancreatic, lung [7, 8] melanoma [9], and renal cell carcinoma [10]. In most studies, B7-H4 was determined to be either located in the cytoplasm or at the plasma membrane protein by immunohistochemistry [9–13]. B7-H4 expression in ovarian cancer cell lines, B7-H4 expression was also reported to be mainly intracellular by flow cytometry [6, 7].

Bin Liu (ed.), Yeast Surface Display: Methods, Protocols, and Applications, Methods in Molecular Biology, vol. 1319, DOI 10.1007/978-1-4939-2748-7_2, © Springer Science+Business Media New York 2015

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We have recently shown that B7-H4 is present at the surface of uncultured tumor cells derived from ovarian cancer samples and its expression is diminished when tumors are cultured in vitro. The broad presence of B7-H4 in various cancers and its known function as negative regulator of T cell activation suggest a specific role in down-regulation of antitumor immunity. In fact, ovarian cancerderived B7-H4+ TAMs suppress HER2-specific T-cell proliferation and cytotoxicity, and the blocking of B7-H4 expression on macrophages using morpholino antisense oligonucleotides improved tumor-associated antigen T-cell responses in vitro and in vivo [6]. Altogether, these results ascribe a translational value to B7-H4 as a target molecule for antitumor immunotherapy. In an effort to develop alternate means for blocking B7-H4 activity for development for clinical applications, we sought to isolate anti-B7-H4 recombinant antibodies (scFvs). Single chain Fragments variables (scFvs) are recombinant antibodies expressing single antigen-binding domain constituted by peptide-linked variable domains of heavy and light immunoglobulin chains. ScFvs small size, versatility, and amenability to affinity maturation, make them particularly interesting for in vivo targeting, in vivo imaging after conjugation with radioisotopes, and for therapeutic purposes after conjugation with endotoxins or nanoparticles [14] or fused to T cell signalling domains to engineer modified T cell receptors [15]. We first derived a yeast-display scFv library from tumorinfiltrating B cells and PBMCs of 11 ovarian cancer patients. AntiB7-H4 scFvs were isolated by selection for specific binding to both soluble B7-H4 recombinant protein (rB7-H4) expressed by mammalian cells and B7-H4+ cancer cells. Then, to identify scFvs that could reverse the B7-H4 mediated T cell inhibition, we set up in vitro systems to model T cell inhibition mediated by presentation of B7-H4 and we tested the ability of the newly isolated anti-B7-H4 scFvs to reverse nonspecific and antigen-specific T cell inhibitions in vitro and in a humanized mouse model of ovarian cancer [16].

2

Materials

2.1 Construction of Yeast-Display scFv Library Derived from Ovarian Cancer Patients

Using the vector pAGA2 [17] that enables homologous recombination in yeast, we built up a library of recombinant antibodies derived from B cells isolated from ascites (n = 10) and PBMCs (n = 1) of ovarian cancer patients (stages III or IV). 1. Yeast strain: EBY100. 2. Yeast growth media: SD-CAA and SD-CAA agar [18]. 3. Yeast induction media: SGR-CAA [18]. 4. PBE buffer: Phosphate buffer saline supplemented with 5 g/L of BSA fraction V and 10 mM EDTA, pH 8.

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5. Pretreating solution: 25 mM DTT, 0.6 M sorbitol, 20 mM Hepes, pH = 7.5. 6. Sorbitol 1 M. 7. Gene Pulser (Bio-Rad), 0.2 cm cuvette. 8. Incubator shaker at 30 °C. 9. Incubator shaker at 20 °C. 10. Yeast-display expression pAGA2 vector, available upon request to N. Scholler ([email protected]) pending MTA. 11. Ficoll-Paque PLUS (Life-Sciences) to isolate human lymphocytes from ascites and peripheral blood of ovarian cancer patients. 12. CD19 magnetic beads (Miltenyi), magnetic stand and LD columns (Miltenyi) to purify B cells from human lymphocytes isolated from cancer patients. 13. mRNA purification kit (Qiagen). 14. SuperScript II reverse transcriptase (Invitrogen). 15. Oligo(dT)12–18 Primer (Promega). 16. Platinum Taq DNA polymerase (Invitrogen). 17. dNTPs Master mix. 18. Forward and reverse primer sequences for amplification of human subfamilies of VH and VL gene fragments and cloning by in vivo homologous recombination in pAGA2 vector are listed in Tables 1 and 2. 19. DNA electrophoresis apparatus. 20. Thermocycler (PCR machine). 2.2 Isolation of Yeast-Display scFv Binding to B7-H4

We performed two rounds of screening of the yeast-display library. First, we selected yeast-display scFvs that bound to B7-H4 recombinant protein. Materials and methods for selection of yeast display recombinant antibodies with recombinant proteins were detailed in ref. [18]. We then proceeded to a second round of selection by differential panning of the selected yeast-display scFvs on B7-H4+ vs. B7-H4− cell lines that were plastic-immobilized. 1. Macrophages expressing B7-H4 [16]. 2. Trizol (Sigma). 3. Oligo (dT)12–18 Primer (Promega). 4. Reverse-Transcription PCR kit (Promega). 5. Primers for B7-H4 amplification and cloning into pTT28 expression vector [19]: B7-H4 For: 5′-gttctggtggtggaggttctggtggtggtggatctgagtttggtatttcagggagacactccatca 3′ B7-H4 Rev: 5′ agaccgaggagagggttagggataggcttaccgtcgacagaa gcctttgagtttagcagctgtag-3′.

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Table 1 Forward primer sequences Forward primers name

Sub-families amplified

Primer sequence

VHF1

VH1/7

cag gts cag ctg gtr cag tct gg

VHF2

VH2

cag rtc acc ttg arg gag tct gg

VHF3

VH3

gar gtg cad ctg gtg gag tc

VHF4

VH4

cag stg cag ctg cag gag tcg ggc

VHF5

VH4

cag gtg cag cta car cag tgg ggc

VHF6

VH5

gar gtg cag ctg gtg cag tcy gg

VKF1

VK1/4

ghc atc cdg wtg acc cag tct cc

VKF2

VK2/6

gat rtt gtg atg ach cag wct cca

VKF3

VK3

gaa atw gtg wtg acr cag tct cca

VKF4

KV5

Gaa acg aca ctc acg cag tct cca g

VKF5

KV6

Gaa att gtg ctg act cag tct cca g

VLF1

VL1

cag tct gtg btg ack cag ccr cc

VLF2

VL2

car tct gcc ctg act cag cc

VLF3

VL3

tcc tat gag ctg ctg acd cag cya

VLF4

VL3–19

tct tct gag ctg act cag gac cc

VLF5

LV3–32

tcc tct ggg cca act cag gtg cc

VLF6

VL4/5

cwg cct gtg ctg act car yc

VLF7

LV6

aat ttt atg ctg act cag ccc c

VLF8

VL7/8

cag rct gtg gtg acy cag gag cc

VLF9

LV/10

cag scw gkg ctg act cag cca cc

VLF10

LV911

cgg ccc gtg ctg act cag cc

VHF

VKF

VLF

6. 293F cells for protein expression (Invitrogen). 7. OptiMEM (Invitrogen). 8. Lipofectamine (Invitrogen). 9. Snake skin dialysis membrane (Pierce). 10. Nickel sepharose beads (Sigma/resin).

Anti-B7-H4 scFvs from Yeast-Display Library

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Table 2 Reverse primer sequences Reverse primers VHR VHR1

JH1/2/4/5

Tga gga gac rgt gac cag gg

VHR2

JH3

Tga aga gac ggt gac cat tgt cc

VHR3

JH6

Tga gga gac ggt gac cgt ggt cc

VKR1

JK1

Ttt gat ttc cac ctt ggt ccc

VKR2

Jk2/4

Ttt gat ctc cas ctt ggt ccc

VKR3

Jk3

Ttt gat atc cac ttt ggt ccc agg

VKR4

Jk5

Ttt aat ctc cag tcg tgt ccc ttg

VLR1

JL1

Tag gac ggt gac ctt ggt ccc

VLR2

JL2/3/5

Tag gac ggt cag cty ggt ccc

VLE3

JL4

Taa aat gat cag ctg ggt tcc tcc

VLR4

JL6

Gag gac ggt cac ctt ggt gcc

VLR5

JL7

Gaa gtc ctg tgc gag gca gc

VKR

VLR

M

R

W

S

Y

K

V

H

D

B

N

a/c

a/g

a/t

c/g

c/t

g/t

a/c/t

a/c/t

a/g/t

c/g/t

a/c/g/t

11. Equilibrium buffer (EQ buffer): 0.3 M NaCl, 0.05 M NaH2PO4, pH 8. 12. Washing buffer: 0.3 M NaCl, 0.05 M NaH2PO4, 20 mM imidazole. 13. Elution buffer (EB): 0.3 M NaCl, 0.05 M NaH2PO4, 300 mM Imidazole, pH 8. 14. NanoOrange Protein Quantification kit (Invitrogen). 15. HIS-probe (Santa Cruz Biotechnology). 16. Polyclonal anti-B7-H4 antibody (Abcam). 17. CO2 incubator at 37 °C. 18. Sterile hood for cell culture. 19. Cell culture media. 20. Phase-contrast microscope.

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21. Wild type ovarian cancer cell lines (OVCAR3, C30) and ovarian cancer cell line expressing B7-H4 after viral transfection. 22. Cell medium. 23. RNA easy kit (Qiagen). 24. Ready-to-go you-prime First-Strand Beads (GE Healthcare). 25. Primers for B7-H4 amplification and cloning into pTT28 vector in frame with 6XHIS Tag. B7-H4-for: 5′-ggttctggtggtggaggttctggtggtggtggatctggtttgg tatttcagggagacactccatca-3′ B7-H4-rev: 5′-agaccgaggagagggttagggataggcttaccgtcgacagaag cctttgagtttagcagctgtag-3′. 26. Primers for B7-H4 amplification and cloning into Xba1/Sal1 linearized lentiviral vector [16]. B7H4-F: 5′-ACGCTCTAGAATGGCTTCCCTGGGGCAGA TCCTCT-3′ B7H4-R: 5′-ACGCGTCGACTTATTTTAGCATCAGGTAA GGGCTG-3′. 27. Self-inactivating lentiviral expression vectors pVSV-G (VSV glycoprotein expression plasmid), pRSV.REV (Rev expression plasmid), pMDLg/p.RRE (Gag/Pol expression plasmid), and pELNS transfer plasmid [20]. 28. Express Inn (Open Biosystems). 29. 293T human embryonic kidney cells. 30. QIAGEN Endo-free Maxi prep kit. 31. Ultracentrifuge and Beckman SW28 rotor (Beckman Coulter, Fullerton, CA). 32. Poly-L-Lysine coated 6-well plates. 33. Rotator. 34. Monoclonal anti-B7-H4 PE conjugated antibody (AbD Serotec). 2.3 Conversion of the Selected Yeast Display Anti-B7-H4 scFv into a Soluble Form and Screening in Systems Modeling B7-H4-Mediated Inhibition of T-Cell Functions

Anti-B7-H4 yeast-display scFvs were transferred from the display vector pAGA2 into its companion vector for yeast secretion, p416-BCCP [17] as detailed in ref. [18]. p416-BCCP vector is available upon request to N. Scholler ([email protected]), pending MTA. 1. T2 APC, wild type or transduced to express B7-H4. 2. Cancer cell lines, wild type or transduced to express B7-H4 and present tumor-associated peptide. 3. Primary human T cells expressing antigen-specific TCR. 4. Selected Anti-B7-H4 scFvs (final concentration 5 μg/ml). 5. IFN-γ quantification ELISA kit (BioLegend).

Anti-B7-H4 scFvs from Yeast-Display Library

3

43

Methods

3.1 Yeast Display Library Construction

3.1.1 B Cell Isolation and scFv Domain Amplification

The insertion of VH-VL fragments encoding the scFv in pAGA2 vector was performed by in vivo homologous recombination between three partners: VH PCR fragments, VL PCR fragments, and linearized pAGA2 vector (Fig. 1). Resulting scFvs were composed of VH (Fig. 1, orange fragment) and VL fragments (Fig. 1, green fragment) attached together by GGSSRSSSSGGGGSGGGG linker (Fig. 1, blue fragment). Sequences were added to each primer ends for enabling annealing with pAGA2 vector. 1. Isolate B cells from peripheral blood by centrifugation of diluted blood on Ficoll gradient (see Note 1) followed up by two washed in medium and magnetic separation with CD19 magnetic beads as recommended by the manufacturer 2. Isolate B cells from ascites after resuspension of the leukocytes in medium (5 × 106 cells/ml) by Ficoll gradient and magnetic separation with CD19 magnetic beads as in step 1. 3. Combine B cells and isolate mRNA (see Note 2)

Fig. 1 Schematic representation of the cloning by in vivo homologous recombinations of VL and VH fragments in pAGA2 vector. pAGA2 vector was linearized with Nhe1 and Xho1. VL fragments (green domain, Table 1) were flanked in 5′ with pAGA2 homologous sequence (black domains, 5′-ggtggtggaggttctggtggtggtggatctgtc-3′) for recombination with pAGA2 in 5′ end, and in 3′ end with homologous sequences (blue upper domain, 5′-cgctgccaccgccgccgctggaacttgacct agaggatccgcc-3′) for recombination with the linker (5′-ggcggatcctctaggtcaagttccagcggcggcggtggcagcggaggcggcggt-3′). VH fragments (orange domain, Table 2) were flanked in 5′ with homologous sequences (blue lower domain, 5′-ctaggtcaagttccagcggcggcggtgg-cagcggaggcggcggt-3′) for recombination with the linker and pAGA2 in 3′ end (grey domains, 3′-gtcttcttcagaaataagcttttgttcggatccctcgaa-5′) (Color figure online)

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Denarda Dangaj and Nathalie Scholler

4. Perform reverse transcription reactions to permit PCR amplification of VH and VL fragments (see Note 3) 5. PCR amplify the products of retrotranscription using the primers described in Tables 1 and 2. Set up the PCR mix as described in ref. [18] and use the following cycling conditions: 94 °C 5 min at, (94 °C 1 min; 55 °C 1 min; 72 °C 1 min) × 25; 72 °C 7 min. 3.1.2 Yeast Transformation

1. Linearize pAGA2 vector with Nhe1 and Xho1 2. Separate linearized vector and PCR products by gel electrophoresis 3. Purify separated DNA fragments by gel extraction kit 4. Transfect the mixture of linearized pAGA2 vector, VH and VL into EBY100 yeast cells at a 1/3/3 ratio and proceed to electroporation. 5. Day 1: inoculate a single colony of EBY100 into 5 ml YEPD, cultured overnight (ON), 30 °C, 200 rpm. 6. Day 2: Inoculate appropriate volume (5, 10, 20, 40, 80 μl) saturated cells into 200 ml YEPD, and cultured ON, 30 °C, 200 rpm. 7. Day 3: Competent cell preparation and yeast electroporation. Identify the culture where the cell density is between 5 × 106 and 2 × 107 cells/ml. Resuspend these cells at 1 × 107/ml in SD-CAA. 8. Day 3 (continued): Spin down and resuspend the cells in ¼ volume pretreating solution. 9. Day 3 (continued): Incubate the cells at 30 °C for 30 min with rotation. 10. Day 3 (continued): The following steps are performed in ice or ice chilled solution. 11. Day 3 (continued): spin and wash the cells with ½ volume of 1 M sorbitol (ice chilled solution) 12. Day 3 (continued): spin and resuspend cells with 2–3 volume 1 M sorbitol (ice chilled solution). The cells are now competent for electroporation. 13. Day 3 (continued): mix linearized pAGA2 (NheI/XhoI) (1–10 μg) and 2–10 times scFv PCR fragments with 100 μl of competent cells (on ice) 14. Day 3 (continued): Perform electroporation at 1.5 kV. Resuspend the cells in 1 ml SD-CAA and inoculate into 1 L SD-CAA (ice-chilled solution) 15. Day 3 (continued): At the same time, serially dilute 100 μl with TE and plate on SD + CAA agar plate for library size assessment.

Anti-B7-H4 scFvs from Yeast-Display Library

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16. Day 3 (continued): Grow the liquid culture at 30 °C, ON with agitation (250 rpm). After 16–20 h, the absorbance at 600 nm should read between 1 and 2 (A600 = 1 corresponds at 107 yeast/ ml). Grow the plated yeast up to 48 h at 30 °C. 17. Day 4: harvest the library and proceed to induction immediately (see Note 4), or store the culture up to 3 weeks at 4 °C (see Note 5). For long-term storage, add 15 % glycerol in the yeast culture and freeze 1 ml aliquots. 3.1.3 Induction of scFv Expression at the Yeast Cell Surface for Screening

1. Induce at room temperature (22–24 °C) with agitation (250 rpm), ON. A600 must be 0.5 after yeast resuspension in SGR-CAA, and 1–1.5 after ON culture. 2. Pellet induced yeast by centrifugation, resuspend in 50 ml of PBE, and pellet again (see Note 6). The pellet should measure about 5 ml. 3. Resuspend the pellet in an equal volume of PBE. Proceed to sorting/panning immediately, or store the induced library up to 2 weeks at 4 °C (see Note 7). 4. Selection by magnetic and flow sorting of yeast-display antiB7-H4 scFv using B7-H4 recombinant protein, as detailed in ref. [18].

3.2 Generation of B7-H4 Recombinant Protein and B7-H4 Expresser Cell Lines 3.2.1 B7-H4 Recombinant Protein

1. Isolate total RNA from tumor associated-macrophages. 2. Synthesize in vitro cDNA using oligo-dT. 3. PCR-amplify the extracellular domain of B7-H4 (IgC + IgV) from in vitro synthesized cDNA for cloning into pTT28 with the primers B7-H4-for and B7-H4-rev. 4. Purify plasmid DNA using the QIAGEN Endo-free Maxi prep kit. 5. Transfect 293-F mammalian cells with B7-H4-pTT28 vector using Express-Inn transfection reagent according to manufacturer’s instructions. 6. Harvest serum free supernatants containing soluble B7-H4 after 3 and 6 days of culture. 7. Dialyze 50–500 ml of cell supernatant containing soluble B7-H4 protein in 5 L of 1×PBS twice using snake-skin membrane. 8. Washing of Nickel sepharose resin preparation: centrifuge at 750 × g for 5 min to remove the preservative solution, wash once with 5 volumes of ddH2O, and twice with 5 volumes EQ buffer for 15 min at room temperature with gentle rotation. 9. Mix 1–1.5 ml of washed Nickel sepharose resin with 100 ml of SN and incubate at 4 °C for 1.5–2 h with rotation.

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Denarda Dangaj and Nathalie Scholler

10. Remove SN containing unbound proteins by centrifugation. 11. Wash twice the resin containing bound B7-H4 with washing buffer for 5 min with gentle rotation (e.g., 50 ml for 500 ml initial SN). 12. Elute resin with EB buffer for 15 min at room temperature with gentle rotation. Repeat once. 13. Dialyze the eluted proteins against 1× PBS and repeat twice. 14. Quantify the protein concentration with the NanoOrange kit. 3.2.2 Generation of B7-H4 Cell Lines

1. Isolate total RNA from OVCAR-3 ovarian tumor cells and reverse transcript with the kit “ready-to-go you-prime FirstStrand Beads”. 2. PCR-amplify the full length B7-H4 using the in vitro synthesized cDNA as a template for PCR reaction with the primers B7-H4-F and B7-H4-R. 3. Digest the PCR products with XbaI and SalI enzymes and gel purify 4. Clone the digested PCR products into XbaI/SalI linearized pELNS, a third generation self-inactivating lentiviral expression vector [20], in which the transgene expression is driven by the EF-1a promoter, to obtain pELNS-B7-H4. 5. Purify plasmid DNA using the QIAGEN Endo-free Maxi prep kit. 6. Seed human embryonic kidney cells 293T at a density of 10 × 106 cell per T-150 tissue culture flask 24 h before transfection. 7. Transfected cells with 7 μg of pVSV-G (VSV glycoprotein expression plasmid), 18 μg of pRSV.REV (Rev expression plasmid), 18 μg of pMDLg/p.RRE (Gag/Pol expression plasmid), and 15 μg of pELNS transfer plasmid using Express Inn [20]. 8. Harvest viral supernatant at 24 and 48 h post-transfection. 9. Concentrate viral particles by ultracentrifugation for 3 h at 25,000 rpm using a Beckman SW28 rotor and resuspend in 4 ml of RPMI full medium. 10. Transduce the cancer cell lines C30 (1.5 × 105 in a six-well plate) with 1.5 ml and T2 cells (3 × 105 cells in a 24 well plate) with 3–5 ml pELNS-B7-H4 lentiviral particles. 11. Replace lentivirus containing medium with fresh RPMI 24 h after transduction 12. Check the expression of surface B7-H4 5–6 days post transduction using a monoclonal anti-B7-H4 PE conjugated antibody.

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Anti-B7-H4 scFvs from Yeast-Display Library

3.3 Selection of Yeast Display Recombinant Antibodies by Differential Cell Panning

Further select the subpopulation of yeast-display scFvs by cell panning: 1. Grow in monolayer to 90 % confluence on poly-L-Lysinecoated dishes C30 ovarian cancer cell line transduced with pELNS-B7-H4 or with pELNS-GFP as negative control. 2. Induce recombinant sub-library.

B7-H4-selected

yeast-display

scFv

3. Wash twice with 50 ml PBE. 4. Deplete for nonspecific binding by two incubations with GFP+ C30 cells at a ratio of (30–60):1 yeast to C30 cells for 30 min at RT with gentle rotation to prevent clumping (1–2 speed). 5. Harvest unbound yeast cells and incubate with plasticimmobilized B7H4+ C30 cells for 30 min at RT with gentle rotation. 6. Wash plastic-immobilized B7H4+ C30 cells with bound yeast twice with PBS for 5 min at RT and examine under microscope. 7. Harvest yeast clusters that are still binding to cells and grow on the cell plate overnight. 8. The next day, transfer to a new flask for further amplification. 9. Repeat yeast panning four times. 10. Convert yeast displayed scFvs into soluble forms [18] and select for best binding scFv clones as described in refs. [17, 21]. 3.4 Screening in Systems Modeling B7-H4-Mediated Inhibition of T-Cell Functions

Test soluble anti-B7-H4 scFv in the following in vitro model systems. 1. Resuspend GFP- or B7-H4-transduced T2 APCs at 10 × 106 cells/ml and loaded with HER-2 or MART-1 peptides at 0.05 μM for 2 h at 37 °C. 2. Wash cells twice with RPMI medium. 3. Coculture peptide-loaded APCs with HER-2 TCR specific T cells at 1:1 ratio of 1 × 105 T2 APCs and 1 × 105 T cells, in 200 μl of RPMI medium in round bottom 96-well tissue culture plates. Use MART-1 peptides as irrelevant peptides for the mock stimulations of HER-2 TCR T cells. 4. Add 5 μg/ml of anti-B7-H4 or control scFvs in cocultures. 5. Analyze IFN-γ production of T cells cocultured with APC expressing B7-H4 or control GFP, in the presence or in the absence of recombinant antibodies (as indicated) and responses after 48 h using an IFN-γ quantification kit (Fig. 2).

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Denarda Dangaj and Nathalie Scholler GFP+T2

HER-2 TCR T cells

B7-H4+ T2

IFN-Y Concentration (pg/ml)

18000 16000 14000 12000 10000 8000 6000 4000 2000 0

untr

irrel scFv CscFv

26

56

Protein-based sorted anti-B7H4 scFvs

1#12

2#29

2#69

3#35

3#54

3#68

Cell-based sorted anti-B7H4 scFvs

Fig. 2 Screening of anti-B7-H4 scFvs that reverse B7-H4-mediated T cell inhibition. IFN-γ production of HER-2 TCR T cells pulsed with HER-2 or irrelevant peptide (MART-1) and cocultured with GFP (GFP+ T2, black bars) or B7-H4 transduced T2 APCs (B7-H4+ T2, grey bars), in the absence or in the presence of various anti-B7-H4 scFvs. IFN-γ production was measured with ELISA assay

4

Notes 1. Ficoll gradient: 15 ml of blood diluted with 15 ml of PBS is layered on 12.5 ml of Ficoll-Paque and centrifuge for 30 min at 1,200 rpm without brake at room temperature 2. Use 20–25 μg of mRNA 3. Up to 50 reverse transcription maybe necessary to reach the quantity template necessary for VH and VL fragment amplification. 4. The display of antibody fragments is induced by culture of the grown library in presence of galactose 5. For storage, yeast can be pelleted by 5 min of centrifugation at 3,000 × g and resuspended in 50 ml of SD-CAA. However, before induction yeast should be freshly passaged. 6. Yeast culture should look opalescent white and have a fresh smell. If the color is yellowish and the smell sour, suspect a contamination and consider thawing another library aliquot. We do not recommend using antibiotic or antifungic so early in the screening process as it can reduce the library diversity. 7. Yeast resuspension can be difficult after centrifugation and necessitate moving the pipette around rather than aspirating up and down.

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References 1. Sica GL, Choi IH, Zhu G et al (2003) B7-H4, a molecule of the B7 family, negatively regulates T cell immunity. Immunity 18:849–861, doi:S1074761303001523 [pii] 2. Prasad DV, Richards S, Mai XM et al (2003) B7S1, a novel B7 family member that negatively regulates T cell activation. Immunity 18:863–873, doi:S107476130300147X [pii] 3. Zang X, Loke P, Kim J et al (2003) B7x: a widely expressed B7 family member that inhibits T cell activation. Proc Natl Acad Sci U S A 100:10388– 10392. doi:10.1073/pnas.1434299100, 1434299100 [pii] 4. Kryczek I, Wei S, Zou L et al (2006) Cutting edge: induction of B7-H4 on APCs through IL-10: novel suppressive mode for regulatory T cells. J Immunol 177:40–44, doi:177/1/40 [pii] 5. Tringler B, Zhuo S, Pilkington G et al (2005) B7-h4 is highly expressed in ductal and lobular breast cancer. Clin Cancer Res 11:1842–1848. doi: 10.1158/1078-0432.CCR-04-1658 , 11/5/1842 [pii] 6. Kryczek I, Zou L, Rodriguez P et al (2006) B7-H4 expression identifies a novel suppressive macrophage population in human ovarian carcinoma. J Exp Med 203:871–881. doi:10.1084/ jem.20050930, jem.20050930 [pii] 7. Choi IH, Zhu G, Sica GL et al (2003) Genomic organization and expression analysis of B7-H4, an immune inhibitory molecule of the B7 family. J Immunol 171:4650–4654 8. Sun Y, Wang Y, Zhao J et al (2006) B7-H3 and B7-H4 expression in non-small-cell lung cancer. Lung Cancer 53:143–151. doi:10.1016/j. lungcan.2006.05.012, S0169-5002(06)002315 [pii] 9. Quandt D, Fiedler E, Boettcher D et al (2011) B7-h4 expression in human melanoma: its association with patients’ survival and antitumor immune response. Clin Cancer Res 17: 3100–3111. doi:10.1158/1078-0432.CCR10-2268, 1078-0432.CCR-10-2268 [pii] 10. Krambeck AE, Thompson RH, Dong H et al (2006) B7-H4 expression in renal cell carcinoma and tumor vasculature: associations with cancer progression and survival. Proc Natl Acad Sci U S A 103:10391–10396. doi:10.1073/ pnas.0600937103, 0600937103 [pii] 11. Jiang J, Zhu Y, Wu C et al (2010) Tumor expression of B7-H4 predicts poor survival of patients suffering from gastric cancer. Cancer Immunol Immunother 59:1707–1714. doi:10.1007/s00262-010-0900-7 12. Miyatake T, Tringler B, Liu W et al (2007) B7-H4 (DD-O110) is overexpressed in high

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Isolation and Validation of Anti-B7-H4 scFvs from an Ovarian Cancer scFv Yeast-Display Library.

B7-H4 (VTCN1, B7x, B7s) is an inhibitory modulator of T-cell response implicated in antigen tolerization. As such, B7-H4 is an immune checkpoint of po...
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