METHODS AND KITS FOR GENERATING AND SELECTING A VARIANT OF A BINDING PROTEIN WITH INCREASED BINDING AFFINITY AND/OR SPECIFICITY

Abstract

Somatic hypermutation promotes affinity maturation of antibodies by targeting the cytidine deaminase AID to antibody genes, followed by antigen-based selection of matured antibodies. Given the importance of antibodies in medicine and research, developing approaches to reproduce this natural phenomenon in cell culture is of some interest. The inventors use here the CRISPR-Cas 9 based CRISPR-X approach to target AID to antibody genes carried by expression vectors in HEK 293 cells. This directed mutagenesis approach, combined with a highly sensitive antigen-associated magnetic enrichment process, allowed rapid progressive evolution of a human antibody against the Human Leucocyte Antigen A*0201 allele. Starting from a low affinity monoclonal antibody expressed on Ag-specific naïve blood circulating B cells, they obtained in approximately 6 weeks antibodies with a two log increase in affinity and which retained their specificity. The strategy for in vitro affinity maturation of antibodies is applicable to virtually any antigen. It not only allows to tap into the vast naive B cell repertoire but could also be useful when dealing with antigens that only elicit low affinity antibodies after immunization. Accordingly as defined by the claims, the present invention relates to methods and kits for generating and selecting a variant of antibody binding protein with increased binding affinity and/or specificity.

Claims

1. A method of generating and selecting a variant of a binding protein with increased binding affinity and/or specificity for a binding domain comprising subjecting a population of cells that express the binding protein to at least one round of mutagenesis coupled to an affinity/specificity-based cell selection and immunomagnetic enrichment.

2. The method of claim 1 wherein the population of cells express an antibody at the cell surface.

3. The method of claim 1, wherein the population of cells is a population of B cells or a population of eukaryotic cells engineered for expressing an antibody.

4. The method of claim 3 wherein the antibody that is expressed by the population of cells is a whole antibody having 2 light chains and 2 heavy chains.

5. The method of claim 1 wherein the mutagenesis comprises contacting the population of cells that express the binding protein with a gene editing platform (a) a defective CRISPR/Cas nuclease engineered for sequence targeting, (b) a non-nuclease DNA modifying enzyme and (c) a plurality of RNA molecules for guiding the defective CRISPR/Cas nuclease and the non-nuclease DNA modifying enzyme to a plurality of target sequences in a DNA nucleic acid molecule coding for the binding domain of the binding protein.

6. The method of claim 5 wherein the defective CRISPR/Cas nuclease is a mutant Cas9 protein from S. pyogenes.

7. The method of claim 5 wherein the defective CRISPR/Cas nuclease comprises the amino acid sequence as set forth in SEQ ID NO: 1.

8. The method of claim 5 wherein the non-nuclease DNA modifying enzyme has the activity of cytosine deaminases, adenosine deaminases, DNA methyltransferases, and/or DNA demethylases.

9. The method of claim 8 wherein the non-nuclease DNA modifying enzyme derives from Activation Induced cytidine Deaminase (AID).

10. The method of claim 8 wherein the non-nuclease DNA modifying enzyme is the AID*Δ that has the amino acid sequence as set forth in SEQ ID NO:2.

11. The method of claim 5 wherein the non-nuclease DNA modifying enzyme is fused to an RNA-binding domain.

12. The method of claim 11 wherein the RNA-binding domain derives from a protein selected from the group consisting of the telomerase Sm7, MS2 Coat Protein, PP7 coat protein (PCP), and SfMu phage Com RNA binding protein.

13. The method of claim 11 wherein the RNA-binding domain is the MS2 coat protein variant having an amino acid sequence as set forth in SEQ ID NO:3.

14. The method of claim 5 wherein the plurality of RNA molecules are designed for targeting a plurality of sequences in the DNA nucleic acid molecule encoding for a binding domain of the binding protein.

15. The method of claim 5 wherein the plurality of RNA molecules comprises a programmable guide RNA motif, a CRISPR RNA motif, and a recruiting RNA motif.

16. The method of claim 15 wherein the recruiting RNA motif comprises the telomerase Ku binding motif, the telomerase Sm7 binding motif, the MS2 phage operator stem-loop, the PP7 phage operator stem-loop, or the SfMu phage Com stem-loop.

17. The method of claim 15 wherein the RNA recruiting motif comprises the MS2 Phage Operator Stem Loop as set forth in SEQ ID NO:5.

18. The method of claim 1 wherein the affinity/specificity-based cell selection and immunomagnetic enrichment comprises a step of contacting a post-mutagenesis population of cells with a plurality of multimers made by mixing specific and unspecific target molecules for the binding protein.

19. The method of claim 18 wherein the plurality of multimers are tetramers.

20. The method of claim 19 wherein the tetramers comprise an epitope peptide that is specifically recognized by an antibody that is loaded in a soluble peptide MHC monomer tetramerized with three other soluble peptide MHC monomers that include at least one non-specific MHC peptide monomer.

21. The method of claim 18 wherein the plurality of multimers are conjugated with a label.

22. The method of claim 21 wherein the label is a fluorescent molecule.

23. The method of claim 18 wherein the immunomagnetic enrichment is carried out using magnetic particles to allow cell enrichment by contacting multimers that bind to cells with said magnetic particles.

24. The method of claim 21 wherein the surfaces of the magnetic particles are functionalized to attach binding molecules that bind selectively the label.

25. The method of claim 18 wherein a first round of contacting is carried out with a plurality of multimers having a determined number of specific target molecules and a second round of contacting is carried out with a plurality of multimers having a decreased number of specific target molecules.

26. The method of claim 25 wherein the concentration of the plurality of multimers is progressively decreased between the first round and the second round to increase selection stringency.

27. The method of claim 1, wherein the population of cells express the binding protein at the cell surface.

Description

FIGURES

[0133] FIG. 1: Isolation and characterization of human mAb A2Ab. (A) Sorting strategy used to isolate HLA-A2-specific B lymphocytes from donor NO. Cells with the following phenotypic characteristics: CD3−, CD19+ (left panel), both PE and APC labeled HLA-A2 tetramers+(middle panel), HLA-B7 tetramer BV421− (right panel) were isolated and used to produce recombinant antibodies. (B) A2Ab Ab in FIG. 1B and a control anti-pp65-HLA-A*0201 human mAb (Ac-anti pp65-A2) were tested by ELISA against the following peptide-MHC recombinant monomers: pp65-HLA-A*0201 (pp65-A2), Me1A-HLA-A*0201 (Me1A-A2) and pUV-HLA-B*0701 (pUV-B7). C) The specificity of A2Ab was assessed in a Luminex single antigen bead assay. Results are shown in terms of interval MFI. Positivity threshold was set at 1000. (D) The affinity of A2Ab was measured by surface plasmon resonance by flowing various concentrations of pp65-A2 complex over CM5 chip-bound A2Ab.

[0134] FIG. 2: Schematic illustration of CRISPR-X. (A) dCas9 associated with a sgRNA containing MS2 hairpins recruits AID* fused to MS2 coat protein leading to localized mutations (stars). Mutations can be induced in the sgRNA binding site or upstream or downstream from it, though only downstream mutations are illustrated here. (B) Binding sites for the nine sgRNAs used on the A2Ab heavy chain variable domain coding sequence are shown. Blue and orange colors indicate complementarity to non-coding and coding strands respectively.

[0135] FIG. 3: Generation and selection of HEK 293 cells expressing affinity-matured antibodies. (A) Overall strategy for antibody affinity maturation. HEK 293 cells expressing the initial Ab are subjected to CRISPR-X mutagenesis. Cells expressing variant antibodies of higher avidity are enriched using Stringent Tetramer-Associated Magnetic Enrichment (S-TAME) and expanded in vitro (R for “enriched population”, subscript n for round of mutation/selection). Enriched cells are separated by FACS into tetramer positive-staining (R+) and tetramer negative-staining (R−) populations. Multiple rounds of mutation/selection can be performed successively as indicated. (B) Staining of A2Ab-expressing HEK 293 cells with 4A2-tetramers or 3A2/1B7 tetramers as marked after 3 successive transfections for CRISPR-X mutagenesis. Results shown are before the S-TAME step. (C) Staining of cells with tetramer 3A2/1B7 after S-TAME. Results are shown for cells transfected with dCas9, sgRNAs and MS2 AID* (R1+ cells, left panel), AID* alone (middle panel) and sgRNA alone (right panel). (D) Cells from the R1 population staining positive with the 3A2/1B7 tetramer were isolated by FACS (R1+ cells). Staining of these cells with tetramers 3A2/1B7 (upper left panel) and 1A2/3B7 (upper right panel) is shown. R1+ cells were subjected to a second round of mutagenesis, S-TAME and FACS selection to generate R2+ cells. Staining of R2+ cells with tetramers 3A2/1B7 (lower left panel) and 1A2/3B7 lower right panel) is shown. The number of cells within marked gates is shown between brackets as a percentage of the total cells analysed.

[0136] FIG. 4: Web Logo representation of amino acid mutations in the A2Ab heavy chain. WT: starting sequence. R1+, R2+: sequences after one or two rounds of mutation/selection respectively. The height of each letter is proportional to the preference for that amino acid at that site, and letters are colored by amino-acid hydrophobicity. Residue positions are numbered starting from the first amino acid of the leader peptide of the heavy chain. Major mutation sites are indicated by arrows.

[0137] FIG. 5: Characterization of evolved antibodies against HLA-A2. (A) ELISA dose-response curves of R2+ mutated mAbs C4.4 and C4.18 compared to A2Ab. (B) The affinity of C4.18 was measured by surface plasmon resonance by flowing various concentrations of pp65-A2 complex over CM5 chip-bound C4.18. (C) Top panel: staining of 721 221 cells which either express HLA-A2 (721 221(A2)) or do not express it (721 221) by A2Ab, C4.4 and C4.18 at 20 μg/mL. MFI are indicated. Lower panel: dose response staining of A2Ab, C4.4 and C4.18 against BLCL HEN expressing HLA-A2. MFI obtained with various concentrations of C4.18 and C4.4 are indicated. (D) The specificity of mutated R2+ mAbs was assessed in a Luminex single antigen bead assay. Results are shown in terms of interval MFI. Positivity threshold was set at 1000.

EXAMPLE

Methods

Donors

[0138] Human peripheral blood samples were obtained from anonymous adult donors after informed consent in accordance with the local ethics committee (Etablissement Français du Sang, EFS, Nantes, procedure PLER NTS-2016-08).

Cell Lines and Culture Conditions

[0139] Human embryonic kidney 293A cells were obtained from Thermo Fisher Scientific, San Jose, Calif., USA (R70507). Cells were grown as adherent monolayers in DMEM (4.5g/l glucose) supplemented with 10% FBS, 1% Glutamax (Gibco) and 1% penicillin (10 000 U/ml)/streptomycin (10 000 U/ml) (a mixture from Gibco). The BLCL cell lines HEN (HLA-A*0201/HLA-A*0101), B721 221 and stably transfected HLA-A2 B721 221 (B721 221 A2) were grown in suspension in RPMI medium supplemented with 10% FBS, 1% Glutamax (Gibco) and 1% penicillin (10 000 U/ml)/streptomycin (10 000U/ml) (a mixture from Gibco).

Plasmid Constructions

[0140] Plasmids for mutagenesis were obtained from Addgene: pGH335_MS2-AID*Δ-Hygro (catalogue n° 85406), pX330S-2 to 7 from the Multiplex CRISPR/Cas9 Assembly System kit (n° 1000000055) and pX330A_dCas9-1×7 from the multiplex CRISPR dCas9/Fok-dCas9 Accessory pack (n° 1000000062). The sgRNA scaffolds in the seven latter plasmids were replaced by the sgRNA_2MS2 scaffold from pGH224_sgRNA_2xMS2_Puro (Addgene n° 85413) and guide sequences then introduced into their BbsI sites before Golden Gate assembly. SgRNA design was performed online using Sequence Scan for CRISPR software (http://crispr.dfci.harvard.edu/SSC/). Final plasmids for mutagenesis thus obtained contain expression cassettes for dCas9 and seven sgRNAs. For production of antibodies, VH and VL regions from human antibodies were subcloned respectively in an IgG-Abvec expression vector (FJ475055) and an Iglambda—AbVec expression vector (FJ51647) as previously described[8]. For mammalian display of antibodies as IgG1, VH and VL regions were subcloned into home-made expression vectors derived from the OriP/EBNA1 based episomal vector pCEP4. The VH and VL expression vectors contain a hygromycin B or Zeocin resistance marker respectively, and a transmembrane region encoding sequence exists in the C gamma constant region sequence.

IgG1 Mammalian Cell Display

[0141] Heavy and light chain expression vectors were co-transfected into the 293A cell line at a 1:1 ratio using JetPEI (PolyplusTransfection, Cat. 101-10N) and cultured for 48 h. Selection of doubly transfected cells was performed using Hygromycin B and Zeocin. Antibody surface expression on the selected cells was confirmed by flow cytometry analysis after staining with a PE-labeled goat-anti-human IgG Fc (Jackson ImmunoResearch).

Peptide MHC Tetramer

[0142] The HLA-A*0201—restricted peptides Pp65.sub.495 (human CMV [HCMV], NLVPMVATV) and MelA27 (melanoma Ag, ELAGIGILTV) and the HLA-B*0702-restricted UV-sensitive peptide (AARGJTLAM; where J is 3-amino-3-(2-nitro)phenyl-propionic acid) were purchased from GL Biochem (Shanghai, China). Soluble peptide MHC monomers used in this study carried a mutation in the α3 domain (A245V), that reduces CD8 binding to MHC class I. Biotinylated HLA-A*0201/MelA.sub.27 (HLA-A2/MelA), HLA-A*0201/Pp65.sub.495 (HLA-A2/Pp65), HLA-B*0702/UV sensitive peptide (HLA-B7/pUV) monomers were tetramerized with allophycocyanin (APC)-labeled premium grade streptavidins (Molecular Probes, Thermo Fischer Scientific, ref S32362) at a molar ratio of 4:1. Where applicable, the avidity of the tetramer for its specific antibody was decreased by mixing specific (ie peptide HLA-A2) and unspecific (ie peptide UV-sensitive HLA-B7) biotinilated monomers before tetramerization with APC-labeled streptavidins at different molar ratios.

Flow Cytometry Analysis

[0143] The specificity and avidity of IgG expressing HEK 293 cells was analysed by flow cytometry. Cells were first stained in PBS containing 0.5% BSA with Ag tetramers for 30 min at room temperature. Anti-PE human IgG was then added at a 1/500 dilution for 15 mn on ice without prior washing. The binding of mutant antibodies was evaluted on 150 000 BLCL cells. Cells were incubated with various concentrations of large-scale purified mAbs diluted in 25 ml of PBS containing 0.5% BSA for 30 min at room temperature. Anti-PE goat anti-human IgG was then added at a 1/500 dilution for 15 min on ice without prior washing.

Mutagenesis

[0144] 4×10.sup.6 anti HLA-A2 IgG-expressing cells were seeded the day before transfection in a 175 cm flask. For each round of mutation, cells were transiently transfected using JET-PRIME (PolyplusTransfection, Cat. 101-10N) with pGH335_MS2-AID*Δ-Hygro together with two other plasmids allowing expression of a total of 9 different sgRNAs along with dCas9 at a ration 1:1:1.

Affinity-Based Cell Selection and Immunomagnetic Enrichment

[0145] After a round of mutagenesis, transfected cells were expanded until confluency over a week. For selection, 10-20×10.sup.6 cells were washed, resuspended in 0.2 mL of PBS containing 2% BSA and the antigen (i.e. APC HLA-A2 tetramers or mixed APC HLA-A2/HLA-B7 tetramers) and incubated for 30 min at room temperature. The tetramer-stained cells were then positively enriched using APC Ab-coated immunomagnetic beads and columns as previously described[8]. The resulting enriched fraction was stained with an anti human IgG-PE. IgG PE+ and tetramer APC+ were collected on an ARIA cell sorter. The adopted strategy for evolution of mAb A2Ab was as follows: 1) three rounds of mutagenesis; 2) magnetic enrichment with 3A2/1B7 tetramer; 3) FACS sorting of positive cells. Positively selected and sorted mutated HEK 293 underwent two new rounds of mutation using the same sgRNAs before selection with the 1A2/3B7 tetramer.

Antibody Production

[0146] Small and large scale productions were performed as previously described.sup.8.

ELISA

[0147] HLA-A2/Pp65 monomers were coated O/N at 4° C. in 100 μL of reconstituted ELISA/ELISPOT coating buffer 1× (Affymetrix) at a final concentration of 2 μg/mL in 96-well ELISA plates (Maxisorp, Nunc). Wells were blocked with 10% FBS DMEM medium (Thermo Fischer Scientific) for 2 h at 37° C. Purified mAbs were serially diluted in PBS (starting concentration:100 μg/ml; dilution factor: 3) and incubated for 2 h at RT. An anti-human IgG-HRP Ab (BD Biosciences) was used at 1 μg/mL for detection after incubation for 1 h at RT. The reaction was visualized by the addition of 50 μL chromogenic substrate (TMB, BD biosciences) for 20 min. ODs were read at 450 nm.

Anti-HLA Antibody Testing (Luminex)

[0148] The specificity analysis of the antibody variants was performed using Single Antigen Flow Bead assays according to the manufacturer's protocol (LabScreen single-antigen LS1A04, One Lambda, Inc., Canoga Park, Calif.), exploring 97 class I alleles. The fluorescence of each bead was detected by a Luminex 100 analyser (Luminex, Austin, Tex.), and recorded as the mean fluorescence intensity (MFI). The positivity threshold for the bead MFI was set at 1000 after removal of the background as previously reported[25]. Clinical relevance of pre-transplant donor-specific HLA antibodies was detected by single-antigen flow-beads.

Surface Plasmon Resonance

[0149] Surface Plasmon Resonance (SPR) experiments were performed at 25° C. on a Biacore 3000 apparatus (GE Healthcare Life Sciences, Uppsala, Sweden) on CM5 sensorchips (GE Healthcare). Capture mAbs were immobilized at 10μg/mL by amine coupling using a mixture of N-hydroxysuccinimide and N-ethyl-N′-dimethylaminopropyl carbodiimide, according to the manufacturer's instructions (GE Healthcare), after a 20-fold dilution in 10 mM sodium acetate buffer pH 5. Then, ethanolamine (1M, pH 8.5, GE Healthcare) was injected to deactivate the sensor chip surface. Purified HLA-A*0201 molecules containing the Pp65.sub.495 peptide were injected at various dilutions over the capture antibodies for 180s at 40 μL/min. A flow cell left blank was used for referencing of the sensorgrams.

Bioinformatics Analysis

[0150] Amplicon preparation: total RNA was purified from 5×10.sup.6 HEK 293 cells and 1 μg of total RNA was reverse transcribed using Superscript reverse transcriptase (ThermoFisher). cDNA was subsequently amplified using Q5 DNA polymerase and primers targeting VH sequences. Sense and antisense primers include target sequences suitable for nextera indexage. Barcodes were further introduced by PCR with indexed nextera and the amplicons were sequenced at the IRIC's Genomics Core Facility at Montreal. Paired-end MiSeq technology (Miseq Reagent Nano kit v2 (500 cycles) from Illumina, Inc. San Diego, Calif., USA) was used, with a 2×250 bp setup.

Pretreatment and Sequence Clustering

[0151] For each chip generated, approximately one million reads were obtained for all the samples. The quality and length distribution of the reads were checked using the FASTQ tool (v0.11.7). After that, for each sample, the paired-end sequences were assembled using the PEAR software (v0.9.6) while keeping only the sequences whose Phred score was greater than 33 and whose overlap was at least 10 nucleotides. Then 30000 sequences were randomly selected to normalize samples. Next, for each sample, full length VH sequences were grouped according to their identity and counted and clusters were formed as described in the text. Mutations observed in the mock control (gRNA only) experiment were then eliminated in order to distinguish site-directed mutations from RT-PCR or sequence errors. Only clusters representing more than 0.1% of the total number of sequences were retained.

Alignment and Mutation Analysis

[0152] For each sample, the generated clusters were annotated by aligning each sequence cluster against the reference sequence using Biostring library (v2.48.0) in a custom R script, to generate a counting table. The generated data were filtered by subtracting the mutations detected in the mock sample. A position matrix was then generated to create a Weblogo using the ggseqlogo library (v0.1). All statistical analyses were performed in a custom R script.

Results

Isolation of a Low Affinity Human Antibody Against HLA-A*0201

[0153] A human HLA-A*0201 molecule (hereafter referred to as HLA-A2) was selected as a target for antibody discovery and maturation as it is easy to obtain blood samples from donors not previously immunized against this MHC allele. In addition, various recombinant HLA molecules were readily available in our laboratory. PBMCs from three HLA-A2-negative donors with negative serology for HLA-A2 circulating antibodies were tested for the presence of blood circulating B cells specific for HLA-A2. This was done by flow cytometry sorting of B cells that bound HLA-A2 tetramers labeled with two different fluorochromes but did not bind HLA-B7 tetramers, using a technique described previously[8, 10]. B lymphocytes stained specifically by HLA-A2 tetramers could be identified in PBMC from all three donors (see FIG. 1A for an example) and were isolated as single cells. We attempted RT-PCR amplification of sequences coding for the variable regions of the heavy and light chains of four B lymphocytes isolated from one donor (NO) using a recently published protocol[8, 10]. A pair of heavy and light chain V region coding sequences was obtained for one of the four cells. After cloning these gene segments into eukaryotic expression vectors in phase with human heavy and light chain constant domains, the corresponding antibody (A2Ab) was successfully produced in the supernatant of transfected HEK cells and tested for its specificity. A2Ab recognizes HLA-A2 but not HLA-B7 in ELISA tests and this recognition does not depend on the peptide loaded into the HLA pocket (FIG. 1B). A single HLA antigen flow bead assay analysis confirmed that A2Ab can recognize HLA-A*0201, but also showed that A2Ab recognizes closely related alleles belonging to the HLA-A*02 supertype (HLA-A*0203, A*0206 and A*6901) and weakly cross-reacts with other MHC A alleles. However, B or C alleles are not recognized (data not shown, results summarized in FIG. 1C). Finally, the affinity of A2Ab for the pp65/HLA-A2 complex was determined by surface plasmon resonance (SPR) to be in the low micromolar range (Kd=8.10.sup.−6, FIG. 1D). This is consistent with the HLA-A2-specific B cells being isolated from a naive/non-immune blood circulating B cell repertoire.

CRISPR-X Targeted Mutagenesis of A2Ab and Screening for Higher Avidity Antibodies

[0154] We used the CRISPR-X approach[24] (FIG. 2A) to mutate the A2Ab sequence. Our overall procedure using iterative mutation and selection is summarized in FIG. 3A. HEK 293 cells were engineered to express cell surface A2Ab by stable transfection of episomal vectors expressing its heavy and light chains (HC and LC, respectively). For induction of mutations, these cells were then transiently transfected with a plasmid coding for AID*Δ fused to MS2 coat protein, and plasmids coding for dCas9 and nine different sgRNAs spanning the sequence coding for the A2Ab HC variable domain (FIG. 2B). AID*Δ is an AID mutant with increased SHM activity whose Nuclear Export Signal (NES) has been removed[24]. It has significantly increased mutation activity compared to wild-type AID without a NES[24]. Three successive transient transfections were performed before cells were screened for expression of mutant antibodies with increased avidity for HLA-A2.

[0155] Cells we started from stably expressed cell surface A2Ab and thus were able to bind tetramers comprising four HLA-A2 molecules. These cells were subjected to three successive transfections. We expected cells expressing higher avidity antibodies post-mutagenesis to be able to bind tetramers containing fewer HLA-A2 molecules. We thus sought to identify cells in the mutated polyclonal population using labeling with a tetramer made up of 3 HLA-A2 molecules and one B7 molecule (3A2/1B7). As shown in FIG. 3B, we were unable to detect any 3A2/1B7-labeled cells in the mutated polyclonal population by flow cytometry, while all cells expressing IgG were labeled with the initial tetramer (4A2) as expected.

[0156] We suspected that 3A2/1B7-labeled cells might be too rare to be detectable in the fraction of the mutated polyclonal population we tested, so we tried to enrich them before analysis. The mutated poylconal population was first incubated with the 3A2/1B7 tetramer coupled to APC, then subjected to positive selection using paramagnetic beads coupled to anti-APC antibodies. After magnetic enrichment, we observed a small proportion of cells clearly labeled by the 3A2/1B7 tetramer (FIG. 3C, left dot-plot). Notably, no such cells were detected when our protocol was carried out using A2Ab-expressing HEK 293 cells transfected with a hyperactive non-guided AID (FIG. 3C, middle dot-plot), or with guide RNAs alone (“mock”, FIG. 3C, right dot-plot). This first “positive” population (R1) was purified by cell sorting and expanded in vitro to yield population R1+ (>95% pure). In marked contrast to the starting population, the R1+ population bound tetramers with just 3 HLA-A2 molecules (3A2/1B7, FIG. 3D, upper left dot-plot).

[0157] To complete a further round of mutagenesis/selection, we exposed the R1+ population to two successive transfections for mutagenesis using the same batch of sgRNAs as above, before selection was performed. This time we used a more stringent enrichment process with tetramers containing only one HLA-A2 molecule (1A2/3B7). A new population of tetramer positive cells was obtained (R2+), with a 2.2 fold increase in the 3A2/1B7 tetramer mean fluorescence intensity compared to R1+ (FIG. 3D, bottom left dot-plot). The R2+ population was also stained by tetramer 1A2/3B7, in marked contrast to R1+ cells (FIG. 3D, compare upper and lower right dot-plots). Each round of mutation and selection thus increases the avidity of the antibodies.

Antibody Sequence Evolution During Mutagenesis and selection Rounds

[0158] As described above, we were unable to detect cells capable of binding to the 3A2/1B7 tetramer after one round of mutagenesis until we used magnetic enrichment. This enrichment generated the R1 population. FACS sorting of this population yielded the R1+ population capable of binding 3A2/1B7 tetramers and the R1− population incapable of binding this tetramer. We used next generation sequencing (NGS) to search for heavy chain sequences enriched in the R1+ population relative to the R1− population and which could contain mutations responsible for the increased affinity of the R1+ population antibodies. 30,000 randomly selected reads from each population were analyzed. Reads represented more than 50 times were placed into a read-specific cluster, while reads represented less than 50 times were grouped together in a category we termed “small clusters”. For the R1+ population, two large clusters representing together 42.5% of reads were detected, in addition to a third large cluster representing WT sequences (Table I). Six other clusters representing together 5.2% of reads were also detected, together with numerous reads in the small cluster category. Seven of these eight non-WT clusters were clearly under-represented in the R1− population, where the WT cluster and small clusters predominated. Mutations observed in the seven clusters were located in the FR3 and CDR3 regions (FIG. 4). They were often shared between different clusters, suggesting that they contribute to the increased affinity of R1+ population antibodies.

[0159] That WT and small cluster sequences represent 52.6% of R1+ reads might seem surprising. However, in the HEK 293 cells subjected to mutagenesis, antibody genes are present on episomal vectors, with several vector copies per cell[26]. Cells selected with the 3A2/1B7 tetramer may contain only one gene copy with a mutation leading to an antibody of increased affinity. All the other copies could contain either no mutation or neutral or even deleterious mutations, yet they will be co-enriched with the copy carrying the affinity-increasing mutation.

[0160] The second round of mutation/selection led to a drastic decline in WT reads (from 13.6% for R1+, to 0% for R2+), while in the R2+ population a cluster representing nearly half of the NGS reads emerged, corresponding to HCs accumulating six mutated amino acids: D74H/S80T/W102L/M112I/G121D/R124P (Table II, FIG. 4). Interestingly, the CDR2 D74H mutation was not detected in the R1+ population. Nine of the thirteen R2+ clusters (a cluster contains more than 50 reads of the cluster-specific sequence) differ only very slightly from this main sequence, underlining a strong convergence of most of the R2+ clusters. The W102, M112I, G121D and R124P mutations were already well represented in the R1+ population (Table I). The second round of mutation/selection led to emergence of two new R2+-specific mutations: D74H in the CDR2 and S8OT in the FR3 region.

Characterization of Evolved Antibodies against HLA-A2

[0161] The R2+ antibodies C4.4 and C4.18 (Tables I and II) were produced as recombinant proteins for comparison of their affinity and specificity to those of the initial A2Ab. As shown in FIG. 5A, C4.4 and C4.18 mAbs show clearly increased reactivity against HLA-A*0201 compared to A2Ab in an ELISA. We next determined C4.18's affinity for HLA-A*0201 by SPR: Kd=10.sup.−7 (FIG. 5B). This is an almost two log increase over that of the initial A2Ab (Kd=8×10.sup.−6). We were unable to make enough C4.4 for SPR studies.

[0162] These results demonstrate that our matured antibodies bind with higher affinity to antigen than A2Ab in fully in vitro tests. But can they bind to antigen expressed on the surface of cells, a prerequisite for biological activity? The initial A2Ab was not of sufficient affinity to bind to two HLA-A2 expressing cell lines tested, 721.221 B cells made HLA-A2 positive by transfection (721.221(A2)), and naturally HLA-A2 expressing BLCL HEN. However, the increased affinity of C4.4 and C4.18 led to ready detection of such binding (FIG. 5C). Binding to 721.221(A2) B cells was HLA-A2 dependent, as no binding was observed to the parental HLA-A2 negative 721.211 B cells. A single HLA antigen flow bead assay analysis confirmed that C4.4 and C4.18 had higher affinity than A2Ab for HLA-A*0201 and also showed a gain in specificity, as they had significantly less crossreactivity against other HLA-A alleles (compare FIG. 5D to FIG. 1C).

Discussion

[0163] We show that starting from a low affinity antibody, CRISPR-X targeting of AID to antibody genes can be used to obtain affinity-matured human antibodies in cellulo in about 6 weeks. Thus we increased the affinity of a fully human anti-HLA-A*0201 mAb to sufficient levels for biological activity and without loss of specificity in just 2 cycles of mutation/selection (each cycle consisting of several successive mutagenesis transfections prior to the selection steps). The low affinity antibody we started from was expressed by naive B cells. Our procedure thus mimics in vitro antibody maturation in secondary lymphoid organs, where naive B lymphocytes stimulated by Ag recognition via specific BCRs of limited affinity go on to generate receptors optimized for Ag recognition.

[0164] Using SHM for in vitro affinity maturation of antibodies is an attractive strategy and has been used previously in a variety of cell lines [2, 27-30]. Some recently described technologies to affinity-mature antibodies in vitro rely on the integration of a library of CDR3 domains using CRISPR Cas9 technology[31] or mutagenesis of only the most permissive CDR positions[32]. Prior to these approaches, the Bowers group pioneered the coupling of AID-induced somatic hypermutation with mammalian cell surface display in the easily transfectable HEK 293 cells for in vitro maturation of mAbs[15]. We have extended this latter approach to include specific targeting of AID to the immunoglobulin genes to be mutated using a combination of dCas9-AID fusions and specific guide RNAs. We have also introduced a magnetic enrichment step prior to FACS sorting of mutated cells to facilitate isolation of cells expressing higher affinity antibodies. These modifications proved necessary to obtain our affinity matured anti-HLA antibodies after only 2 rounds of mutation/selection. Indeed, we were unable to detect any cells carrying higher affinity antibodies when AID activity was not targeted to the Ig sequences, and we could only detect and isolate them after the first mutation round if magnetic enrichment preceded FACS sorting.

[0165] While this manuscript was in preparation, Liu et al. described a variety of diversifying base editors and showed that they retained their intrinsic nucleotide preferences when recruited to DNA as MS2 coat fusions. They also demonstrated that it was possible to use diversifying base editors to affinity mature a previously studied murine anti-4-hydroxy-3-nitrophenylacetyl (NP) antibody called B1-8.sup.31. The matured antibodies they obtained contained various mutations that had already been observed after subjecting B1-8 to SHM in a mouse in vivo immunization model. The effect of these point mutations was tested separately, and it was not clear whether any of their antibodies contained multiple mutations. In our study, we define previously unknown combination of mutations that are required to increase the affinity of a human antibody against HLA-A2, without loss of specificity. As might be expected, “beneficial” mutations could be found in the CDR2 and CDR3. Interestingly, CDR3 mutations appeared after the first round of mutation/selection, while CDR2 mutations only appeared after the second round. In addition to the CDR2 and CDR3 mutations, some mutations also appeared in the FR3. In particular, the C4.18 mAb obtained after the second round of mutagenesis differs from the first round C3.9 mAb by only two additional mutated amino acids located in FR3. This is interesting as antibody in vitro evolution studies have suggested that mutations leading to higher affinity often correspond to residues distant from the antigen binding site and that affinity maturation of antibodies occurs most effectively by changes in second sphere residues rather than contact residues[33, 34]. It is also interesting to note that increasing the affinity of our antibodies for HLA-A*0201 also led to an increase in their specificity: they progressively lost their crossreactivity against non-HLA-A*02 alleles.

[0166] The progressive evolution of A2Ab we observed, with a gradual accumulation of combinations of mutations, is probably necessary for the maturation of the affinity of most antibodies. The combination of CDR and FR mutations could result from CRISPR-X allowing simultaneous targeting of multiple sites all along the Ig variable sequence and potentially represents an important advantage over other recently described technologies limiting mutagenesis to the CDR3[31] or to the most permissive CDR positions[32].

[0167] Our CRISPR-X based approach can readily be developed further to increase the potential for antibody diversification. We used the same 9 gRNAs for both rounds of mutagenesis. Further rounds of mutagenesis could be carried out using different gRNAs. The CRISPR-X approach using S. pyogenes dCas9 requires the presence of an NGG PAM immediately downstream from the gRNA binding site. Cas9 variants with relaxed PAM requirements could also be used in this approach, including the recently described variant using a PAM reduced to NG. This would lift almost all constraints on gRNA choice. We focused on mutating the Ig heavy chain gene alone, but both heavy and light chain genes were present in cells subjected to mutagenesis. We did not detect any light chain mutations after transfection of the heavy chain gRNAs (data not shown), demonstrating the specificity of the targeting approach. However, AID could be targeted simultaneously to both heavy and light chain genes by cotransfecting cells with a mixture of heavy and light chain gRNAs, increasing the diversification possibilities by association of mutated heavy and light chains in different combinations.

[0168] The A2Ab mAb used here served as an initial proof of concept for antibody maturation in vitro using CRISPR-X. However, the fully human mAbs specific for the HLA-A*0201 allele we generated could have direct clinical applications, notably in the context of mismatch HLA-A2 organ transplantation. Two recent studies described the efficacy of anti-HLA-A2-specific CARs of murine origin in the control of graft rejection in animal models[35, 36]. Using fully human antibodies could be an important step forward for implementation of such strategies to humans. Furthermore, the availability of a series of mAbs of increasing affinity (derived from different rounds of mutation/selection) could be useful to study the impact of CAR affinity on biological activity and could also help to improve predictive algorythms for antibody maturation.

[0169] In conclusion, we describe a new approach for progressive and controlled antibody evolution. This procedure should allow us to obtain antibodies of high affinity and specificity against virtually any Ag, if available in a recombinant form, starting directly from circulating naïve B cells, which represent a vast pool of Ag-specific antibodies to tap into. Our approach may prove particularly useful when fully human antibodies are required: when first isolated from non-immunized individuals, they are often of insufficient affinity for therapeutic or research purposes. Many Ag of interest for the treatment of pathologies such as cancer are in this category and thus represent potential targets for this approach. In addition, our approach can be adapted to optimize antibody specificity by addition of a simple negative selection step to eliminate antibodies with undesired interactions. This could be useful for improving the specificity of currently existing murine, chimeric or humanized antibodies.

TABLES

[0170]

TABLE-US-00005 TABLE I CRISPR-X-mediated evolution of A2Ab: NGS analysis, round 1 R1 mAb % R1+ % R1− Cluster name name (counts) (counts) G121E C3.2 31.8 (9542) 0.3 (94) WT A2Ab 13.6 (4103) 52.6 (15788) W102L//M112I//G121D//R124P C3.9 10.7 (3197) 0 G121E//V140L 1.1 (340) 0 G121D C3.3 1.1 (316) 0.3 (95) S103N//G121D C3.5 0.9 ( 261) 0 W1021//D109A//M112I// 0.8 (239) 0 G121D//R124P M112I//G121D//R124P 0.7 (209) 0 V140L 0.6 (168) 1.5 (448) R117S 0 0.5 (148) Y114S 0 0.4 (124) D109A 0 0.4 (119) S103R 0 0.2 (67) S108A 0 0.2 (66) G137R 0 0.2 (61) R119S 0 0.2 (60) P60A 0 0.2 (54) V123G 0 0.2 (54) small clusters R1+ (number) C3.4 38.7 (11625) small clusters R1− (number) 42.7 (12817) total 100 (30000) 100 (30000)

TABLE-US-00006 TABLE II CRISPR-X-mediated evolution of A2Ab: NGS analysis, round 2 R2 mAb % R2+ % R2− Cluster name name (counts) (counts) D74H//S8OT//W102L//M112I// C4.4 49.2 (14755) 9.2 (2756) G121D//R124P D74H//S8OT//W102L//D109A// 2.4 (733) 0.2 (73) M112I//G121D//R124P D74H//S8OT//M112I// 2.2 (650) 0 G121D//R124P G121E 1.7 (496) 38.9 (11670) D74H//S8OT//F83S//W102L// 0.7 (223) 0.4 (112) M112I//G121D//R124P D74H//S8OT//A98P//W102L// 0.6 (182) 0 M112I//G121D//R124P D74H//S8OT//W102L//M112I// 0.6 (173) 0 G121D//R124P//V140L D74H//W102L//M112I// C4.18 0.5 (163) 0 G121D//R124P G121D//R124P 0.2 (74) 0.9 (274) D74H//S8OT//W102L//S104T// 0.2 (72) 0 M112I//G121D//R124P D74H//S8OT//W102L//G121E 0.2 (68) 0 W102L//M112I//G121D//R124P 0.2 (63) 4.2 (1247) D74H//S8OT//W102L//L105R// 0.2 (59) 0 M112I//G121D//R124P W52C//G121E 0 1.1 (333) G121E//V140L 0 0.7 (209) R47S//R57H//G121E 0 0.7 (204) W102L//G121E 0 0.7 (203) WT A2Ab 0 0.6 (193) M112I//G121D//R124P 0 0.5 (137) W102L//M112I//G121E 0 0.4 (128) H101Q//G121E 0 0.4 (122) I39M//H101Q//G121E 0 0.4 (119) P60S//G121E 0 0.4 (81) C41Y//G121E 0 0.2 (66) I39M//G121E 0 0.2 (56) W102C//G121E 0 0.2 (56) small clusters R+ (number) 41 (12289) small clusters R2− (number) 39.9 (11961) total 100 (30000) 100 (30000)

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