WO2024249320A1

WO2024249320A1 - In vivo optimization of biologics

Info

Publication number: WO2024249320A1
Application number: PCT/US2024/031053
Authority: WO
Inventors: Michael R. Farzan; Wenhui He; Tianling OU; Andi PAN; Yiming YIN
Original assignee: University of Florida; University of Florida Research Foundation Inc
Current assignee: University of Florida; University of Florida Research Foundation Inc
Priority date: 2023-05-26
Filing date: 2024-05-24
Publication date: 2024-12-05
Anticipated expiration: 2025-11-26

Abstract

The disclosure provides methods for promoting affinity maturation, and in particular in vivo affinity maturation, of non-antibody peptides. The disclosure also provides a system of affinity maturation of a non-antibody peptide of interest as well as compositions comprising peptides generated from methods described herein and polynucleotides encoding such systems.

Description

IN VIVO OPTIMIZATION OF BIOLOGICS

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of and priority under 35 U.S.C. § 119(e) to U.S. Provisional Application Number 63/504,709, filed May 26, 2023, titled “IN VIVO OPTIMIZATION OF BIOLOGICS,” the contents of which are incorporated herewith by reference in their entirety.

REFERENCE TO AN ELECTRONIC SEQUENCE LISTING

The contents of the electronic sequence listing (U119770222WO00-SEQ-CEW.xml; Size: 56,598 bytes; and Date of Creation: May 24, 2024) is herein incorporated by reference in its entirety.

GOVERNMENT SUPPORT

The invention was made with government support under Grant Nos. AI171954, AI129868, and DA056771 awarded by the National Institutes of Health. The government has certain rights in the invention.

BACKGROUND

A number of protein therapeutics have been developed to treat a wide range of diseases. Current approaches for improving a protein therapeutic are often cumbersome and time consuming. Accordingly, there is a need for new approaches to improve existing protein therapeutics.

SUMMARY

The present disclosure provides methods for promoting affinity maturation, and in particular, in vivo affinity maturation of a peptide of interest (e.g., a therapeutic peptide), wherein the peptide of interest is not an antibody. In some embodiments, these methods involve CRISPR-based editing of a native coding locus of the B cell receptor (BCR). They involve the replacement of a portion of this locus in a B cell obtained from a subject (such as a mouse) with a heterologous construct encoding the peptide of interest. In accordance with these methods, B cells (e.g., murine B cells) can be edited so that their heavy or light variable chains can be directly replaced by a heterologous construct encoding the peptide of interest, while preserving the organization and regulation of the B-cell receptor locus. The edited B cells can be subsequently adoptively transferred to a mouse subject for somatic hypermutation (SMH) and affinity maturation. BCRs edited by any of the methods described herein are able to affinity mature, which may facilitate development of more potent peptides of interest, and enable engineered B cells to respond adaptively to diverse or changing antigens. The present disclosure thus also provides methods of engineering B cells, and engineered B cells.

In one aspect, the present disclosure provides a method comprising: introducing a nucleic acid comprising a nucleotide sequence encoding a peptide of interest into a genomic locus encoding the endogenous B cell receptor (BCR) in a B cell, thereby generating an engineered BCR; and administering the B cell comprising the engineered BCR to a mammalian subject, wherein the peptide of interest is not an antibody.

In one aspect, the present disclosure provides a system of affinity maturation of a peptide of interest, comprising: a B cell, a nucleic acid comprising a nucleotide sequence encoding a peptide of interest, for generating an engineered BCR in the B cell; and an injection mechanism for administering the B cell comprising the engineered BCR to a mammalian subject, wherein the peptide of interest is not an antibody.

In some embodiments, the peptide of interest is grafted onto the endogenous heavy chain constant region in the engineered BCR. In some embodiments, the peptide of interest is grafted onto the endogenous light chain constant region in the engineered BCR.

In some embodiments, the nucleic acid comprising the nucleotide sequence encoding a peptide of interest further comprises a nucleotide sequence encoding a variable region of a heterologous antibody.

In some embodiments, the peptide of interest is inserted between FR3 and FR4 of the variable region in the heavy chain or light chain in the engineered BCR. In some embodiments, the nucleotide sequence encoding the peptide of interest is inserted into a sequence encoding a variable region of a heterologous antibody between the framework 3 region and framework 4 region, in the nucleic acid being introduced.

In one aspect, the present disclosure provides a method of comprising: contacting a B cell with (i) a homology-directed repair (HDR) template comprising a nucleotide sequence encoding a peptide of interest, (ii) a Cas protein, and (iii) a guide RNA, wherein the B cell comprises heavy and light chain genomic loci encoding a B cell receptor (BCR), and wherein the guide RNA comprises a sequence having complementarity to a region of heavy chain or light chain genomic locus encoding the BCR; whereby the Cas protein introduces a doublestrand DNA break immediately adjacent to a target site in the heavy chain or light chain genomic locus, and the target site is replaced with the nucleotide sequence encoding the peptide of interest through HDR, thereby generating an engineered BCR; and administering the B cell comprising the engineered BCR to a mammalian subject, wherein the peptide of interest is not an antibody.

In one aspect, the present disclosure provides a system of affinity maturation of a peptide of interest, comprising: a B cell, wherein the B cell comprises heavy chain and light chain genomic loci encoding a B cell receptor (BCR), a homology-directed repair (HDR) template comprising a nucleotide sequence encoding a peptide of interest, a Cas protein, and a guide RNA, wherein the guide RNA comprises a sequence having complementarity to a region of the heavy chain or light chain genomic locus encoding the BCR, for generating an engineered BCR in the B cell, and an injection mechanism for administering the B cell comprising the engineered BCR to the subject, wherein the peptide of interest is not an antibody.

In some embodiments, the target site is in the heavy chain genomic locus. In some embodiments, the target site is in the light chain genomic locus.

In some embodiments, the HDR template further comprises a nucleotide sequence encoding a variable region of a heterologous antibody. In some embodiments, the HDR template comprises, from 5’ to 3’, nucleotide sequences encoding the peptide of interest, a linker, and the variable region of the heterologous antibody.

In some embodiments, the HDR template further comprises a nucleotide sequence encoding a heterologous light chain constant domain. In some embodiments, the HDR template comprises, from 5’ to 3’, nucleotide sequences encoding the peptide of interest, a first linker, the heterologous light chain constant domain, and a second linker.

In some embodiments, the peptide of interest is inserted between FR3 and FR4 of the variable region in the heavy chain or light chain in the engineered BCR. In some embodiments, the nucleotide sequence encoding the peptide of interest is inserted into a sequence encoding a variable region of a heterologous antibody between the framework 3 region and framework 4 region, in the HDR template. In some embodiments, the HDR template comprises, from 5’ to 3’, nucleotide sequences encoding FR1 to FR3 of the variable region of the heterologous antibody, a first linker, the peptide of interest, a second linker, and FR4 of the variable region of the heterologous antibody.

In some embodiments, the HDR template further comprises a nucleotide sequence encoding a heterologous light chain constant domain. In some embodiments, the HDR template comprises, from 5’ to 3’, nucleotide sequences encoding the peptide of interest, a linker, the heterologous light chain constant domain, and a cleavage site.

In some embodiments, the peptide of interest is inserted between FR3 and FR4 of the variable region in the heavy chain or light chain in the engineered BCR. In some embodiments, the nucleotide sequence encoding the peptide of interest is inserted into a sequence encoding a variable region of a heterologous antibody between the framework 3 region and framework 4 region, in the HDR template. In some embodiments, the HDR template comprises, from 5’ to 3’, nucleotide sequences encoding FR1 to FR3 of the variable region of the heterologous antibody, a linker, the peptide of interest, a cleavage site, and FR4 of the variable region of the heterologous antibody. In some embodiments, the cleavage site is a P2A cleavage site.

In some embodiments, the HDR template comprises a 3’ homology arm that is homologous to a region proximal to the 3’ region of a J segment and a 5’ homology arm that is homologous to the 5’ region of a V segment.

In one aspect, the present disclosure provides a method, comprising: contacting a B cell with (i) a homology-directed repair (HDR) template comprising a nucleotide sequence encoding a peptide of interest and a nucleotide sequence encoding a variable region of a heterologous antibody, (ii) a Cas protein, and (iii) a guide RNA, wherein the B cell comprises heavy and light chain genomic loci encoding a B cell receptor (BCR), and wherein the guide RNA comprises a sequence having complementarity to a region of heavy chain or light chain genomic locus encoding the BCR; whereby the Cas protein introduces a double-strand DNA break immediately adjacent to a target site in the heavy chain or light chain genomic locus, and the target site is replaced with the nucleotide sequence encoding the peptide of interest and the nucleotide sequence encoding the variable region of the heterologous antibody through HDR, thereby generating an engineered BCR; and administering the B cell comprising the engineered BCR to a mammalian subject, wherein the peptide of interest is not an antibody. In one aspect, the present disclosure provides a system of affinity maturation of a peptide of interest, comprising: a B cell, wherein the B cell comprises heavy chain and light chain genomic loci encoding a B cell receptor (BCR), a homology-directed repair (HDR) template comprising a nucleotide sequence encoding a peptide of interest and nucleotide sequence encoding a variable region of an antibody, a Cas protein, and a guide RNA, wherein the guide RNA comprises a sequence having complementarity to a region of the heavy chain or light chain genomic locus encoding the BCR, for generating an engineered BCR in the B cell, and an injection mechanism for administering the B cell comprising the engineered BCR to the subject, wherein the peptide of interest is not an antibody.

In some embodiments, the variable region of the heterologous antibody is a heavy chain variable region and the guide RNA comprises a sequence having complementarity to a region of the heavy chain genomic locus. In some embodiments, the nucleotide sequence encoding the heavy chain variable region of the heterologous antibody comprises recombined germline VDJ segments.

In some embodiments, the variable region of the heterologous antibody is a light chain variable region and the guide RNA comprises a sequence having complementarity to a region of the light chain genomic locus. In some embodiments, the nucleotide sequence encoding the light chain variable region of the heterologous antibody comprises recombined germline VJ segments.

In some embodiments, the peptide of interest is connected to the N-terminus of the variable region of the heterologous antibody. In some embodiments, the peptide of interest is connected to the variable region of the heterologous antibody via a linker. In some embodiments, the linker is between 5-30 amino acids in length. In some embodiments, the linker is a glycine- serine linker. In some embodiments, the linker is a (G+Sja (SEQ ID NO: 8) linker.

In some embodiments, the replacement of the target site with the sequence encoding the peptide of interest does not result in integration of any exogenous genetic regulatory elements into the heavy chain or light chain genomic locus encoding the BCR.

In some embodiments, the mammalian subject is a rodent. In some embodiments, the mammalian subject is a wild-type mouse. In some embodiments, the mammalian subject is not a transgenic mouse.

In some embodiments, the Cas protein is Cas9, Casl2a or Cas 13. In some embodiments, the method generates an affinity-matured peptide of interest in the subject that is a variant of the peptide of interest.

In some embodiments, the method results in somatic hypermutation and affinity maturation of the peptide of interest in the subject. In some embodiments, the method provides rates of somatic hypermutation of about 0.1%-50% in the peptide of interest.

In some embodiments, the affinity-matured peptide of interest has an enhanced biological property relative to the peptide of interest. In some embodiments, the biological property is bioavailability. In some embodiments, the biological property is binding affinity to a target. In some embodiments, the biological property is inhibition of a target.

In some embodiments, the peptide of interest is an FDA-approved therapeutic peptide. In some embodiments, the peptide of interest binds to a target selected from a soluble protein, a transmembrane protein, and a pathogenic antigen. In some embodiments, the peptide of interest binds to a viral antigen, optionally, an HIV antigen. In some embodiments, the peptide of interest is human CD4 domain 1 and 2 (D1D2) or a variant thereof. In some embodiments, the peptide of interest is a blood factor, a chemokine, a cytokine, a soluble receptor, a thrombolytic agent, a hormone, a hematopoietic growth factor, an interferon, an interleukin, or an enzyme, a nanobody, an adnectin, or a DARPin.

In some embodiments, the HDR template is comprised within a double-stranded DNA (dsDNA) vector. In some embodiments, the HDR template is comprised within an adeno-associated viral (AAV) vector. In some embodiments, the AAV vector is encapsidated in an AAV6 or AAV-DJ capsid.

In some embodiments, the guide RNA comprises a sequence of between 15 and 200 nucleotides that is complementary to a region of the heavy chain or light chain genomic locus.

In one aspect, the disclosure provides an affinity-matured variant of the peptide of interest generated using the methods of the disclosure.

In one aspect, the disclosure provides an engineered mature B cell generated using the methods of the disclosure.

In one aspect, the present disclosure provides a B cell comprising an affinity-matured variant generated using the methods of the disclosure.

In one aspect, the present disclosure provides a population of B cells comprising one or more affinity-matured variants generated using the methods of the disclosure. In one aspect, the disclosure provides a method of administering an affinity-matured variant or a B cell or B cells of the disclosure, to a subject. In some embodiments, the subject is a human.

In one aspect, the disclosure provides a nucleic acid molecule encoding an engineered murine B cell receptor (BCR) comprising a peptide of interest, wherein the peptide of interest is not an antibody, and wherein the nucleic acid molecule comprises endogenous murine BCR regulatory elements.

In one aspect, the disclosure provides a nucleic acid molecule encoding an engineered murine B cell receptor (BCR) comprising a peptide of interest and a variable region of a heterologous antibody, wherein the peptide of interest is not an antibody, and wherein the nucleic acid molecule comprises endogenous murine BCR regulatory elements.

In one aspect, the disclosure provides a pharmaceutical composition comprising an affinity-matured variant or a B cell according to the disclosure.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 shows a schematic of the insertion of a D1D2 cassette into native IgH loci. The insertion of the cassette grafts D1D2 onto BCRs in vitro. In one embodiment, Mb2Cpfl recognizing a CTTA PAM sequence generated a cleavage at IgH J4 under the guidance of a gRNA. rAAV-DJ delivered HDRTs that targets 600bp VH1-26 promoter and its signal peptide, and an intron downstream IgH J4. The D1D2 cassette ended with a splice donor that allowed linkage to endogenous HC constant region and expression of a D1D2-BCR, differentiation into memory B cells and plasma cells upon subsequent antigen-induced activation.

FIG. 2 shows the structure of a BCR-based D1D2 expression cassette. In one embodiment, D1D2 (173 amino acids) is connected by a (GGGGS)3 (SEQ ID NO: 8) linker to N terminus of the variable region of the heavy chain of mouse monoclonal antibody 0KT3. After insertion of the cassette in the heavy chain locus, an endogenous intron proceeding D1D2 separates it from an endogenous signal peptide. D1D2 is connected by a (GGGGS)3 (SEQ ID NO: 8) linker to N terminus of full-length HC of mouse monoclonal antibody 0KT3. Antibodies displayed on edited B cells consists of D1D2-OKT3 and an endogenous constant domain paired with an endogenous light chain.

FIGs. 3A and 3B show that engineered B cells expressed D1D2 and could bind to

HIV-1 gpl20. FIG. 3A shows scatter plots of primary mouse B cells that were isolated from spleen 36 hours before electroporation and transduced with 2 x 10⁴ M.O.I. rAAV DJ. Control cells received the same dose of rAAV but no gRNA. Edited cells were stained 48 hours after electroporation. B cells were stained by anti-IgM antibodies, anti-human CD4 antibodies or dimeric ConM gpl20. They were first gated on singlet viable IgM+ cells. FIG. 3B shows a schematic of the D1D2 construct expressed by the cells of FIG. 3A.

FIGs. 4A and 4B show a different D1D2 display design that also enabled engineered B cells to express D1D2 and bind to HIV-1 gpl20. FIG. 4A shows schematics of different D1D2 display designs. Compared to D1D2-OKT3 construct of FIG. 2, this construct grafted D1D2 onto Kappa LC and connects LC to HC constant domain 1 with a (648)3 (SEQ ID NO: 8) linker. FIG. 4B shows scatter plots of cells expressing the D1D2 constructs stained by anti-IgM antibodies, anti -human CD4 antibodies or dimeric ConM gpl20, and gated on singlet viable cells. The structure of the expression cassette is shown in more detail in FIG. 13.

FIG. 5 depicts an embodiment of the overall workflow of the BCR-based in vivo evolution platform. 5 x 10⁶ engineered CD45.1 B cells with 1% to 15% editing efficiency were adoptively transferred to CD45.2 wild type mice in lOOpl PBS retro-orbitally. Mice were immunized subcutaneously and intramuscularly one day after engraftment and every two weeks. Blood was collected one week after each immunization. Spleen and lymph nodes were harvested after five times of immunizations for B cell isolation. Target cell population was enriched by cell sorting and lysed for mRNA extraction. cDNA libraries were sequenced in 2 x 300bp Miseq platform. Amino acid mutations were identified and ranked by an inhouse pipeline. CD4-Ig variants were produced and their potency, breadth, thermostability, in vivo half-life and autoreactivity were evaluated via Tzm-bl neutralization assays, thermal shift assays, in vivo pharmacokinetics in immunocompromised mice and Hep2 cell assays.

FIG. 6 shows that engineered B cells generated a neutralizing responses in recipient mice after 3 immunizations with monomeric or decameric gpl20. Mouse serum in 96-well plates were incubated with pseudovirus at 37 °C for an hour. TZM-bl cells were then added to the wells at 10,000 cells/well. Cells were then incubated for 48 hours at 37°C. At 48 hours post infection, cells were lysed in wells and luciferase expression was determined. The positive control was 200 pg/ml CD4-Ig. The negative control was from a mouse that was immunized but received unedited B cells. FIG. 7 shows that engineered CD45.1+ B cells persisted, class switched, and bound to gpl20 in immunized mice. These mice received 5 million B cells, the editing efficiency of which was 1%. Primary mouse B cells were isolated from spleen and lymph nodes four days after the 3^rd immunization. They were stained by DAPI, anti CD45.1 antibodies, anti IgG antibodies, and dimeric ConM gpl20. Data shown were gated on singlet viable IgG+ cells. Percent gpl20 reactive cells among CD45.1+ cells ranged from 1% to 2%. Sorted cells were lysed immediately and stored in -80°C for later mRNA extraction.

FIGs. 8A and 8B show amino acid mutations in CD45.1+ IgG+ gpl20+ B cells after five immunizations. 3,000 to 50,000 B cells were isolated from mice four days after the 5^th immunization. Sorted cells were lysed for RNA extraction and deep sequencing. Mutations were identified by an in-house pipeline. Mutation rate represents occurrence among all unique mRNA molecules.

FIG. 9 shows a model with the top ranked amino acid mutations in D1D2. Mutated residues in D1D2 are labeled in red in the structural representation. Three mutations were predicted to be in contact with gpl20. D1D2 binding to CD4 binding site of a protomer of trimeric HIV-1 gpl60, modified from PDB 5U1F is shown.

FIGs. 10A-10C show that mutations in CD4-Ig improve potency against multiple HIV-1 isolates in the 12-isolate global panel. CD4-Ig variants in 96-well plates were incubated with pseudovirus at 37°C for an hour. TZM-bl cells were then added to the wells at 10,000 cells/well. Cells were then incubated for 48 hours at 37°C. At 48 hours post infection, cells were lysed in wells and luciferase expression was determined. IC50 values are represented in colored circles, with geometric mean values indicated by a line. Fold change is calculated by dividing IC50 of CD4-Ig vl by IC50 of CD4-Ig variants. Significant differences from CD4-Ig vl input were determined by paired t-test (***p<0.001).

FIGs. 11A-11I show that mutations in CD4-Ig did not increase autoreactivity to human proteins. The anti-HIV-1 antibody 2F5 served as a positive control. Autoreactivity was measured following the protocol from Zeus Scientific. Briefly, HEp-2 cells slides were incubated with 200 pg/mL CD4-Ig for 30 minutes following two PBS rinses. Immunofluorescence on HEp-2 slides was captured by a BioRad fluorescence microscope. Higher fluorescence indicated higher autoreactivity. FIG. 11A shows 2F5 200 pg/ml; FIG. 11B shows 2F5 20 pg/ml; FIG. 11C shows CD4-Ig vl; FIG. 11D shows CD4-Ig vl.l; FIG. HE shows CD4-Ig vl.2; FIG. HF shows CD4-Ig vl.3; FIG. 11G shows wildtype (wt) CD4-Ig; FIG. 11H shows human serum containing autoreactive antibodies; and FIG. Ill shows human serum containing non-autoreactive antibodies.

FIGs. 12A and 12B show CD4-Ig variants had similar half-life. 10 mg/kg CD4-Ig variants were formulated in 200ul and injected into hFcRn immunocompromised mice (n=6). Sera were collected on Day 1, Day 3, Day 6, Day 14, Day 18 and Day 31 after injection. CD4-Ig vl.3 showed similar half-life to input version. FIG. 12A shows CD4-Ig concentration as determined by ELISA with anti-CD4 antibodies and corresponding CD4-Ig standards. FIG. 12B shows the half-life calculated by fitting a one-phase decay model. (* p<0.05, ** p<0.01, ***p<0.001, ****<0.0001).

FIG. 13 shows the structure of a BCR-based D1D2 expression cassette. In one embodiment, a signal peptide is connected to D1D2, which is connected by a linker to an Ig Kappa light chain constant region, which is further connected to a (GGGGSja (SEQ ID NO: 8) linker. After insertion of the cassette into the heavy chain locus, D1D2 is grafted onto the Kappa light chain constant region, which is connected to the endogenous heavy chain constant domain 1 with a (648)3 (SEQ ID NO: 8) linker.

FIG. 14 shows the genetic framework of a heavy chain variable region CDR3-based display.

FIG. 15 shows a schematic of a strategy for evolving higher affinity mincle using CDR3-based display.

FIG. 16 shows an illustration of an embodiment of a BCR-based mincle expression cassette, mincle-OKT3-CDR3KO.

FIGs. 17A-17D show engineering of primary murine B cells to express a B-cell receptor with CD4 domains 1 and 2 (D1D2). FIG. 17A shows an exemplary schematic of an engineered BCR with a potency and half-life enhanced form of CD4 D1D2 fused through a linker (e.g., (648) (SEQ ID NO: 8) linker) to the amino-terminus of the heavy-chain variable region of the mouse antibody OKT3 (D1D2-OKT3-VH). The OKT3 heavy chain pairs with an endogenous mouse light chain. FIG. 17B shows introduction of D1D2-OKT3-VH (the “D1D2 cassette”) at the murine heavy-chain locus. The CRISPR effector protein Mb2Casl2a targets the J4 coding region 5’ of a CTTA PAM sequence. An rAAV-delivered homology directed repair template (HDRT) containing the D1D2 cassette complements the 5’ UTR of a VH segment and the intron 3’ of JH4 with 576 bp and 600 bp homology arms, respectively. The VDJ-recombined heavy chain is replaced with the D1D2 cassette encoding D1D2- OKT3-VH. FIG. 17C shows expression of D1D2-OKT3-VH in primary mouse B cells. Expression of D1D2 in the edited cells was measured by flow cytometry with monomeric HIV-1 gpl20. The representative flow cytometry plots show B cells edited with HDRT targeting the 5’ UTR of VH1-34 or VHI-64, generated 48 hours after electroporation. The HDRT were delivered to cells with rAAV transduced at 10⁴ multiplicity of infection (MOI). Controls include cells electroporated with Mb2Casl2a ribonucleoproteins (RNP) without rAAV (No HDRT) or without gRNA but transduced with HDRT-encoding rAAV (No gRNA). Plots were gated on viable singlet B cells. FIG. 17D shows quantitation of the editing efficiency in FIG. 17C from independent experiments. Each dot represents an average from two biological replicates. Error bars represent the standard error of mean (SEM). Statistical significance was determined by two-way ANOVA followed by H-Sidak’s multiple comparisons (****p < 0.0001). “NS” refers to not statistically significant.

FIGs. 18A-18E show neutralizing responses generated by engineered B cells in immunized mice. FIG. 18A shows exemplary experimental designs of immunization and blood collections from mice containing transferred engineered B cells.. Naive B cells from CD45.1 donor mice were engineered ex vivo and 5 million cells per mouse were adoptively transferred to CD45.2 recipient mice 24 h later. Mice were immunized with native-like transmembrane envelope trimers (SOSIP-TM; 16055-ConM-8.1) mRNA-LNP on two-week (2 wk, Days 2, 16, and 30; n = 5; top schematic) or four- week (4 wk, Days 2, 30, 58; n = 5; bottom schematic) intervals, and serum was collected seven days after each immunization. Spleens and lymph nodes were harvested four days after the final immunization, and B cells isolated from these tissues were analyzed by flow cytometry and next-generation sequencing (NGS). FIGs. 18B-18C show neutralization of BG505 HIV-1 pseudovirus in TZM-bl cell assays using sera from mice immunized with SOSIP-TM mRNA. Sera were assessed from mice immunized at two-week (FIG. 18B) or four-week (FIG. 18C) interval. Sera from mice (n = 3) engrafted with unedited CD45.1 B cells and immunized on two-week interval (“mock”) served as negative controls. 100 pg/ml CD4-Ig-vO combined with normal mouse serum served as a positive control. Dots and error bars indicate the median and interquartile range for each group. FIG. 18D shows a summary of the 50% inhibitory dilutions (ID50) of sera from each immunized mouse in FIG. 18B and FIG. 18C. For each experimental design, the columns of data points correspond to, from left to right, samples obtained following the 1^st, 2^nd, and 3^rd immunizations. Statistical significance was determined using repeated- measures two-way ANOVA with Geisser-Greenhouse correction. FIG. 18E shows serum concentrations of CD4-OKT3-IgG after each immunization, measured by ELISA with an anti-CD4 antibody and CD4-OKT3-IgG as the standard. For each experimental design, the columns correspond to, from left to right, samples obtained following the 1^st, 2^nd, and 3^rd immunizations.

FIGs. 19A and 19B show that engineered B cells persist in vivo following immunization. FIG. 19A shows quantification of CD45.1 -positive donor B cells in immunized mice. Four days after the final immunization of the mice characterized in FIGs. 18A-18E, B cells were isolated from their lymph nodes and spleens. B cells were analyzed by flow cytometry. The plot shows the percent of CD45.1 -positive donor cells from mice immunized at two-week (2 wk, n = 4) or four-week intervals (4 wk, n = 5), as compared to control mice engrafted with unedited cells and immunized in parallel (Mock, n = 3). For gating strategies, see FIG. 25A. FIG. 19B shows a greater proportion of CD45.1 donor cells from immunized mice were found to bind to HIV-1 gpl20. The cells analyzed in FIG. 19A were measured for binding to HIV-1 gpl20. See FIG. 26B for source flow cytometry analysis. Error bars indicate SEM. Statistical significance was determined by generalized linear mixed model followed by Tukey HSD pairwise comparisons (*p < 0.05; **p < 0.01; ***p < 0.001).

FIGs. 20A-20E show that DlD2-expressing B cells hypermutated and class switched in vivo. FIG. 20A shows nucleotide mutation frequency across the DlD2-encoding region. mRNA isolated from CD45+ IgG+ gpl20-binding B cells from each of 10 mice was analyzed by NGS and the mean frequency of nucleotide mutations is plotted for each position. Triangles represent the most frequent coding mutations. Codons for R59 and K90 are indicated. FIG. 20B shows distribution of synonymous (Syn) and non- synonymous (Non- syn) mutation frequency across D1D2. Each dot represents the mutation frequency at one nucleotide position, averaged on five mice in each group. The top two dots indicate the nucleotide mutations leading to R59 and K90 mutations. The horizontal line indicates the median. FIG. 20C shows the average number of accumulated mutations per unique sequence. The significance was determined by two-tailed unpair t test. FIG. 20D shows the frequency of synonymous mutations within domains 1 (DI) and 2 (D2) for mice immunized at two- week (2 wk) and four-week (4 wk) intervals. For both experimental design, the left box corresponds to DI, and the right box corresponds to D2. The center line indicates the mean, and the boxes denote quartile range. Repeated measure mixed effects analysis with H-Sidak’s multiple comparisons (*p < 0.05). FIG. 20E shows the distribution of accumulated nucleotide mutations per unique D1D2 sequence. The center line indicates mean, and boxes denote quartile range. Statistical significance in FIG. 20B and FIG. 20E was determined by mixed effects analysis with H-Sidak’s multiple comparisons (*p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001). “NS” refers to not statistically significant.

FIGs. 21A-21C show diverse and convergent amino-acid mutations in engrafted mice. FIG. 21A shows the frequency of amino acid changes across D1D2 sequences from each mouse (Ml through M10). Three amino acids with the highest mutation rate are labeled (N30, R59, and K90). Samples for M6 and M7 were combined in the sequencing analysis. FIG. 21B shows amino acid changes found in the eight most frequently mutated D1D2 residues from mice Ml (left panel) and M2 (right panel) immunized at two- week intervals. The data for remaining mice are shown in FIGs. 28A and 28B. FIG. 21C shows minimum spanning trees of D1D2 sequences from individual mice (Ml, top tree and sequences; M2, bottom tree and sequences). Each tree presents the inferred lineage and all amino-acid mutations found in Ml and M2. The central black dot represents the inferred ancestral sequence which corresponds to the input sequence. Each circle indicates a distinct amino-acid sequence. Circle size is proportional to the number of distinct nucleotide sequences with the same translation. The additional circles mark translations encoded by the eighteen largest number of distinct sequences, with the rank order indicated by number. Branch length corresponds to evolutionary distance, defined as the number of amino-acid differences. The figures for the remaining mice are provided in FIGs. 29A-29P.

FIGs. 22A-22C show that in vivo hypermutations in D1D2 improve the neutralization potency of CD4-Ig-vO. FIG. 22A shows neutralization potency of CD4-Ig-vO and its variants R59K, K90R, or both R59K K90R against the indicated isolates in TZM-bl assays. Curves showing the neutralization potency are presented in FIG. 30A. Statistical significance was determined by two-way ANOVA with Dunnett’s multiple comparisons. FIG. 22B is a representation showing the location of selected D1D2 mutations (arrows) shown on a structure of CD4 (ribbon structure) bound to an HIV-1 Env trimer (light grey space filled structure). Structure was adapted from pdb:5UlF. FIG. 22C shows representative neutralization curves of CD4-Ig, CD4-Ig-vO and vl-v4 variants bearing combinations of mutations shown in Table 3, against a 12-isolate global panel of HIV- 1 pseudo viruses. All curves were fitted with a variable slope four parameters dose response model. Each dot represents an average of two independent experiments. Center lines indicate median.

FIGs. 23A-23C show a comparison of three display strategies for expressing a biologic (e.g., D1D2) in the native coding locus of the B cell receptor (BCR). FIG. 23A shows the structure of edited BCRs showing three designs of biologic- e.g., CD4 D1D2) presenting B-cell receptors. Below each structure is a representation of its corresponding cassette. D1D2 was attached to the N terminus of the 0KT3 heavy chain variable region (CD4-0KT-VH, left schematic) or the 0KT3 kappa light chain constant region (KC, center and right schematics). The 0KT3 kappa light chain constant region was linked to heavy chain constant region via a GS linker (CD4-CK-GS, center schematic), or non-covalently associated with heavy chain constant region by P2A cleavage (CD4-CK-P2A, right schematic). FIG. 23B shows the exemplary editing strategy used to express constructs in FIG. 23A in primary murine B cells. In this case, homology arms of the repair template complement an intronic region immediately downstream of JH4 (see, e.g., Yin et al. bioRxiv, 2023.2010.2020.563154 (2023); Hartweger et al. J. Exp. Med. 216: 1301-1310 (2019); Huang et al. Nat. Commun. 11: 5850 (2020); and Nahmad et. al. Nat. Commun. 11: 5851 (2020)), and a cassette including a poly- A tail that terminates transcription of the VDJ region, a splice acceptor (SA), a heavy-chain promoter, a D1D2 construct shown in FIG. 23A, and a splice donor (SD). FIG. 23C shows flow cytometric analysis of singlet viable B cells edited using the strategies in FIG. 24B. Cells were stained with anti-IgM antibodies and either antihuman CD4 antibodies (top row) or gpl20 (bottom row) at 48 h post-editing. HDRTs were delivered by 10⁵ MOI rAAV-DJ with homology arms targeting J4 and the intron immediate downstream J4. FIGs. 24A and 24B are created with BioRender.com.

FIGs. 24A-24D show neutralization of mouse serum and CD4-OKT-IgG to various pseudoviruses and immunization regimen of mice receiving protein immunogens. FIG. 24A shows the neutralizing potency against HIV-1 CE1176 pseudovirus. Sera were collected from mice (n = 5) engrafted with edited cells and immunized twice by mRNA-LNP at two-week interval and then were tested in TZM-bl assays, described herein, and represented as individual curves. Sera from mice (n = 3) engrafted with unedited CD45.1 B cells and immunized on two-week interval (“mock”) served as negative controls. Dots and error bars indicate median and interquartile range. CD4-Ig-vO was used as a positive control and was mixed with normal mouse serum at 125 pg/ml. Error bars represent SEM. FIG. 24B shows neutralizing potency of CD4-OKT3-Ig against BG505 (top plot) and TRO11 (bottom plot) pseudoviruses. CD4-OKT3-Ig (solid line)) purified by size exclusion chromatography (SEC) was compared with CD4-Ig-vO (dashed line) for IC50 for BG505 and TRO11 pseudoviruses (PVs). FIG. 24C shows the engraftment and immunization regimen for additional mice containing cells edited according to the same method as FIGs. 17A-17D but vaccinated with protein antigens. Mice were engrafted with 15,000 (Mi l through M15) or 500,000 (M16 through M24) successfully edited B cells, as determined by flow cytometry analysis. Mice were vaccinated with protein antigens (monomeric or decametric ConM and TRO 11 gpl20) at two-week interval. The schedule of blood and tissue collection was the same as in FIG. 18A. Decameric antigens are indicated with an asterisk FIG. 24D shows the BG505 pseudovirus neutralizing response of sera from mice engrafted and immunized according to FIG. 24C. Dots and error bars indicate median and interquartile range. Sera from mice (n = 3) engrafted with unedited CD45.1 B cells and immunized with decametric ConM gpl20 (“Mock 3^rd”) serve as negative controls. Note that the protein immunization only induced a neutralizing response in groups engrafted with a higher number of cells.

FIGs. 25A and 25B show analysis of antigen binding B cells after three immunizations. FIG. 25A shows the gating strategy for flow cytometry analysis of donor and recipient antigen binding B cells and germinal center (GC) B cells in recipient mice. For FIGs. 19A and 19B, mature B cells were gated as singlet viable IgG+ cells. For FIGs. 19C and 19D, GC B cells were gated as singlet viable CD19+ CD1381ow cells. FIG. 25B shows flow cytometry analysis of antigen binding B cells in singlet viable IgG+ donor (CD45.1) and recipient (CD45.2) population for FIGs. 19A and 19B.

FIG. 26 shows analysis of activation-induced deaminase (AID) motif and convergence of the top nucleotide mutations among 10 mRNA-immunized mice. The first two columns show the original amino acid and nucleotide at the indicated position. AID hotspot motifs are defined as DGYW / WRCH (R=A/G, Y=C/T, and W=A/T). Mutation frequency is calculated as an average from 10 mice immunized by mRNA-LNP every two or four weeks. Blue bars indicate percentage of mutation. Y/N indicates whether the mutation is located at AID motifs. Coding mutations are indicated with shading; silent mutations are shown without shading.

FIGs. 27A and 278B show diverse and convergent amino-acid mutations in engrafted mice. FIG. 27A shows the mutation frequency at eight residues with the highest mutation rate across D1D2 from additional mice that were not presented in FIG. 21B (M3 through M10) immunized with mRNA-LNP at two-week and four-week intervals. FIG. 27B shows the mutation frequency at eight residues with the highest mutation rate from additional mice (Ml 6 through M24) immunized with adjuvanted proteins at two-week and four-week intervals, as shown in FIG. 24C. Note that mice Ml 1-M15 were engrafted with low numbers of successfully edited B cells (15,000) like M3-M10; M16-M24, engrafted with 500,000 edited B cells, were immunized with protein antigens. No gpl20-binding donor cells were isolated from mice Ml 1-M15, and they were therefore excluded from NGS analysis.

FIGs. 28A-28P show minimum spanning trees and mutations for mice immunized with mRNA-LNP or adjuvanted protein. Minimum spanning trees of D1D2 sequences are shown for additional mice immunized by mRNA-LNP every two or four weeks (M3 through MIO), similar to those presented in FIG. 21C, or by adjuvanted protein every two weeks (M16 through M24). M6 and M7 were combined into one sample. FIG. 28A shows mouse M3; FIG. 28B shows mouse M4; FIG. 28C shows mouse M5; FIG. 28D shows combined results from mice M6 and M7; FIG. 28E shows mouse M8; FIG. 28F shows mouse M9; FIG. 28G shows mouse MIO; FIG. 28H shows mouse M16; FIG. 281 shows mouse M17; FIG. 28J shows mouse M18; FIG. 28K shows mouse M19; FIG. 28L shows mouse M20; FIG. 28M shows mouse M21; FIG. 28N shows mouse M22; FIG. 280 shows mouse M23; and FIG. 28P shows mouse M24. Each tree presents the inferred lineage and all amino-acid mutations found in each mouse. The central black dot represents the inferred ancestral sequence which corresponds to the input sequence. Each circle indicates a distinct amino-acid sequence. The circle size is proportional to the number distinct nucleotide sequences with the same translation. Colored circles mark the translations encoded by the largest number of distinct sequences, with the rank order indicated by number. Branch length corresponds to evolutionary distance, defined as the number of amino-acid differences.

FIGs. 29A-29C show neutralizing potency of CD4-Ig variants with the indicated single or double mutations against a panel of HIV-1 isolates. FIG. 29A shows neutralization curves of CD4-Ig variants modified with R59K, K90R, or R59K K90R against the indicated HIV-1 pseudoviruses. IC 50 values are presented in FIG. 22A. FIG. 29B shows neutralization curves of CD4-Ig N30H, a recurring mutation from NGS analysis, against three HIV-1 pseudoviruses (CNE55, left panel; 25710, center panel; and 398F1, right panel). FIG. 29C shows neutralization curves of CD4-Ig variants against BG505 (right panels) and TRO11 (left panels) in an initial screening for potent CD4-Ig variants. All curves were fitted with a variable slope four parameters dose response model.

FIGs. 30A-30C show that the affinity matured CD4-Ig variants retained bioavailability. FIG. 30A shows the polyreactivity of CD4-Ig variants measured by immunofluorescence assays using HEp-2 cells and 200 pg/ml of each antibody. The autoreactive antibody 2F5 served as a positive control (“pc”). Baseline (dashed line) was determined as the fluorescence intensity of negative human serum. Each bar is an average of four independent measurements. Error bars indicate SEM. All CD4-Ig variants were significantly less polyreactive than 2F5, as determined by two-way ANOVA with Dunnett’s multiple comparison (*p < 0.05; ****p < 0.0001). FIG. 30B shows thermostability of the indicated CD4-Ig variants, as measured by differential scanning fluorimetry. Each bar represents an average of two independent experiments, and significance was determined by two-way ANOVA with Dunnett’s multiple comparison (****p < 0.0001). FIG. 30C shows pharmacokinetic studies of CD4-Ig variants in immunocompromised hFcRn mice. 8 mg/kg of the indicated CD4-Ig variants was infused intravenously into six nine- week old mice per group, and sera were collected at days 1, 3, 6, 14, 21 and 31. CD4-Ig concentrations were measured by ELISA with anti-CD4 antibodies. The half-life of the CD4-Ig variants was calculated by fitting a one-phase model. Each dot represents the half-life of a CD4-Ig variant in one mouse. Significance was determined two-way ANOVA with Sidak’s multiple comparisons (*p < 0.05; ****p < 0.0001). “NS” refers to not statistically significant.

FIG. 31 shows IC50 values of CD4-Ig variants.

DETAILED DESCRIPTION

The following detailed description is made by way of illustration of certain aspects of the disclosure. It is to be understood that other aspects are contemplated and may be made without departing from the scope or spirit of the present disclosure. The following detailed description, therefore, is not to be taken in a limiting sense. Scientific and technical terms used herein have meanings commonly used in the art unless otherwise specified. The definitions provided herein are to facilitate understanding of certain terms used frequently herein and are not meant to limit the scope of the present disclosure.

The present disclosure describes an in vivo protein evolution system that utilizes affinity maturation to improve a peptide of interest (e.g., a therapeutic peptide), wherein the peptide of interest is not an antibody. Although engineering B cells to express antibodies in vivo has been previously described ^1-6, the power of affinity maturation in these engrafted B cells has not been fully utilized. The BCR based in vivo evolution system described herein efficiently combines mutagenesis and screening for desired characteristics at once. It is widely applicable to a broad range of peptides in terms of size as well as target. It can positively select for mutations that improve affinity, neutralization breadth, and/or bioavailability, yet avoid deleterious mutations that result in protein misfolding, susceptibility to proteases, self-aggregation, off-target binding, or instability at physiologic temperature. This selection against these undesirable properties obviates the need for time-consuming, trial- and-error optimization for bioavailability necessary with most engineered or in vitro selected biologies. Combined with a relatively rapid developmental cycle, this approach carries distinct advantages over structure-based rational design and in vitro selection techniques including phage or yeast display methods.

As a proof-of-principle, human CD4 domain 1 and 2 (D1D2) was improved using this platform. D1D2 serves as a critical component of HIV-1 entry inhibitor eCD4-Ig. eCD4-Ig is a fusion of D1D2, Fc of human IgGl, and a tyrosine sulfated coreceptor-mimetic sulfopeptide⁷ and is a potential monotherapy to replace cocktails of broadly neutralizing antibodies (bNAbs). The D1D2 variants obtained using the methods and compositions of the disclosure showed ten-fold higher neutralization potency, greater neutralization breadth against HIV-1 viruses, high thermostability, and/or low autoreactivity.

Thus, when a nucleic acid encoding a non-antibody peptide of interest (e.g., a therapeutic peptide) is inserted into a genomic locus encoding the endogenous BCR in a B cell, the peptide of interest undergoes in vivo affinity maturation that efficiently selects for more potent and/or bioavailable variants, enabling rapid development of more effective diagnostic and therapeutic proteins.

Definitions

The terms “peptide” and “protein” are used interchangeably to refer to a naturally derived or recombinant product expressed in cells that is a polymer of amino acid residues linked by peptide bonds, and for the purposes of the instant disclosure, have a minimum length of at least 5 amino acids. Both full-length proteins and fragments thereof greater than 5 amino acids are encompassed by the definition. The terms also include peptides that have co- translational (e.g., signal peptide cleavage) and posttranslational modifications of the peptide, such as, for example, disulfide-bond formation, glycosylation, acetylation, phosphorylation, proteolytic cleavage (e.g., cleavage by furins or metalloproteases), and the like. Furthermore, as used herein, a “peptide” or “protein” refers to a protein that includes modifications, such as deletions, additions, and substitutions (generally conservative in nature as would be known to a person in the art) to the native sequence, as long as the peptide or protein maintains the desired activity relevant to the purposes of the described methods.

As used herein, the term “antibody” refers to a protein that includes a heavy (H) chain variable region (abbreviated herein as VH), and a light (L) chain variable region (abbreviated herein as VL). In another example, an antibody includes two heavy (H) chain variable regions and two light (L) chain variable regions. An antibody can have the structural features of IgA, IgG, IgE, IgD, IgM (as well as subtypes thereof).

The VH and VL regions can be further subdivided into regions of hypervariability, termed “complementarity determining regions” (“CDR”), interspersed with regions that are more conserved, termed “framework regions” (“FR”). The extent of the framework region and CDRs has been precisely defined (see, Kabat, E. A., et al. (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, U.S. Department of Health and Human Services, NIH Publication No. 91-3242, and Chothia, C. et al. (1987) J. Mol. Biol. 196:901-917, see also www.hgmp.mrc.ac.uk). Kabat definitions are used herein. Each VH and VL is typically composed of three CDRs and four FRs, arranged from amino-terminus to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.

The VH or VL chain of the antibody can further include a heavy or light chain constant region, to thereby form a heavy or light immunoglobulin chain, respectively. In one embodiment, the antibody is a tetramer of two heavy immunoglobulin chains and two light immunoglobulin chains, wherein the heavy and light immunoglobulin chains are interconnected by, e.g., disulfide bonds. In IgGs, the heavy chain constant region includes three immunoglobulin domains, CHI, CH2 and CH3.

As used herein, the term “subject” includes any human or nonhuman animal. Except when noted, the terms “patient” or “subject” are used interchangeably. Non-human animals include all vertebrates, e.g., mammals and non-mammals, such as mice, rats, non-human primates, sheep, dogs, cats, cows, horses, chickens, amphibians, and reptiles.

As used herein, the terms “having affinity for” or “specifically binds to” a target are well understood in the art. An antibody or peptide of interest is said to exhibit “specific binding” if it reacts more frequently, more rapidly, with greater duration, greater avidity, and/or with greater affinity with a particular target than it does with alternative targets. It is also understood with this definition that, for example, an antibody or peptide of interest that specifically binds to a first target ligand or antigen may or may not specifically or preferentially bind to a second target. As such, “specific binding” or “preferential binding” does not necessarily require (although it can include) exclusive binding.

Binding affinity (or binding specificity) can be determined by a variety of methods including equilibrium dialysis, equilibrium binding, gel filtration, ELISA, surface plasmon resonance, or spectroscopy (e.g., using a fluorescence assay). These techniques can be used to measure the concentration of bound binding protein as a function of target protein concentration. The concentration of bound binding protein ([Bound]) is generally related to the concentration of free target protein ([Free]) by the following equation:

[Bound] = [Free]/(KD+[Free])

It is not always necessary to make an exact determination of KA or KD though, since sometimes it is sufficient to obtain a quantitative measurement of affinity, e.g., determined using a method such as ELISA or FACS analysis, is proportional to KA or KD, and thus can be used for comparisons, such as determining whether a higher affinity is, e.g., two-fold higher, to obtain a qualitative measurement of affinity, or to obtain an inference of affinity, e.g., by activity in a functional assay, e.g., an in vitro or in vivo assay.

The term “regulatory element,” as used herein, refers to a region or regions of a nucleic acid sequence that regulates transcription. Exemplary regulatory elements include, but are not limited to, promoters, enhancers, post-transcriptional elements, transcriptional control sequences, and such like.

As used herein, the term “variant” refers to a peptide having characteristics that deviate from a base or starting peptide. A “variant” may be at least about 80% identical, at least about 85% identical, at least about 90% identical, at least about 91% identical, at least about 92% identical, at least about 93% identical, at least about 94% identical, at least about 95% identical, at least about 96% identical, at least about 97% identical, at least about 98% identical, at least about 99% identical, at least about 99.5% identical, or at least about 99.9% identical to a base or starting peptide. Variants of a peptide may contain modifications to the amino acid sequence relative to the base or starting peptide, which arise from point mutations in the nucleic acid sequence encoding the base or starting peptide. Variants of a peptide may also contain modifications to the amino acid sequence relative to the base or starting peptide, which arise from addition or deletion of one or more nucleotides relative to the nucleic acid sequence encoding the base or starting peptide. Modifications can include chemical modifications as well as truncations, such as truncations at the N- or C-terminus of a protein sequence.

“Percent (%) identity” refers to the extent to which two sequences (nucleotide or amino acid) have the same residue at the same positions in an alignment. For example, “an amino acid sequence is X% identical to SEQ ID NO: Y” refers to % identity of the amino acid sequence to SEQ ID NO: Y and is elaborated as X% of residues in the amino acid sequence are identical to the residues of sequence disclosed in SEQ ID NO: Y. Generally, computer programs are employed for such calculations. Exemplary programs that compare and align pairs of sequences, include ALIGN (Myers and Miller, 1988), FASTA (Pearson and Lipman, 1988; Pearson, 1990) and gapped BLAST (Altschul et al., 1997), BLASTP, BLASTN, or GCG (Devereux et al., 1984).

The percentage of sequence identity may be calculated over the entire length of the sequences to be compared, or may be calculated by excluding small deletions or additions which total less than about 25 percent or so of the chosen reference sequence. The reference sequence may be a subset of a larger sequence.

When highly-homologous fragments are desired, the extent of percent identity between the two sequences will be at least about 80%, preferably at least about 85%, and more preferably about 90% or 95% or higher, as readily determined by one or more of the sequence comparison algorithms well-known to those of skill in the art, such as e.g., the FASTA program analysis described by Pearson and Lipman (1988) and blastn computer program based on the algorithm of Brutlag et al. (Comp. App. Biosci. 6:237-245 (1990). A preferred method for determining the best overall match between a query sequence (e.g., a sequence of the present disclosure) and a subject sequence, also referred to as a global sequence alignment, can be determined using the FASTA or blastn. In a sequence alignment the query and subject sequences are either both nucleotide sequences or both amino acid sequences. The result of said global sequence alignment is expressed as percent identity. Preferred parameters used in a FASTA amino acid alignment are: Matrix=PAM 0, k-tuple=2, Mismatch Penalty=l, Joining Penalty=20, Randomization Group Length=0, Cutoff Score=l, Window Size=sequence length, Gap Penalty=5, Gap Size Penalty=0.05, Window Size=500 or the length of the subject amino acid sequence, whichever is shorter. Whether a nucleotide is matched/aligned is determined by results of the FASTA sequence alignment. This percentage is then subtracted from the percent identity, calculated by the above FASTA program using the specified parameters, to arrive at a final percent identity score. This final percent identity score is what is used for the purposes of the present disclosure.

As used herein, “AAV” is adeno-associated virus, and may be used to refer to the naturally occurring wild-type virus itself or derivatives thereof. The term covers all subtypes, serotypes and pseudotypes, and both naturally occurring and recombinant forms, except where required otherwise. As used herein, the term “serotype” refers to an AAV which is identified by and distinguished from other AAVs based on capsid protein reactivity with defined antisera, e.g., serotypes including AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, and AAV11. For example, serotype AAV6 is used to refer to an AAV which contains capsid proteins encoded from the cap gene of AAV6 and a genome containing 5' and 3' ITR sequences from the same AAV6 serotype. Pseudotyped AAV refers to an AAV that contains capsid proteins from one serotype and a viral genome including 5'-3' ITRs of a second serotype. Pseudotyped rAAV would be expected to have cell surface binding properties of the capsid serotype and genetic properties consistent with the second serotype. The abbreviation “rAAV” refers to recombinant adeno-associated viral particle or a recombinant AAV vector (or "rAAV vector"). An “AAV virus” or “AAV viral particle” refers to a viral particle composed of at least one AAV capsid protein (preferably by all of the capsid proteins of a wild-type AAV) and an encapsidated polynucleotide. If the particle comprises a heterologous polynucleotide (i.e., a polynucleotide other than a wild-type AAV genome such as a transgene to be delivered to a mammalian cell), it is typically referred to as “rAAV.”

The term “treating” or “alleviating” includes the administration of compounds or agents (e.g., pharmaceutical compositions comprising variant of a peptide of interest) to a subject to prevent or delay the onset of the symptoms, complications, or biochemical indicia of a disease, alleviating the symptoms or arresting or inhibiting further development of the disease, condition, or disorder, such as an infectious disease.

Subjects in need of treatment include those already suffering from the disease or disorder as well as those being at risk of developing the disorder. Treatment may be prophylactic (to prevent or delay the onset of the disease, or to prevent the manifestation of clinical or subclinical symptoms thereof) or therapeutic suppression or alleviation of symptoms after the manifestation of the disease.

As used herein, a “vector” is a nucleic acid with or without a carrier that can be introduced into a cell. Vectors capable of directing the expression of genes encoding for one or more polypeptides are referred to as “expression vectors.” Examples of vectors suitable for the invention include, e.g., viral vectors, plasmid vectors, liposomes and other gene delivery vehicles.

As used herein, “B cells” are B lymphocytes, a type of white blood cell of the lymphocyte subtype and engineered B-cells. Examples of B-cells include plasmablasts, plasma cells, lymphoplasmacytoid cells, memory B cells, B-2 cells, B-l cells, regulatory B- cells. As used herein, “somatic hypermutation” (SHM) refers to the increased mutation of B-cell receptor loci gene regions encoding variable regions of the light and heavy chains in B lymphocytes following antigen stimulation. SHM generally introduces point mutations in the rearranged Ig genes.

Engineered. BCRs and Affinity Maturation

The BCR is a transmembrane receptor protein located on the outer surface of B cells. The receptor's binding moiety is composed of a membrane-bound antibody. The present disclosure provides B cell editing methods. Described herein is a method for rewriting the BCR of mature B cells such that a peptide of interest, which is not an antibody, is expressed on the receptor’s binding moiety. The recombined murine heavy- or light-chain variable region is replaced by a nucleic acid comprising a sequence encoding a peptide of interest, without displacement or additional regulatory elements. Thus, provided herein are methods to directly replace recombined heavy chain- or light chain-variable genes with a nucleic acid encoding a peptide of interest, leaving each locus otherwise unmodified. Expressing the peptide of interest on the receptor’s binding moiety enables affinity maturation of the peptide of interest in vivo upon exposure to its target. The peptide of interest can be any suitable nonantibody peptide. The methods of the disclosure can be used for the affinity maturation of peptides of a wide size range that have a wide range of targets. In some embodiments, the peptide of interest is a therapeutic peptide.

In one aspect, the disclosure provides a method comprising introducing a nucleic acid comprising a nucleotide sequence encoding a non-antibody peptide of interest into a genomic locus encoding the endogenous BCR in a B cell, thereby generating an engineered BCR. In some embodiments, the method further comprises administering the B cell comprising the engineered BCR to a mammalian subject. The peptide of interest may be introduced into a genomic locus encoding the endogenous BCR by any suitable means known in the art.

In some embodiments, the methods of the disclosure graft the peptide of interest onto the endogenous heavy chain constant region or the endogenous light chain constant region in the engineered BCR, such that the peptide of interest is expressed on the surface of the B cell. In some embodiments, the methods of the disclosure graft the peptide of interest onto the endogenous heavy chain constant region in the engineered BCR. In some embodiments, the methods of the disclosure graft the peptide of interest onto the endogenous light chain constant region in the engineered BCR. As used herein, “grafted onto” refers to a connection between the peptide of interest and the endogenous heavy or light chain constant region either directly or through one or more additional peptide segments. For example, in some embodiments wherein the peptide of interest is grafted onto the endogenous heavy chain constant region, the peptide of interest is connected to the N-terminus of the heavy chain variable region of a heterologous antibody that is in turn connected to the endogenous heavy chain constant region.

In some embodiments, the methods of the disclosure introduce the peptide of interest into the heavy chain variable region or light chain variable region of the engineered BCR such that the peptide of interest is expressed on the surface of the B cell. In some embodiments, the peptide of interest is introduced into the heavy chain variable region of the engineered BCR. In some embodiments, the peptide of interest is introduced into the light chain variable region of the engineered BCR. In some embodiments, the engineered BCR comprises a heavy chain variable region or light chain variable region from a heterologous antibody. Thus, in some embodiments, the nucleic acid comprising the nucleotide sequence encoding a peptide of interest further comprises a nucleotide sequence encoding a variable region of a heterologous antibody. A heterologous antibody as used herein refers to an antibody that is not encoded by the native or endogenous genomic loci of the B cell being modified. The heterologous antibody can be any suitable antibody, e.g., any suitable mouse antibody, that is not autoreactive.

In some embodiments, the peptide of interest is connected to the N-terminus of the heavy chain variable region of the engineered BCR. In some embodiments, the peptide of interest is connected to the N-terminus of the heavy chain variable region of a heterologous antibody in the engineered BCR. In such an embodiment, the light chain of the engineered BCR may be the endogenous light chain. In some embodiments, the peptide of interest is connected to the N-terminus of the light chain variable region of the engineered BCR. In some embodiments, the peptide of interest is connected to the N-terminus of the light chain variable region of a heterologous antibody in the engineered BCR. In such an embodiment, the heavy chain of the engineered BCR may be the endogenous heavy chain. In some embodiments, the peptide of interest is fused directly to the N-terminus of the variable region. In some embodiments, the peptide of interest is connected to the N-terminus of the variable region via a linker. Any suitable linker known in the art may be used. In some embodiments, the linker is 5-30 amino acids in length (e.g., 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 amino acids in length). In some embodiments, the linker is 5-10, 10-15, 15-20, 20-25, or 25-30 amino acids in length. In some embodiments, the linker comprises Gly and Ser. In some embodiments, the linker is a Gly4- Ser (SEQ ID NO: 10) repeat linker. In some embodiments, the linker is a (Gly4-Ser)_n (SEQ ID NO: 9) linker, where n = 1, 2, 3, 4, 5, or 6.

In some embodiments, the peptide of interest is inserted between framework region 3 (FR3) and framework region 4 (FR4) of the heavy chain or light chain variable region of the engineered BCR. In some embodiments, the peptide of interest is inserted into the complementarity-determining region 3 (CDR3) of the heavy chain or light chain variable region of the engineered BCR. In some embodiments, the peptide of interest replaces the CDR3 of a variable region of the engineered BCR. In some embodiments, the peptide of interest is inserted between FR3 and FR4 of the heavy chain or light chain variable region of a heterologous antibody in the engineered BCR. Thus, in some embodiments, the methods of the disclosure comprise introducing into a B cell a nucleic acid comprising a nucleotide sequence encoding the peptide of interest inserted into a sequence encoding a variable region of a heterologous antibody between FR3 and FR4. In some embodiments, the nucleotide sequence encoding the peptide of interest is inserted into the CDR3 of a nucleotide sequence encoding a variable region of a heterologous antibody, in the nucleic acid being introduced. In some embodiments, the nucleotide sequence encoding the peptide of interest replaces the CDR3 in a nucleotide sequence encoding a variable region of a heterologous antibody, in the nucleic acid being introduced. In some embodiments, the variable region is a heavy chain variable region. In some embodiments, the variable region is a light chain variable region. Methods of determining the CDR and FR regions of a BCR or antibody are well known in the art. In some embodiments, the N- and C-termini of the peptide of interest inserted between FR3 and FR4 of a variable region are within about 15 A of each other. In some embodiments, the N- and C-termini are within about 14 A, 13 A, 12 A, 11 A, 10 A, 9 A, 8 A, 7 A, 6 A, or 5 A of each other. In some embodiments, the peptide of interest inserted between the FR3 and FR4 of a variable region is between about 5-500 amino acids in length. In some embodiments, the peptide of interest inserted between FR3 and FR4 of a variable region is between about 5-50, 50-100, 100-150, 150-200, 200-250, 250-300, 300-350, 35-400, 400- 450, or 450-500 amino acids in length.

In some embodiments, the peptide of interest is connected to a heterologous light chain constant region, which is in turn connected to the endogenous heavy chain constant region in the engineered BCR. Thus, in some embodiments, the methods of the disclosure comprise introducing into a B cell a nucleic acid comprising a nucleotide sequence encoding the peptide of interest and a nucleotide sequence encoding a heterologous light chain constant domain. In some embodiments, the peptide of interest is connected to the heterologous light chain constant region via a linker. In some embodiments, the heterologous light chain constant region is further connected to the endogenous heavy chain constant region through a second linker. The two linkers may be different (e.g., different amino acids, or different lengths) or they may be the same. Any suitable linkers known in the art may be used. In some embodiments, a linker is 5-30 amino acids in length (e.g., 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 amino acids in length). In some embodiments, a linker is 5-10, 10-15, 15-20, 20-25, or 25-30 amino acids in length. In some embodiments, a linker comprises Gly and Ser. In some embodiments, a linker is a Gly4-Ser (SEQ ID NO: 10) repeat linker. In some embodiments, a linker is a (Gly4-Ser)_n (SEQ ID NO: 9) linker, where n = 1, 2, 3, 4, 5, or 6.

Other designs wherein the peptide of interest is expressed on the surface of the engineered BCR are also contemplated herein.

In some embodiments, the disclosure provides a method of introducing a nucleic acid comprising a nucleotide sequence encoding a peptide of interest into a genomic locus encoding the endogenous BCR in a B cell, using CRISPR-based homology-directed repair (HDR). Accordingly, in some embodiments, the method comprises contacting a B cell obtained from a mammalian subject with (i) a homology-directed repair (HDR) template comprising a nucleotide sequence encoding a non-antibody peptide of interest, (ii) a clustered regularly interspaced short palindromic repeats (CRISPR)-associated protein (Cas), and (iii) a guide RNA, wherein the B cell comprises heavy and light chain genomic loci encoding a B cell receptor (BCR), and wherein the guide RNA comprises a sequence having complementarity to a region of heavy chain or light chain genomic locus encoding the BCR, whereby the Cas protein introduces a double-strand DNA break immediately adjacent to a target site in the heavy chain or light chain genomic locus, and the target site is replaced with the nucleotide sequence encoding the peptide of interest through HDR, thereby generating an engineered BCR. In some embodiments, the method further comprises administering the B cell comprising the engineered BCR to a mammalian subject. In some embodiments, the target site in the heavy chain genomic locus of the B cell. Thus, in some embodiments, the guide RNA comprises a sequence having complementarity to a region of the heavy chain genomic locus. In some embodiments, the target site is in the light chain genomic locus of the B cell. Thus, in some embodiments, the guide RNA comprises a sequence having complementarity to a region of the light chain genomic locus.

In some embodiments, the peptide of interest is introduced into a variable region of the engineered BCR. In some embodiments, the HDR template comprises a nucleotide sequence encoding a peptide of interest and a nucleotide sequence encoding a heavy chain or light chain variable region of a heterologous antibody. In some embodiments, the variable region is a heavy chain variable region and the nucleotide sequence encoding the heavy chain variable region of the heterologous antibody comprises recombined germline VDJ segments. In some embodiments, the variable region is a light chain variable region and the nucleotide sequence encoding the light chain variable region of the heterologous antibody comprises recombined germline VJ segments. In some embodiments, the peptide of interest is connected to the N-terminus of the variable region of the heterologous antibody. In some embodiments, the peptide of interest is connected to the N-terminus of the heavy chain variable region of a heterologous antibody in the engineered BCR. In such an embodiment, the light chain of the engineered BCR may be the endogenous light chain. In some embodiments, the peptide of interest is connected to the N-terminus of the light chain variable region of a heterologous antibody in the engineered BCR. In such an embodiment, the heavy chain of the engineered BCR may be the endogenous heavy chain. In some embodiments, the peptide of interest is connected to the variable region of the heterologous antibody via a linker. In some embodiments, the HDR template comprises, from 5’ to 3’, nucleotide sequences encoding the peptide of interest, a linker, and the variable region of the heterologous antibody. Any suitable linker known in the art may be used. In some embodiments, the linker is 5-30 amino acids in length (e.g., 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 amino acids in length). In some embodiments, the linker is 5-10, 10-15, 15-20, 20-25, or 25-30 amino acids in length. In some embodiments, the linker comprises Gly and Ser. In some embodiments, the linker is a Gly4-Ser (SEQ ID NO: 10) repeat linker. In some embodiments, the linker is a (Gly4-Ser)_n (SEQ ID NO: 9) linker, where n = 1, 2, 3, 4, 5, or 6. The heterologous antibody may be any suitable heterologous antibody, e.g., any suitable mouse antibody, that is not autoreactive.

In some embodiments, the peptide of interest is inserted between FR3 and FR4 of the variable region in the heavy chain or light chain in the engineered BCR. In some embodiments, the nucleotide sequence encoding the peptide of interest is inserted between FR3 and FR4 of a sequence encoding a variable region of a heterologous antibody, in the HDR template. In some embodiments, the variable region is a heavy chain variable region. In some embodiments, the variable region is a light chain variable region. In some embodiments, the HDR template comprises, from 5’ to 3’, nucleotide sequences encoding FR1 to FR3 of the variable region of a heterologous antibody, a first linker, the peptide of interest, a second linker, and FR4 of the variable region of the heterologous antibody. The two linkers may be different (e.g., different amino acids, or different lengths) or they may be the same. Any suitable linkers known in the art may be used. In some embodiments, the linker is 5-30 amino acids in length (e.g., 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 amino acids in length). In some embodiments, the linker is 5-10, 10-15, 15-20, 20-25, or 25-30 amino acids in length. In some embodiments, the linker comprises Gly and Ser. In some embodiments, the linker is a Gly4-Ser (SEQ ID NO: 10) repeat linker. In some embodiments, the linker is a (Gly4-Ser)_n (SEQ ID NO: 9) linker, where n = 1, 2, 3, 4, 5, or 6. The heterologous antibody may be any suitable heterologous antibody, e.g., any suitable mouse antibody, that is not autoreactive. In some embodiments, the peptide of interest to be inserted between FR3 and FR4 of the heavy chain or light chain variable region is a peptide where the N- and C-termini are in proximity to each other in the tertiary structure. In some embodiments, the N- and C-termini of the peptide of interest are within about 15 A of each other. In some embodiments, the N- and C-termini are within about 14 A, 13 A, 12 A, 11 A, 10 A, 9 A, 8 A, 7 A, 6 A, or 5 A of each other. In some embodiments, the peptide of interest inserted between the FR3 and FR4 of a variable region is between about 5-500 amino acids in length. In some embodiments, the peptide of interest inserted between FR3 and FR4 of a variable region is between about 5-50, 50-100, 100-150, 150-200, 200-250, 250-300, 300-350, 35-400, 400-450, or 450-500 amino acids in length.

In some embodiments, the peptide of interest is connected to a heterologous light chain constant domain in the engineered BCR, which is in turn connected to the endogenous heavy chain constant region in the engineered BCR. In some embodiments, the peptide of interest is connected to the heterologous light chain constant region via a linker. In some embodiments, the heterologous light chain constant region is further connected to the endogenous heavy chain constant region through a second linker. In some embodiments, the HDR template comprises a nucleotide sequence encoding a peptide of interest and a nucleotide sequence encoding a heterologous light chain constant domain. In some embodiments, the HDR template comprises, from 5’ to 3’, nucleotide sequences encoding the peptide of interest, a first linker, the heterologous light chain constant domain, and a second linker. The two linkers may be different (e.g., different amino acids, or different lengths) or they may be the same. Any suitable linkers known in the art may be used. In some embodiments, the linker is 5-30 amino acids in length (e.g., 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 amino acids in length). In some embodiments, the linker is 5-10, 10-15, 15-20, 20-25, or 25-30 amino acids in length. In some embodiments, the linker comprises Gly and Ser. In some embodiments, the linker is a Gly4-Ser (SEQ ID NO: 10) repeat linker. In some embodiments, the linker is a (Gly4-Ser)_n (SEQ ID NO: 9) linker, where n = 1, 2, 3, 4, 5, or 6.

In some embodiments, the second linker is a cleavage site, such as a P2A peptide cleavage site.

The heterologous antibody may be any suitable heterologous antibody, e.g., any suitable mouse antibody, that is not autoreactive.

An HDR template of the disclosure further comprises 3’ and 5’ homology arms that target the HDR to the relevant genomic locus, e.g., the heavy chain or light chain genomic locus of the BCR in the B cell. In some embodiments, the HDR template comprises a 3’ homology arm that targets a region proximal to the 3’ region of a J segment and a 5’ homology arm that targets the 5’ region of a V segment. In some embodiments, the 3’ homology arm of an HDR template targeting to the heavy chain genomic locus comprises a sequence that targets a region immediately downstream of the most 3’ JH segment, e.g., IgH J4 in mouse cells. In some embodiments, the 3’ homology arm of an HDR template targeting the light chain genomic locus targets a region immediately downstream of the most 3’ JK segment, e.g., IgK J5 in mouse cells. The 5’ homology arm of an HDR template can target a region 5’ of any IgH V gene. In some embodiments, the 5’ homology arm of an HDR template targeting the heavy chain genomic locus targets a region 5’ of IgH VI -26, Ig VI -34, IgH Vl-64, IgH Vl-80, or IgH Vl-85 in mouse cells. In some embodiments, the 5’ homology arm of an HDR template targeting the heavy chain genomic locus targets the promoter and/or signal peptide of IgH Vl-26, Ig Vl-34, IgH Vl-64, IgH Vl-80, or IgH Vl-85, in mouse cells.

Exemplary HDR templates for displaying D1D2 and mincle are shown in Table 2. An HDR template of a disclosure can comprise a sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to any one of SEQ ID NOs. 2, 4, 6, or 7. In some embodiments, the methods of the disclosure do not result in integration of any exogenous genetic regulatory elements into the heavy chain or light chain genomic locus encoding the BCR. The endogenous regulatory elements of the BCR genomic loci are relied upon for the expression of the engineered BCR.

In some embodiments, the endogenous recombined VDJ is replaced by the nucleic acid encoding the peptide of interest. In some embodiments, the endogenous recombined VDJ is not replaced. In some embodiments, the nucleic acid encoding the peptide of interest is inserted into an intron.

The disclosure further provides engineered B cells, in particular engineered B cells adapted for SHM in an animal model, such as a murine model. Provided herein are engineered primary B cells, such as engineered fully mature murine B cells. In some embodiments, B cells obtained from a wild-type murine subject are transduced with an HDR template of the disclosure

In various embodiments, the disclosed BCR editing methods are performed ex vivo in a B cell obtained from subject. In some embodiments, an engineered B cell containing an engineered BCR is generated by any of the disclosed methods. In some embodiments, the engineered B cell is transferred into the subject from which it was obtained. In some embodiments, the engineered B cell is transferred into a subject other than the subject from which the B cell was obtained. In various embodiments, following this step of adoptive transfer, the BCR is allowed to undergo rapid SHM, class switching, and ultimately, affinity maturation. The disclosed methods may result in a rate of somatic hypermutation in the peptide of interest of about 0.1%-50% (e.g., 0.1%-10%, 10%-20%, 20%-30%, 30%-40%, or 40%-50%), across the length of the peptide of interest. As will be understood by the skilled person, the rate of somatic hypermutation in the nucleic acid may be higher. In some embodiments, the subject is a mammalian subject, e.g., a rodent. In some embodiments, the mammalian subject is a mouse (e.g., a wild-type mouse). In some embodiments, the mammalian subject is not a transgenic mouse. In some embodiments, the mammalian subject is a rat.

In various embodiments, the engineered BCRs of the disclosure are not integrated into the genome of the animal model.

The disclosed methods may be used to generate variants of the peptide of interest that have an enhanced biological property relative to the peptide of interest. Examples of relevant biological properties include but are not limited to, bioavailability and potency. The disclosed methods may be used to generate peptide variants that have a higher potency than the unmutated base or starting peptide of interest. In some embodiments, the peptide variants have higher binding affinity to a target, higher binding avidity to a target, or show greater inhibition of the target than the base or starting peptide of interest. The disclosed methods may be used to generate peptide variants that have higher bioavailability in a mammalian subject, such as a human subject, than the unmutated base or starting peptide of interest. The disclosed methods may be used to generate peptide variants that have greater half-lives than the unmutated base or starting peptide of interest.

Peptides of Interest

The disclosed methods can be applied to promote the affinity maturation of any peptide of interest. Peptides or proteins include molecules with a wide range of size and function, including antibodies and nanobodies. In some embodiments, the disclosed methods can be applied to promote the affinity maturation of any peptide of interest, wherein the peptide of interest is not an antibody. In some embodiments, the peptide of interest is therapeutic peptide. In some embodiments, the peptide of interest is a diagnostic peptide. In some embodiments, the peptide of interest is a reporter peptide. In some embodiments, the peptide of interest is a commercial and/or FDA-approved non-antibody protein biologic. In some embodiments, the peptide of interest is a newly discovered non-antibody protein biologic.

Therapeutic peptides of the present disclosure may be used to, for example, replace a protein that is deficient or abnormal, augment an existing biological pathway, inhibit a target, provide a novel function or activity, in a cell, tissue, organ, or subject. The therapeutic peptide may also be used to elicit an immune response.

Exemplary types of peptides include, but are not limited to, a blood factor, a chemokine, a cytokine, a soluble receptor (e.g., mincle or CD4 D1D2), an adhesin, a thrombolytic agent, a hormone, a serum protein (e.g., anticoagulant), a hematopoietic growth factor, an interferon, an interleukin fe.g., IL-7 or IL-21), or an enzyme, a nanobody, an adnectin, or a DARPin, or a fragment or domain thereof. In some embodiments, the therapeutic peptide is not a nanobody.

A peptide of interest of the disclosure may bind to a target. Exemplary types of targets include, but are not limited to, a soluble protein, a transmembrane protein, and a pathogenic antigen. In some embodiments, the peptide of interest binds an antigen on an infectious agent such as a pathogenic organism. In some embodiments, the peptide of interest binds a viral antigen e.g., an HIV antigen). In some embodiments, the peptide of interest binds and inhibits an activity of the target.

Genomic Editing Tools

Gene editing, or genome editing, is a type of genetic engineering in which DNA is inserted, replaced, or removed from a genome using nucleases. The nucleases may be artificially engineered. Alternately, the nucleases may be found in nature. The nucleases create specific double- stranded breaks (DSBs) at desired locations in the genome. The cell's endogenous repair mechanisms subsequently repairs the induced break(s) by natural processes, such as homologous recombination (HR) and non-homologous end-joining (NHEJ). Nucleases include, for example, Zinc Finger Nucleases (ZFNs), Transcription Activator-Like Effector Nucleases (TALENs), CRISPR nucleases (e.g., a Cas protein), and engineered meganuclease re-engineered homing endonucleases. Cas proteins include, for example, a Type II nuclease (e.g., Cas9), a Type V nuclease (e.g., Cpfl/Casl2a, C2cl/Casl2b, C2c3/Casl2c), or a Type VI nuclease (e.g., C2c2/Casl3).

Described herein are compositions comprising a DNA-binding nuclease that specifically binds to a target site in any B cell gene. In some embodiments, the gene is an immunoglobulin gene, a gene that encodes a protein that enhances antigen presentation, a gene that encodes a protein that suppresses antigen presentation, a gene that includes a sequence that is related to antibody retention or secretion, a gene that encodes a cytokine, a gene that promotes differentiation into a memory B cell, a gene that promotes differentiation into a plasma cell, or a gene that promotes trafficking of a B cell to a lymphoid organ (e.g., lymph node, spleen, bone marrow). In some embodiments, In some embodiments, the target site is in the light chain locus. In some embodiments, the target site in the heavy chain locus.

The disclosed nucleases may mediate homology-directed repair (HDR).

In preferred embodiments, the DNA-binding nuclease comprises a CRISPR/Cas system. The CRISPR (clustered regularly interspaced short palindromic repeats) locus, which encodes RNA components of the system, and the Cas (CRISPR-associated) locus, which encodes proteins (Jansen et al., 2002. Mol. Microbiol. 43: 1565-1575; Makarova et al., 2002. Nucleic Acids Res. 30: 482-496; Makarova et al., 2006. Biol. Direct 1: 7; Haft et al., 2005. PLoS Comput. Biol. 1: e60) make up the gene sequences of the CRISPR/Cas nuclease system. CRISPR loci in microbial hosts contain a combination of CRISPR-associated (Cas) genes as well as non-coding RNA elements capable of programming the specificity of the CRISPR-mediated nucleic acid cleavage.

Accordingly, Cas proteins are provided for use in any of the disclosed genomic HDR editing methods. In some embodiments, the Cas protein is a Cas9. In some embodiments, the Cas protein is a Cas 12a protein. In some embodiments, the Cas protein is a Cas 13 protein.

In some embodiments, “derivatives” or “variants” of a Cas protein are used in the methods of the disclosure. A Cas protein variant shares homology to a Cas protein, or a fragment thereof. For example a Cas protein variant is at least about 70% identical, at least about 80% identical, at least about 90% identical, at least about 95% identical, at least about 96% identical, at least about 97% identical, at least about 98% identical, at least about 99% identical, at least about 99.5% identical, or at least about 99.9% identical to a wild type Cas protein. In some embodiments, the Cas protein variant may have 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 21, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50 or more amino acid changes compared to a wild type Cas protein. In some embodiments, the Cas protein variant comprises a fragment of a Cas protein (e.g., a gRNA binding domain or a DNA-cleavage domain), such that the fragment is at least about 70% identical, at least about 80% identical, at least about 90% identical, at least about 95% identical, at least about 96% identical, at least about 97% identical, at least about 98% identical, at least about 99% identical, at least about 99.5% identical, or at least about 99.9% identical to the corresponding fragment of a wild type Cas protein. In some embodiments, the fragment is at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% identical, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% of the amino acid length of a corresponding wild type Cas protein.

The disclosed native loci editing methods involve the use of guide RNAs (gRNAs) to achieve HDR-directed editing. As such, the methods also involve the introduction of a guide RNA such as a single-guide RNAs (sgRNA) into the cell or the animal model. The guide RNAs (sgRNAs) include nucleotide sequences that are complementary to the target chromosomal DNA. The sgRNAs can be, for example, engineered single chain guide RNAs that comprise a crRNA sequence (complementary to the target DNA sequence) and a common tracrRNA sequence, or as crRNA-tracrRNA hybrids. The sgRNAs can be introduced into the cell or the organism as a DNA (with an appropriate promoter), as an in vitro transcribed RNA, or as a synthesized RNA. In some embodiments, the guide RNA is between 15 and 100 nucleotides long and comprises a sequence of at least 10 contiguous nucleotides that is complementary to a genomic (coding) target sequence encoding a BCR. In some embodiments, the guide RNA is about 100, about 200, about 250, about 300, about 400, or more than about 400 nucleotides long. In some embodiments, the guide RNA is about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 nucleotides long. In some embodiments, the guide RNA comprises a sequence of 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40 contiguous nucleotides that is complementary to the target sequence in the BCR. In some embodiments, the 3' end of the target sequence is immediately adjacent to a protospacer-adjacent motif (PAM) sequence (such as the canonical PAM sequence, NGG).

In some embodiments, the guide RNA comprises a sequence having complementarity to a genomic locus of a BCR. In some embodiments, the guide RNA comprises a sequence having a length of about 10-100 bp, 10-50 bp, 10-40 bp, or 10-30 bp that is complementary to the genomic locus. In some embodiments, the guide RNA has a sequence of 10-30 nucleotides in length that is complementary to the chromosomal target sequences. In some embodiments, the guide RNA has a conserved backbone (or direct-repeat) sequence of about 20 nucleotides. In some embodiments, the length of a guide RNA is dependent on the types of CRISPR effector protein used in the experiment, e.g., Cas9, Casl2a, Casl3. In exemplary embodiments, the guide RNA of the disclosure has a backbone sequence specific for Casl2a. In some embodiments, the guide RNA of the disclosure has a backbone sequence specific for Cas9.

Researchers have invested intense effort to increase the efficiency of HDR and suppress NHEJ. For example, a small-molecule inhibitor of ligase IV, an essential enzyme in the NHEJ pathway, has been shown to increase HDR efficiency. However, this strategy is challenging in post-mitotic cells, which typically down-regulate HDR, and its therapeutic relevance is limited by the potential risks of inhibiting ligase IV in non-target cells. Enhanced HDR efficiency can also be achieved by the timed delivery of Cas9-guide RNA complexes into chemically synchronized cells, as HDR efficiency is highly cell-cycle dependent. Such an approach, however, is limited to research applications in cell culture since synchronizing cells is highly disruptive. Despite these developments, current strategies to replace point mutations using HDR in most contexts are very inefficient (typically ~0.1 to 5%), especially in unmodified, non-dividing cells. In addition, HDR competes with NHEJ during the resolution of double- stranded breaks, and indels are generally more abundant outcomes than gene replacement. These observations highlight the need to develop alternative approaches to install specific modifications in genomic DNA that do not rely on creating double- stranded DNA breaks. A small-molecule inhibitor of ligase IV, an essential enzyme in the NHEJ pathway, has been shown to increase HDR efficiency. However, this strategy is challenging in post-mitotic cells, which typically down-regulate HDR, and its therapeutic relevance is limited by the potential risks of inhibiting ligase IV in non-target cells. Enhanced HDR efficiency can also be achieved by the timed delivery of Cas9-guide RNA complexes into chemically synchronized cells, as HDR efficiency is highly cell-cycle dependent. Such an approach, however, is limited to research applications in cell culture since synchronizing cells is highly disruptive. In some cases, it is possible to design HDR templates such that the product of successful HDR contains mutations in the PAM sequence and therefore is no longer a substrate for subsequent Cas9 modification, increasing the overall yield of HDR products, although such an approach imposes constraints on the product sequences. Recently, this strategy has been coupled to the use of ssDNA donors that are complementary to the nontarget strand and high-efficiency ribonucleoprotein (RNP) delivery to substantially increase the efficiency of HDR, but even in these cases the ratio of HDR to NHEJ outcomes is relatively low (< 2).

In any of the disclosed CRISPR HDR methods, the editing takes about 48 hours, 50 hours, 72 hours, 84 hours, or 96 hours to complete. In any of the disclosed CRISPR HDR methods, the editing takes about 3 days to complete.

The homology arms of the HDR methods of the disclosure may be delivered to the animal subject by a recombinant AAV (rAAV) particle or virion. The rAAV particle of the disclosed methods, may be of any AAV serotype, including any derivative or pseudotype (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 2/1, 2/5, 2/6, 2/8, 2/9, 3/1, 3/5, 3/8, or 3/9). As used herein, the serotype of an rAAV viral vector (e.g., an rAAV particle) refers to the serotype of the capsid proteins of the recombinant virus. In some embodiments, the rAAV particle is not AAV2. In some embodiments, the rAAV particle is not AAV8. Non-limiting examples of derivatives and pseudotypes include rAAV2/l, rAAV2/5, rAAV2/6, rAAV2/8, rAAV2/9, AAV2-AAV3 hybrid, AAVrh.10, AAVrh.74, AAVhu.14, AAV3a/3b, AAVrh32.33, AAV- HSC15, AAV-HSC17, AAVhu.37, AAVrh.8, CHt-P6, AAV2.5, AAV6.2, AAV2i8, AAV- HSC15/17, AAVM41, AAV9.45, AAV6(Y445F/Y731F), AAV2.5T, AAV-HAE1/2, AAV clone 32/83, AAVShHIO, AAV2(Y^F), AAV8 (Y733F), AAV2.15, AAV2.4, AAVM41, AAV-DJ and AAVr3.45. These AAV serotypes and derivatives/pseudotypes, and methods of producing such derivatives/pseudotypes are known in the art (see, e.g., Mol Ther. 2012 Apr;20(4):699-708). The AAV vector toolkit: poised at the clinical crossroads. Asokan Al, Schaffer DV, Samulski RJ.). In some embodiments, the rAAV particle is a pseudotyped rAAV particle, which comprises (a) a nucleic acid segment comprising ITRs from one serotype (e.g., AAV2, AAV3) and (b) a capsid comprised of capsid proteins derived from another serotype (e.g., AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, or AAV 10). Methods for producing and using pseudotyped rAAV vectors are known in the art (see, e.g., Duan et al., J. Virol., 75:7662-7671, 2001; Halbert et al., J. Virol., 74:1524- 1532, 2000; Zolotukhin et al., Methods, 28:158-167, 2002; and Auricchio et al., Hum. Molec. Genet., 10:3075-3081, 2001).

Additional serotypes of the rAAV capsids disclosed herein include capsids include AAV2, AAV6 and capsids derived from AAV2 and AAV6. In addition, such capsids include AAV7m8, AAV2/2-MAX, AAVSHhlOY, AAV3, AAV3b, AAVLK03, AAV7BP2, AAV1(E531K), AAV6(D532N), AAV6-3pmut and AAV2G9. In some embodiments, the homology arms are delivered in an AAV6 capsid. In some embodiments, the homology arms are delivered in an AAV-DJ capsid.

The AAV-DJ capsid is described in Grimm et al., J. Virol., 2008, 5887-5911 and Katada et al., (2019) Evaluation of AAV-DJ vector for retinal gene therapy, PeerJ 7:e6317 each of which is herein incorporated by reference. The AAV-DJ comprises the insertion of 7 amino acids into the HSPG binding domain of the AAV2 capsid and has high expression efficiency in Muller cells following intravitreal injection. The AAV7m8 capsid, which is closely related to AAV-DJ, is described in Dalkara et al. Sci Transl Med. 2013; 5(189): 189ra76, herein incorporated by reference.

Pharmaceutical Compositions and Methods of Administration

In another aspect, the present disclosure provides a composition, e.g., a pharmaceutical composition, containing a variant peptide generated by any of the disclosed methods, formulated together with a pharmaceutically acceptable agent. Such compositions may include one or a combination of (e.g., two or more different) variant peptides of the disclosure. For example, a pharmaceutical composition of the disclosure can comprise a combination of peptides that bind to different regions on the target or that have complementary activities.

Further provided herein are pharmaceutical compositions comprising an engineered primary B cell in accordance with the disclosure. Also provided herein are pharmaceutical compositions comprising a population of engineered primary B cells.

Pharmaceutical compositions of the invention also can be administered in combination therapy, i.e., combined with other agents. Examples of therapeutic agents that can be used in combination therapy are described in greater detail below in the section on uses of the antibodies of the invention.

As used herein, “pharmaceutically acceptable agent” includes any and all carriers, buffers, solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like that are physiologically compatible. Preferably, the agent is suitable for intravenous, intramuscular, subcutaneous, parenteral, spinal or epidermal administration (e.g., by injection or infusion). Depending on the route of administration, the antibody may be coated in a material to protect the compound from the action of acids and other natural conditions that may inactivate the compound.

The pharmaceutical compounds of the invention may include one or more pharmaceutically acceptable salts. A “pharmaceutically acceptable salt” refers to a salt that retains the desired biological activity of the parent compound and does not impart any undesired toxicological effects (see e.g., Berge, S. M., et al. (1977) J. Pharm. Sci. 66:1-19). Examples of such salts include acid addition salts and base addition salts. Acid addition salts include those derived from nontoxic inorganic acids, such as hydrochloric, nitric, phosphoric, sulfuric, hydrobromic, hydroiodic, phosphorous and the like, as well as from nontoxic organic acids such as aliphatic mono- and dicarboxylic acids, phenyl-substituted alkanoic acids, hydroxy alkanoic acids, aromatic acids, aliphatic and aromatic sulfonic acids and the like. Base addition salts include those derived from alkaline earth metals, such as sodium, potassium, magnesium, calcium and the like, as well as from nontoxic organic amines, such as N,N '-dibenzylethylenediamine, N-methylglucamine, chloroprocaine, choline, diethanolamine, ethylenediamine, procaine and the like.

A pharmaceutical composition of the invention also may include a pharmaceutically acceptable anti-oxidant. Examples of pharmaceutically acceptable antioxidants include: (1) water soluble antioxidants, such as ascorbic acid, cysteine hydrochloride, sodium bisulfate, sodium metabisulfite, sodium sulfite and the like; (2) oil-soluble antioxidants, such as ascorbyl palmitate, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), lecithin, propyl gallate, alpha-tocopherol, and the like; and (3) metal chelating agents, such as citric acid, ethylenediamine tetraacetic acid (EDTA), sorbitol, tartaric acid, phosphoric acid, and the like.

Examples of suitable aqueous and nonaqueous carriers that may be employed in the pharmaceutical compositions of the invention include water, ethanol, polyols (such as glycerol, propylene glycol, polyethylene glycol, and the like), and suitable mixtures thereof, vegetable oils, such as olive oil, and injectable organic esters, such as ethyl oleate. Proper fluidity can be maintained, for example, by the use of coating materials, such as lecithin, by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants.

These compositions may also contain adjuvants such as preservatives, wetting agents, emulsifying agents and dispersing agents. Prevention of presence of microorganisms may be ensured both by sterilization procedures, supra, and by the inclusion of various antibacterial and antifungal agents, for example, paraben, chlorobutanol, phenol sorbic acid, and the like. It may also be desirable to include isotonic agents, such as sugars, sodium chloride, and the like into the compositions. In addition, prolonged absorption of the injectable pharmaceutical form may be brought about by the inclusion of agents which delay absorption such as aluminum monostearate and gelatin.

Pharmaceutically acceptable agents include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion. The use of such media and agents for pharmaceutically active substances is known in the art. Except insofar as any conventional media or agent is incompatible with the active compound, use thereof in the pharmaceutical compositions of the invention is contemplated. Supplementary active compounds can also be incorporated into the compositions.

Therapeutic compositions typically must be sterile and stable under the conditions of manufacture and storage. The composition can be formulated as a solution, microemulsion, liposome, or other ordered structure suitable to high drug concentration. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), and suitable mixtures thereof. The proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. In many cases, it will be preferable to include isotonic agents, for example, sugars, polyalcohols such as mannitol, sorbitol, or sodium chloride in the composition. Prolonged absorption of the injectable compositions can be brought about by including in the composition an agent that delays absorption, for example, monostearate salts and gelatin.

Sterile injectable solutions can be prepared by incorporating the active compound in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by sterilization microfiltration. Generally, dispersions are prepared by incorporating the active compound into a sterile vehicle that contains a basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum drying and freeze-drying (lyophilization) that yield a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof.

The compositions described herein may be administered locally or systemically. In certain embodiments, administration will be parenteral administration. In certain embodiments, the pharmaceutical composition is administered subcutaneously, and in certain embodiments intravenously. Preparations for parenteral administration include sterile aqueous or non-aqueous solutions, suspensions, and emulsions.

The amount administered will depend on variables such as the type and extent of disease or indication to be treated, the overall health of the subject, the in vivo potency of the active component, the pharmaceutical formulation, and the route of administration. The initial dosage can be increased beyond the upper level in order to rapidly achieve the desired bloodlevel or tissue level.

Alternatively, the initial dosage can be smaller than the optimum, and the daily dosage may be progressively increased during the course of treatment. Dosing frequency can vary, depending on factors such as route of administration, dosage amount, serum half-life of the antibody, and the disease being treated.

An exemplary route of administration is parenteral, e.g., intravenous infusion. In certain embodiments, a protein or expression vector disclosed herein is lyophilized, and then reconstituted in buffered saline, at the time of administration. Therapeutic Uses

The peptides, expression vectors, compositions and methods disclosed herein can be used to treat a disease or condition in a subject. The method comprises administering to the subject an effective amount of a peptide, expression vector or pharmaceutical composition disclosed herein, either alone or in a combination with another therapeutic agent, to treat a disease or condition in the subject.

The term “administration” or “administering” includes routes of introducing the peptide, expression vector or pharmaceutical composition of the disclosure to a subject to perform its intended function. Examples of routes of administration that may be used include injection (subcutaneous, intravenous, parenterally, intraperitoneally, intrathecal), oral, inhalation, rectal and transdermal. The pharmaceutical preparations may be given by forms suitable for each administration route. For example, these preparations are administered in tablets or capsule form, by injection, inhalation, eye lotion, ointment, suppository, etc. administration by injection, infusion or inhalation; topical by lotion or ointment; and rectal by suppositories. The injection can be bolus or can be continuous infusion. Depending on the route of administration, the peptide, expression vector or pharmaceutical composition of the disclosure can be coated with or disposed in a selected material to protect it from natural conditions which may detrimentally affect its ability to perform its intended function. The peptide or expression vector or pharmaceutical composition of the disclosure can be administered alone, or in conjunction with either another agent. The peptide, expression vector or pharmaceutical composition of the disclosure can be administered prior to the administration of the other agent, simultaneously with the agent, or after the administration of the agent. Furthermore, a peptide of the disclosure can also be administered in a pro-drug form which is converted into its active metabolite, or more active metabolite in vivo.

Any suitable disease, disorder, or condition that is amenable to treatment by a therapeutic peptide may be treated. In some embodiments, the disease, disorder, or condition is a neurodegenerative, proliferative, inflammatory, or autoimmune disease, disorder, or condition. In some embodiments, the disease, disorder, or condition is an infectious disease caused by a pathogenic organism (e.g., a virus).

In some embodiments, the disclosure provides a method of blocking the entry of HIV into a host cell, e.g., a human host cell. The method comprises exposing the host cell to an effective amount of a peptide, expression vector or pharmaceutical composition disclosed herein, either alone or in a combination with another therapeutic agent, to block the entry of HIV into the host cell. The disclosure also provides a method of causing the killing of a host cell, e.g., a human host cell, infected with HIV. The method comprises exposing the host cell to an effective amount of a protein, expression vector or pharmaceutical composition disclosed herein, either alone or in a combination with another therapeutic agent, to cause the killing of the infected host cell. The disclosure also provides a method of causing the inactivation of a viral particle, e.g., an HIV viral particle. The method comprises exposing the viral particle to an effective amount of a peptide, expression vector or pharmaceutical composition disclosed herein, either alone or in a combination with another therapeutic agent, to cause the inactivation of the HIV viral particle. The invention also provides a method of clearing virus particles from the plasma of a subject, e.g., HIV virus particles. The method comprises exposing the subject to an effective amount of a protein, expression vector or pharmaceutical composition disclosed herein, either alone or in a combination with another therapeutic agent, to clear virus particles from the plasma of a subject.

The methods and compositions described herein can be used alone or in combination with other therapeutic agents and/or modalities. The term administered "in combination," as used herein, is understood to mean that two (or more) different treatments are delivered to the subject during the course of the subject's affliction with the disorder, such that the effects of the treatments on the patient overlap at a point in time. In certain embodiments, the delivery of one treatment is still occurring when the delivery of the second begins, so that there is overlap in terms of administration. This is sometimes referred to herein as "simultaneous" or "concurrent delivery." In other embodiments, the delivery of one treatment ends before the delivery of the other treatment begins. In certain embodiments of either case, the treatment is more effective because of combined administration. For example, the second treatment is more effective, e.g., an equivalent effect is seen with less of the second treatment, or the second treatment reduces symptoms to a greater extent, than would be seen if the second treatment were administered in the absence of the first treatment, or the analogous situation is seen with the first treatment. In certain embodiments, delivery is such that the reduction in a symptom, or other parameter related to the disorder is greater than what would be observed with one treatment delivered in the absence of the other. The effect of the two treatments can be partially additive, wholly additive, or greater than additive. The delivery can be such that an effect of the first treatment delivered is still detectable when the second is delivered. The details of one or more embodiments of the invention are set forth in the accompanying Figures, the Detailed Description, and the Examples. Other features, objects, and advantages of the invention will be apparent from the description and the claims.

EXAMPLES

The invention is further illustrated by the following examples which are intended to illustrate but not limit the scope of the invention.

Example 1. In vivo optimization of a non-antibody peptide

The development of an in vivo protein evolution system for improving protein biologies is described. Protein in vivo evolution and characterization comprised four major steps: (1) precise insertion of a protein gene into IgH loci in mouse primary B cells via CRTS PR/Cas 12a, (2) antigen-driven affinity maturation of engineered B cells in vivo, (3) identification of mutations through deep sequencing, and (4) characterization of identified variants. As a proof-of-principle, improvement of human CD4 domain 1 and 2 (D1D2) was tested using this platform.

An IGHV native loci-editing strategy was developed that replaced recombined IgH VDJ genes with, for example, a D1D2 expression cassette. This allowed D1D2 to be expressed on B cell receptors (BCRs) on naive B cell surface utilizing a true endogenous IgH promoter, signal peptide (SP) and enhancer at its native location. FIGs. 1 and 2 show an example of the native-locus editing strategy. A protein display construct was designed where D1D2 was grafted onto variable domain of heavy chain (HC). The D1D2 expression cassette comprises D1D2 connected by a (648)3 (SEQ ID NO: 8) linker to the N terminus of the HC variable region of mouse monoclonal antibody 0KT3 (FIG. 2). This cassette ends with a splice donor that grafts the D1D2-OKT3 transcript to the IgH constant region during alternative splicing. The 0KT3 IgH variable region is interchangeable with any non- autoreactive mouse IgH variable region in this platform. Other designs, each placing the biologic near the heavy-chain or light-chain B cell-receptor locus could also be implemented.

FIG. 1 shows a schematic of the insertion of the D1D2 cassette into native IgH loci to graft D1D2 onto BCRs. To engineer the cells, naive CD45.1 mouse B cells were electroporated with ribonucleoprotein comprising Mb2Cpf 1 endonuclease and a gRNA targeting IgH J4. Immediately after electroporation, homology directed repair templates

(HDRT) were delivered via recombinant AAV-DJ at a multiplicity of infection (M.O.I.) of 2 x 10⁴. The HDRT had a 600 bp 3’ homology arm (HA) that targeted an intron immediately downstream IgH J4, and a 573 bp 5’ HA that targeted IGHV1-26 promoter and SP, to replace the native recombined VDJ with the D1D2 expression cassette. IGHV1-26 is one of the most commonly detected V genes in productive recombination of C57BL/6 mouse splenic IgM+ B cells⁸. This technique also works with comparable efficiency using other murine B chains as targets (for example VH1-80, VH1-64). Since the promoter and SP of mouse V genes are relatively conserved, these HDRT may also target B cells with highly similar promoter and SP of V genes⁹. As shown in cell surface staining, B cells that have undergone IGHV native editing could display D1D2 on BCRs (FIG. 3). Further, edited B cells could bind to HIV-1 gpl20 (FIG. 3).

The editing efficiency of two D1D2 display strategies was also compared. Displaying D1D2 on the N terminus of the OKT3 heavy chain variable region was compared with displaying D1D2 on the N terminus of OKT3 light chain constant domain, which is linked to the heavy chain constant region. Using an intron insertion method, a D1D2 expression cassette was inserted into an intron downstream J4 without replacing VDJ gene. It was also determined that grafting D1D2 on the N terminus of OKT3 HC enabled better expression than grafting D1D2 on Kappa light chain (LC) (FIG. 4). Staining by anti CD4 antibodies showed that D1D2 linked to OKT3 heavy chain variable reached 35% efficiency, and D1D2 linked to the light chain constant region reached 27% efficiency, at the same dose of AAV- DJ.

24 hours after editing, 5 x 10⁶ engineered CD45.1 B cells expressing the D1D2 cassette shown in FIG. 2 were engrafted into immunocompetent CD45.2 mice. To drive D1D2-BCR affinity maturation, these mice were immunized every two weeks for three to five times with lOpg monophosphoryl lipid A, lOpg saponin based adjuvant Quil A, and 5pg monomeric or decameric gpl20 purified by size exclusion chromatography (SEC). ConM gpl20 was used, which was derived from the consensus of all group M HIV-1 isolates, or TRO11 gpl20, which is one of the most CD4-Ig resistant HIV-1 isolates in the 12-isolates global panel¹⁰. Tzm-bl neutralization assay showed that one week after three immunizations, all mice engrafted with edited B cells produced neutralizing responses against TRO11 pseudovirus (PV) (FIG. 6). This suggested that the editing strategy allowed B cells to persist, differentiate and class switch in vivo. To assess whether D1D2 expressing B cells underwent SHM at the D1D2 gene, B cells were isolated from the spleen and lymph nodes, and CD45.1+, IgG+, gpl20 reactive B cells were enriched through fluorescence activated cell sorting (FIG. 7). Sorted cells were lysed for RNA extraction. Libraries were sequenced using Illumina MiSeq 2 x 300bp paired end reads. Sequencing reads were processed by an in-house pipeline. Strikingly, rate of prominent amino acid mutations, such as R59K and K90R/N, reached above 90% in multiple mice (FIGs. 8A-8B). Although B cells were edited with the same HDRT, mutations enriched differently in mice. Notably, some highly mutated common residues were shared among mice. The high mutation rate and common mutations among mice suggested that D1D2 underwent selection and affinity matured in vivo. Differential yet converging selection indicated directed affinity maturation and biological variations of immune system.

CD4-Ig variants bearing one or a combination of mutation candidates H27Y, K59R, D63N, R90K, I138F and T160S were produced and SEC-purified (FIG. 9, Table 1).

Table 1.

CD4-lg version Mutated residues

1.0 Input

1.1 K59R, D63N, R90K

1.2 K59R, D63N, R90K, T160S

1.3 K59R, D63N, R90K, I138F, T160S

1.4 H27Y, K59R, D63N, R90K, I138F, T160S

Half-maximal inhibitory concentration (IC50) of CD4-Ig variants was measured against multiple HIV-1 isolates in the 12-isolates global panel by Tzm-bl neutralization assays. Input version of D1D2 was previously developed by Emmune Inc. and contained several mutations that increased potency and half-life of eCD4-Ig. It was determined that CD4-Ig variants that had new mutations further improved IC50 by an average of ten folds than input (FIGs. 10A- 10C). None of the mutation compromised CD4-Ig neutralization potency. Notably, potency of CD4-Ig variants against resistant HIV-1 isolates, such as TRO11, improved as much as 30 fold. Increased average IC50 suggested that positive selection on D1D2 improved its potency without compromising neutralization breadth. Mutations in CD4-Ig did not induce autoreactivity to human proteins or decreased in vivo half-life, indicating the in vivo protein evolution process could select against reactivity to self proteins, and select for in vivo stability (FIGs. 11A-11I and 12A-12B). This demonstrated the success of the BCR-based in vivo evolution platform. In summary, this BCR based in vivo evolution system is an efficient approach to improve potency, breadth, and stability of protein biologies.

Example 2. CDR3-based peptide display

Another design for displaying peptides, a CDR3-based peptide display, was explored. Any mouse heavy chain framework region can be used to display a peptide with a proximal N and C termini in the CDR3 (FIG. 14). The nucleotide sequence encoding mincle is inserted between the nucleotide sequences encoding FR3 and FR4 of the heavy chain variable region of the mouse antibody 0KT3, to display mincle. A schematic of the experimental plan for evolving higher affinity mincle using CDR3-based display is shown in FIG. 15. FIG. 16 shows an exemplary construct developed for evolving a higher affinity mincle.

Example 3. Exemplary constructs for BCR-based display of non-antibody peptides

The sequences of exemplary HDR templates for displaying D1D2 and mincle on BCRs, with or without the AAV ITRs and a short CMV promoter, are provided in Table 2 below. FIG. 16 shows a construct where mincle is inserted between FR3 and FR4 of the 0KT3.

Table 2. Sequences

References:

1. Moffett, H., et al. (2019) B cells engineered to express pathogen-specific antibodies protect against infection. Sci Immunol. 4(35):eaax0644.

2. Huang, D., Tran, J.T., Olson, A. et al. (2020). Vaccine elicitation of HIV broadly neutralizing antibodies from engineered B cells. Nat Commun 11, 5850.

3. Nahmad, A.D., Raviv, Y., Horovitz-Fried, M. et al. (2020). Engineered B cells expressing an anti-HIV antibody enable memory retention, isotype switching and clonal expansion. Nat Commun. 11, 5851.

4. Voss, J., Gonzalez-Martin, A., Andrabi, R., et al. (2019) Reprogramming the antigen specificity of B cells using genome-editing technologies. Elife. 8:e42995.

5. Hartweger, H., McGuire, A., Horning, M., et al. (2019). HIV-specific humoral immune responses by CRISPR/Cas9-edited B cells. J Exp Med. 216(6): 1301- 1310.

6. Greiner, V., et al. (2019). CRISPR-mediated editing of the B cell receptor in primary human B cells. iScience. 12:369-378.

7. Gardner, M., Kattenhom, L., Kondur, H. et al. (2015) AAV-expressed eCD4-Ig provides durable protection from multiple SHIV challenges. Nature 519, 87-91. 8. Lin, S., et al. (2016). Highly sensitive and unbiased approach for elucidating antibody repertoires. Proc Natl Acad Sci. 113 (28) 7846-7851.

9. Johnston, Colette M et al. (2006) Complete sequence assembly and characterization of the C57BL/6 mouse Ig heavy chain V region. J. Immunol. Res. 176,7: 4221-34.

10. deCamp, A., et al. (2014) Global panel of HIV-1 Env reference strains for standardized assessments of vaccine-elicited neutralizing antibodies. J. Virol., 88(5), 2489-2507.

Example 4. In vivo affinity maturation of the HIV-1 Env-binding domain ofCD4

This Example describes engineering of the B-cell receptor of primary murine B cells to express a human protein biologic without disrupting the ability of the locus to affinity mature. Previously, a number of in vitro approaches for improving the affinity of protein biologies have been developed, including phage, yeast, and mammalian-cell display techniques, as well as structure-guided design²'⁴. Often these improvements associate with properties that impair the bioavailability of these biologies, for example by increasing interactions with serum or cell-surface proteins or cell membranes⁵'¹¹. In contrast, the natural process of affinity maturation in germinal centers of immunized animals selects for higher affinity while eliminating self-reactive, unstable, poorly expressed, or protease- sensitive antibody variants¹²'¹⁵. In addition, in vitro selection methods typically proceed in a small number of discrete steps without ongoing diversification of selected intermediates, whereas diversification and selection in vivo are continuous, coordinated, and sensitive to small affinity differences^{12, 16, 17}. Thus, the affinity maturation in the mammalian germinal centers described herein has critical advantages over in vitro techniques for improving the affinity and bioavailability of protein therapeutics.

It was previously demonstrated that human heavy- and light-chain variable regions can directly replace their murine counterparts in primary mature B cells, a technique described as ‘native-loci’ editing¹⁸. When these CRISPR/Casl2a-engineered cells were adoptively transferred to recipient mice, their B-cell receptors (BCRs) affinity matured in response to antigen, facilitating identification of broader, more potent, and more bioavailable forms of the original human antibody. It was further observed that somatic hypermutation and affinity maturation of the engineered BCR were markedly more efficient when exogenous variable chain genes were introduced at their native-loci rather than in a commonly used intronic region, indicating that the latter insertion disrupted the activities of the engineered cell.

However, many important protein therapeutics are not antibodies. Rather, they were developed as soluble forms of cellular receptors and recombinant forms of cytokines and serum proteins, such as hirudin¹⁹, IL-27²⁰, factor IX²¹, CTLA4^{22, 23}, tumor necrosis factor^{24, 25}, and the HIV-1 receptor CD4^{26, 27}. To determine if in vivo affinity maturation could serve as a useful tool for improving the potency of non-antibody biologies, the domains 1 and 2 (D1D2) of human CD4 were selected as an exemplary molecule for proof-of-principle studies, with the goal of improving a recently developed CD4-Ig variant, CD4-Ig-vO, which was a product of iterative in vitro optimization for half-life, breadth, and potency^{30, 31}. CD4-Ig, the immunoadhesin form of CD4 D1D2, had been evaluated as a potential therapy for HIV-1 infection, but its limited half-life precluded its clinical use^{26, 28, 29}.

As described herein a sequence encoding D1D2 fused to an antibody heavy chain variable region was introduced into the VDJ-recombined locus of primary murine B cells, these cells affinity matured in recipient mice immunized with mRNA lipid nanoparticles (LNP) that expressed the HIV-1 envelope glycoprotein. High frequency somatic hypermutations observed in multiple mice markedly enhanced the neutralization potency of CD4-Ig-vO to below 1 pg/ml while retaining its near absolute breadth, high thermostability and long in vivo half-life. Thus, non-antibody protein therapeutics can be expressed from the BCR locus and affinity matured in mouse germinal centers.

Engineering primary mouse B cells to express a protein biologic ex vivo

It was previously demonstrated that introduction of human antibody heavy chain complementarity determining regions (HCDR3s) or heavy- and light-chain variable genes into their respective murine loci enabled in vivo affinity maturation of the resulting chimeric BCRs^{18, 32}. However, extension of this approach to immunoadhesins such as CD4-Ig is complicated by the absence of the first heavy constant region 1 (CHI) that noncovalently associates with a light-chain constant domain. Three designs of BCRs capable of presenting a half-life and potency optimized form of human CD4 domains 1 and 2³⁰ (D1D2; residues 1- 173 of the mature CD4 protein) were evaluated in primary murine B cells while ensuring that CHI associates with a CK domain (FIGs. 23A and 24B). Among these designs, the highest expression of D1D2 was observed when it was fused to the amino-terminus of the heavy- chain variable domain of the murine antibody 0KT3 (0KT3-VH) through a (648)3 (SEQ ID NO: 8) linker (FIG. 17A; FIG. 23C). In this design, 0KT3 VH and CHI domains associate with an endogenous light chain expressed in these cells. The VDJ-recombined heavy-chain variable (VH) gene of naive mature murine B cells was replaced with a sequence encoding this construct, D1D2-OKT3-VH, while utilizing a native murine variable- segment promoter and a native heavy-chain signal sequence (FIG. 17B). Accordingly, double- stranded break was introduced by CRISPR/Mb2Casl2a ribonucleoproteins (RNPs) into the 3’-most JH segment, JH4 in the mouse genome^{18, 32, 33}. Repair of this break was guided by a homology- directed repair template (HDRT) delivered by recombinant adeno-associated virus DJ (AAV- DJ). This HDRT included homology arms complementary to a 570 bp 5’ untranslated region (UTR) upstream of VH1-34 or VH1-64, and a 600 bp 3’ intronic region immediately downstream of the J4 segment. An insert sequence encoding D1D2-OKT3-VH and terminating with a modified heavy-chain splice donor was located between these homology arms. This resulted in the VH, DH, and JH segments between the targeted VH (VH1-34 or VH1-64) and JH4 segment being replaced with this chimeric heavy-chain gene, and the expressed protein resembles a mouse BCR except that it initiates with D1D2.

The efficiency of HDRTs with 5’ homology arms targeting either VH1-34 or VH1-64 was compared. VH1-64 is upstream of VH1-34 in the heavy-chain locus and more commonly used in C57BL/6 mouse splenic IgM+ B cells^{34, 35}. It was observed that HDRT targeting VH1-34 resulted in an average of 11% editing efficiency, similar to that observed with AAV targeting VH1-64, as indicated by flow cytometry using a fluorescently labeled anti-CD4 antibody or HIV-1 envelope glycoprotein gpl20 (FIGs. 17C-17D). This efficiency is higher than necessary for expansion and affinity maturation of engineered B cells ^{16, 18, 32}. Thus, primary murine B cells can be engineered ex vivo to efficiently express an exemplary biologic, D1D2-OKT3-V_H.

Engineered B cells generated neutralizing sera in immunized mice

To evaluate the in vivo activity of engineered cells, splenic B cells were harvested from B6 CD45.1 mice, and the heavy-chain variable genes were replaced with D1D2-OKT3- VH. Engineered cells were adoptively transferred into wild-type (CD45.2) C57BL/6J mice. Because the observed editing efficiency was higher than necessary for expansion and affinity maturation of engineered B cells^{16, 32} , HDRT levels were adjusted so that roughly 15,000 B cells (0.3%) expressing D1D2-OKT3-VH were engrafted. Mice were then immunized intramuscularly 24 hours post-engraftment and boosted twice at two- or four- week intervals (2 wk, 4 wk) with mRNA lipid nanoparticles (mRNA-LNP) encoding an engineered HIV-1 envelope glycoprotein (Env) trimer, namely the previously described 16055-ConMv8.1 SOSIP-TM³² (FIG. 18A). The neutralization potency of sera was monitored after each immunization using pseudoviruses expressing the BG505 or CE1176 envelop glycoproteins (FIGs. 18B-18D; FIG. 24A). No neutralization was observed using sera from mice engrafted with edited cells one week after the first vaccination, or from mice engrafted with unedited cells throughout the study. Lack of neutralization after priming is consistent with the low number of successfully edited cells³⁶. However, after two immunizations, sera from LNP- vaccinated mice was found to detectably neutralize the heterologous BG505 pseudovirus with average 50% inhibitory dilutions (ID50) ranging from 20 to 45. Note that CD4-Ig-vO neutralizes comparably but less efficiently than an antibody with a D1D2-OKT3-VH variable chain (FIG. 24B). Similar results were observed with an additional group of five mice immunized with mRNA-LNP at four-week intervals (FIGs. 18C-18D). Enzyme-linked immunosorbent assay (ELISA) measurements of sera from immunized mice indicated that, after two or three LNP immunizations, the average DlD2-OKT3-Vn-IgG concentrations ranged from 40 to 75 pg/ml (FIG. 18E). A parallel study with adjuvanted gpl20-based protein antigens was also performed. Specifically, mice were engrafted with 5 million cells, including either 1.5xl0⁴ or 5xl0⁵ DlD2-OKT3-Vn-expressing cells, followed by immunization at two- week interval with multimeric (F10) and monomeric gpl20 constructs (FIG. 24C). Notably, in contrast to mice vaccinated with mRNA-LNP, no neutralizing activity was observed in mice that received 1.5xl0⁴ successfully edited cells but were vaccinated with these protein antigens (FIG. 24D). However, robust responses were observed in mice infused with 5xl0⁵ successfully edited cells. Thus, as previously reported, adoptive transfer of more antigen-reactive B cells can compensate for a less immunogenic vaccine^{16, 36}. Therefore, immunization of mRNA-LNP-expressing transmembrane SOSIP (SOSIP-TM) antigens can generate therapeutic concentrations^{27, 37, 38} of D1D2 in mice engrafted with a limited number of DlD2-OKT3-Vn-edited B cells.

Antigen-induced activation of engineered B cells in vivo

B cells harvested from the spleens and lymph nodes of mRNA-LNP vaccinated mice engrafted with cells expressing D1D2-OKT3-VH or with unmodified B cells were further analyzed. Similar percentages of CD45.1+ donor cells were found in mice vaccinated at two- and four- week intervals, and percentages were modestly but not significantly higher than mice engrafted with unedited cells (FIG. 19A; FIG. 25A and 25B). Higher percentages of antigenreactive cells were found in the CD45.1+ donor-cell population than in the CD45.2+ host-cell population (FIG. 19B). Fewer gpl20-reactive CD45.2+ B cells were observed in mice engrafted with DlD2-expressing cells than in mock engrafted mice, suggesting competition between edited donor cells and host B cells. It was determined that gpl20-reactive donor B cells class-switched and migrated to the spleen and lymph node germinal centers.

Somatic hypermutation of the D1D2 region in engineered B cells in vivo

It was then investigated whether DlD2-expressing gpl20-reactive B cells underwent somatic hypermutation (SHM). Accordingly, 3,000 to 7,000 IgG+ CD45.1+ gpl20-reactive B cells were isolated and the D1D2 region was sequenced. Substantial SHM was observed in all ten mRNA-LNP-immunized mice (FIG. 20A). Despite the heterogeneity of these responses, several mutations were observed at a high frequency across multiple mice. Most notably, two mutations in codons for R59 and K90 in CD4 domain 1 were uniformly present among the 10 mice, with average frequencies of 54% and 64% and ranges of 5 to 89% and 30 to 81%, respectively. The predominance of these mutations is more remarkable given the average mutation frequency across all 516 nucleotides was 1.1%. Several non-coding changes were also observed at high average frequency. The seven most frequent non-coding changes coincided with defined activation-induced deaminase (AID) hotspots (DGYD and WRCH with letters corresponding to the IUPAC nucleotide code), whereas the top three coding mutations emerged away from these hotspot sequences (FIG. 26), suggesting that these latter changes were products of strong selection pressure. Consistent with ongoing purifying selection, non-coding changes were observed more frequently than coding changes, but several coding changes, again including those at R59 and K90, dominated once they emerged (FIG. 20B). The average number of total mutations was the same between mice immunized at two- and four- week intervals (FIG. 20C). However, more non-coding changes were found in mice immunized every four weeks, in particular the region encoding CD4 domain 2 (FIG. 20D). Non-coding changes were distributed evenly across CD4 domains 1 and 2, and thus the proximity to the VH1-34 promotor did not affect SHM rates. Most DlD2-encoding sequences included at least two nucleotide changes, and roughly half had three or more (FIG. 20E). The frequency of unmodified D1D2 sequences varied from 0% to 20%, with fewer unmutated clones in the mice immunized at four-week intervals than in the two-week group. These results indicate that B cells edited to express D1D2-OKT3-VH underwent substantial SHM.

High-frequency amino-acid changes recurred in engrafted mice

Further analysis of coding mutations observed in mRNA-LNP-immunized mice showed mouse-to-mouse variation, but also revealed a number of consistencies (FIG. 21A). For example, in addition to R59 and K90, sequence from most mice encoded changes of at residue N30 (FIG. 21B; FIG. 27A). These N30 mutations, observed in 6 of 10 mice, overlay an AID hotspot motif, whereas R59 and K90 changes, found in 8 and 9 mice, respectively, were not close to any defined hotspots. These changes were also dominant in protein- immunized mice (M16 - M24, FIG. 27B). A minimum spanning tree clustering analysis highlights the underlying diversity of sequences found in each LNP-immunized mouse, and the presence of multiple successful founder sequences that give rise to multiple closely related sequences (FIG. 21C; FIGs. 28A-28P). High-frequency mutations, including again R59 and K90, were consistently observed among the largest clusters in most mice. Of note, trees generated from mice engrafted with 500,000 DlD2-expressing cells and immunized with adjuvanted protein antigens were sparser and contained more unmutated ancestral sequences than mRNA-immunized mice engrafted with far fewer DlD2-expressing cells, suggesting that somatic hypermutation was less robust with protein antigens (FIGs. 28H- 28P). Collectively, these data show that the D1D2 region introduced at the mouse heavychain locus underwent robust and heterogenous SHM in vivo, and that a small subset of these hypermutations were strongly favored in most mice.

In vivo hypermutations markedly improve the potency of the CD4-Ig-vO

Because R59K and K90R mutations were prominent in most immunized mice, we compared CD4-Ig variants with these changes to CD4-Ig-vO for its neutralization potency in TZM-bl cells. (FIG. 22A; FIG. 29A). R59K improved the potency of CD4-Ig-vO against all nine isolates tested, whereas K90R improved the potency against eight. The combination of these two mutations improved the potency against all isolates, with a geometric mean potency 13-fold greater than CD4-Ig-vO. Parallel analysis of N30H indicated that this mutation did not improve neutralization against most assayed isolates (FIGs. 29B-29C), and therefore it was excluded from further analysis. We also characterized a number of less prominent mutations against a CD4-sensitive (BG505) and a CD4-resistant (TR011) HIV isolate (FIG. 29C). Among these, only D63N improved neutralization against both isolates, consistent with its presence at the interface between gpl20 and CD4. Three mutations away from the gpl20- binding interface were also included in the analysis (FIG. 22B). It was postulated that these changes might enhance the in vivo stability of CD4-Ig-vO because they either emerged frequently in multiple mice (H27Y, T160S) or appeared to fill a hydrophobic cavity present in CD4 domain 2 (I138F). Four combinations of these mutations with R59K and K90R were generated (vl-v4) and characterized against a global panel of 12 isolates (FIGs. 22C, Table 3).

Table 3: CD4-Ig variants

As expected CD4-Ig-vO, already modified with potency-enhancing mutations, was 7- fold more potent than WT CD4-Ig. The four variants vl-v4 ranged from 9- to 12-fold more potent than CD4-Ig-vO and 60- to 80-fold more potent than WT CD4-Ig, with v3 modestly more potent than the other three variants. CD4-Ig-vl was also more potent than R59K/K90R, implying that D63N mutation, the sole difference between these variants, contributes to neutralization potency. To evaluate the affinity maturation of individual B cell clones, three naturally emerging D1D2 variants (Ml-1, M3-1, M6/7-1) with the greatest number of progenies in minimal-spanning trees generated from three mice were evaluated (FIG. 21C; FIGs. 28A-28P). The improved potency of vl-v4 and naturally occurring variants demonstrates affinity maturation of DlD2-OKT3-Vn-modified BCRs. Surface plasmon resonance (SPR) was used to evaluate whether these potency enhancements could be explained by higher affinity for Env. It was found that the enhanced potency of these CD4-Ig variants correlates with their slower off-rates and higher affinities for ConM SOSIP. Often when proteins are affinity optimized with in vitro techniques such a phage- or mammalian display, the resulting variants lose properties critical to their in vivo activity, including their thermal stability and half-life. To determine if the half-life optimized CD4-Ig- vO similarly reverted to the poor half-life of CD4-Ig with unmodified DI and D2, the thermal stability and polyreactivity of WT CD4-Ig, CD4-Ig-vO, and vl through v4 were characterized. No differences in polyreactivity were observed between CD4-Ig-vO and the CD4-Ig variants assayed, except in the case of v2, which was modestly but significantly more polyreactive than CD4-Ig-vO (FIG. 30A). Moreover, differential scanning fluorimetry showed that nearly every CD4-Ig variant retained the high thermostability of CD4-Ig-vO, significantly greater than WT CD4-Ig (FIG. 30B). Similarly, the half-lives of all four variants in transgenic mice expressing the human FcRn receptor remained close to CD4-Ig-vO (FIG. 30C). The in vivo half-life of all variants remained significantly greater than wild-type CD4- Ig. Therefore, the CD4-Ig variants bearing potency enhancing mutations selected in vivo retained characteristics important to their in vivo efficacy.

DISCUSSION

It was previously demonstrated that when heavy- and light-chain sequences of HIV-1 and SARS-CoV-2 neutralizing antibodies were introduced at the V(D)J-recombined regions of their respective mouse loci, the resulting BCR affinity matured after immunization^{18, 32}. However, many protein-based therapeutics are not antibodies. Rather they derive from human proteins including cytokines and soluble forms of membrane- associated receptors. To determine if non-antibody biologies could be similarly improved through in vivo affinity maturation, a variant of CD4 domains 1 and 2 (D1D2) that was previously optimized for potency and long half-life when fused to an IgG Fc domain was selected as an exemplary biologic. However, it was surprisingly found that direct replacement of the mouse VDJ- recombined region with a sequence encoding D1D2 resulted in poor BCR expression, perhaps because heavy chains with unpaired CHI domains are actively retained in endoplasmic reticulum³⁹. To overcome this problem, three strategies were developed, and it was found that a fusion of D1D2 with a murine heavy-chain variable region resulted in efficient expression. B cells modified to express this heavy-chain fusion protein class switched, entered germinal centers, and underwent robust somatic hypermutation and affinity maturation, implying that underlying regulation was not significantly disrupted. Affinity maturation of the D1D2 domain allowed the identification of variants with markedly greater neutralizing potency against HIV-1 compared to the initial D1D2. This approach for improving the efficacy of protein therapeutics is qualitatively distinct from in vitro approaches such as phage, yeast, or mammalian display²'⁴. These latter approaches exclusively select for higher affinity for their targets, but they often identify proteins with undesirable properties, limiting their in vivo half-lives and efficacies^{9, 10}. Rational design often led to reduced stability and yield of antibodies¹¹, and CD4-Ig variants rationally designed for greater potency resulted in decreased yield and lower thermostability³¹. This concern is more pronounced with non-antibody biologies which are often short-lived in sera. In contrast, the in vivo selection process in germinal centers described herein bypasses many of these pitfalls. Unstable, easily degraded, and poorly expressed variants are outcompeted by BCRs of comparable affinity but greater surface expression¹². Peripheral tolerance eliminates BCRs that interact with self-proteins, including those with low affinity and high avidity interactions, for example with membranes or extracellular matrices¹². Moreover, unlike in vitro techniques, in vivo affinity maturation occurs continuously and coordinately, building successively on selected high affinity variants, as is clear from the minimum- spanning tree analysis described herein. This process presumably has a strong evolutionary bias to sequences and structures that retain their in vivo activities. Moreover, this approach greatly simplifies affinity maturation of ligands of multi-pass membrane proteins such as G protein- coupled receptors expressed from mRNA. In contrast, in vitro approaches require reconstitution of these proteins in a liposome or nanodisc, complicating antigen production and selection of ligand variants. Thus, the in vivo affinity maturation methods described herein offer a unique way to improve biologies while preserving properties important to their efficacy. Further, this approach helps establish a foundation for the therapeutic use of engineered B cells. It suggests that B cells expressing a non-antibody biologic could adaptively control an evolving pathogen.

In vivo affinity maturation thus allowed the development of CD4-Ig variants with markedly greater potency against a global panel of HIV-1 isolates. CD4-Ig had been previously been evaluated as a promising, difficult-to-escape therapy for HIV-1, with several phase I clinical trials establishing its safety in adults and children^{26, 28, 29}. However, the original CD4-Ig faced three main limitations: it had prohibitively short in vivo half-life, its potency was lower than most HIV-1 broadly neutralizing antibodies^{26, 29}, and it enhanced infection at low concentrations in cell-culture assays, by promoting interaction of the HIV-1 Env to the co-receptor CCR5. Since then, newer variants have been developed, including eCD4-Ig, whose C-terminal sulfopeptide improved potency while blockading binding of Env to CCR5^{27, 31}’³⁷’⁴⁰. In recent years, more potent forms of CD4-Ig and eCD4-Ig have been developed with half-lives approximating many HIV-1 broadly neutralizing antibodies. This Example describes use of one of these newer CD4-Ig variants, referred as CD4-Ig-vO. D1D2 of CD4-Ig-vO was affinity matured in vivo, and several mutations were identified that recurred with high frequency across multiple mice, most notably R59K and K90R. Introduction of these mutations into CD4-Ig-vO substantially improved its potency, while preserving its breadth and in vivo half-life. These CD4-Ig variants neutralized a global panel of HIV-1 isolates with IC50 values well below 1 pg/ml, similar to that of potent broadly neutralizing antibodies while maintaining the greater breadth of CD4-Ig. The most potent of these variants, CD4-Ig-v3, bound SOSIP trimers with a Kaof approximately 50 nM, five-fold slower than CD4-Ig-vO. Moreover, several natural emerging D1D2 variants bound the immunogen SOSIP with even higher affinity. These potency enhanced variants may be more effective in controlling HIV-1 in humans.

Despite these limitations, this work describes a novel approach for refining biologies in vivo, bypassing several important limitations of in vitro methods. It provided the first example whereby non-antibody biologic affinity matures in the germinal center. We demonstrate the potential of this in vivo affinity maturation by markedly improving the potency of CD4-Ig while maintaining its half-life and breadth, suggesting that many protein therapeutics can be similarly improved.

Methods

Mice. Mouse studies were approved and carried out in accordance with approved protocols. 9 to 12 weeks old CD45.1 -positive mice (B6.SJL-Ptprca Pepcb/BoyJ, 002014) from The Jackson Laboratories were used as a source of splenic B cells. Age- and gender- matched CD45.2-positive C57BL/6J mice (Jackson Laboratories, 000664) were used as host mice for B cell transplantation and immunizations. Nine to ten weeks old SCID hFcRn transgenic mice (B6.Cg-Fcgrt^tmlDCTPrkdc^scldTg(FCGRT)32Dcr/DcrJ) were used for pharmacokinetics evaluation. No more than 5 mice or less than 2 mice were housed together. All procedures were performed on animals anesthetized with isoflurane. AAV production. HEK293T cells (CRL-3216) were seeded 18-22 h before transfection and grew to 60%-80% confluency in T225 flasks in Dulbecco's Modified Eagle Medium (DMEM, Thermo Fisher Scientific, 10566016) containing 10% FBS (Thermo Fisher Scientific, 26140-079) at 37°C in 5% CO2. 174 l (1 pg/ul) polyethylenimine (PEI, Polysciences, 49553-93-7) was vortex mixed with 1930 pl Opti-MEM (Thermo Fisher Scientific, 31985062); 58 pg of plasmids encoding the AAV rep and cap genes, adenoviral helper genes, and desired HDRT flanked by AAV ITRs were mixed with 1800 pl Opti-MEM. Opti-MEM containing PEI was added into Opti-MEM containing plasmids drop by drop followed by vertexing. The mixture was incubated at room temperature for 20-30 min. For each T225 flask, 4 ml mixture was added. Culture media was changed 18-22 h after transfection, and AAV was harvested after an additional 48 h. AAV was purified with the AAVpro Purification Kit (Takara, 6666) according to manufacturer’s instruction, and concentrated in PBS. Viral genome number was quantified by real-time PCR with AAVpro Titration Kit Ver.2 (Takara, 6233) according to manufacturer’s instruction, and capsid assembly was assessed by electrophoresis on reducing SDS-PAGE gels.

Protein production, purification, and conjugation. Expi293F cells (Thermo Fisher Scientific, A14527) were maintained in Expi293™ Expression Medium (Thermo Fisher Scientific, A1435102) following the manufacturer’s instructions. Cells were diluted to three million/ml in preheated medium, and then transfected with FectoPRO reagent (Polyplus, 116- 040). To produce gpl20 proteins, plasmids expressing the gpl20 and PDI (protein disulfideisomerase) were co-transfected at 5:1 ratio in total of 80 pg / 100 ml culture. For CD4-Ig production, plasmids were added at 55 pg / 100 ml culture. For CD4-OKT3-IgG production, heavy chain and light chain plasmids were added at 1:1 ratio at 60 pg / 100 ml culture. Five days post-transfection, cell supernatant was harvested, centrifuged, and filtered. Monomeric ConM gpl20 was captured with C-Tag affinity column (Thermo Scientific, 2943072005), ConM gpl20 F10 and SOSIP were captured with house-made PGT145 or CHOI columns, while CD4-Ig was captured by HiTrap Mabselect SuRe columns (Cytiva, 11003493). SOSIP was eluted with gentle elution buffer (Thermo Scientific, 21027), buffer exchanged in desalting columns (Thermo Scientific, 89894) to EQB (lOmM HEPES pH 8.0 in H2O with 500mM NaCl), and concentrated in 100K Amicon Ultra -15 centrifugal filter devices (Sigma, UFC9050). Other proteins were eluted with IgG elution buffer (Pierce, 21004). pH was adjusted with 1/10 elution volume of IM Tris-HCl, pH 9.0 (Thermo Fisher Scientific, J62085.K2). Elute was buffer exchanged and concentrated in PBS with 50K or 100K Amicon Ultra -15 centrifugal filter devices (Sigma, UFC9050). Proteins were purified by size exclusion chromatography in the Superdex 200 Increase 10/300 GL column (Cytiva, 28990944) or HiPrep 26/60 Sephacryl S400 HR column (Cytiva, 28935605), concentrated and stored in PBS at -80°C. Fractions were assessed by SDS-PAGE to be >95% pure. mRNA lipid nanoparticle production. Codon-optimized genes encoding SOSIP variants fused to the Env C-terminal transmembrane domain (TM) sequence were inserted into a pUC vector with 5’ UTR, 3’ UTR, and polyA sequences under T7 promotor. For in vitro transcription (IVT), the DNA templates were linearized by digestion with Hindlll and Seal (NEB) and purified by phenol-chloroform extraction. IVT was then performed using MEGAscript® T7 Transcription Kit (Thermo Fisher Scientific, AMB-1334-5) according to the manufacturer’s instructions with modifications as using the CleanCap® Reagent AG (TriLink, N-7413) and ml-pseudouridine-5’-triphosphate (TriLink, N-1081). Template DNA was digested with Turbo DNase, and synthesized mRNA was purified by LiCl precipitation and 75% ethanol washing. After RNA qualification via electrophoresis in a denaturing agarose gel, double stranded RNA was then removed by cellulose (Sigma, C6288) depletion. The mRNA solution was then precipitated with 3M sodium acetate pH 5.2 and washed with isopropanol and then 75% ethanol. Finally, the RNase free water suspended mRNA were quantified and stored at -80°C before LNP formulation. mRNA-LNP were formulated via mixing cartridges in the NanoAssembr BenchTop instrument (Precision) according to the manufacturer’s instruction. First, mRNA was diluted to 0.1-0.35 mg/ml in RNase free water with 25 mM sodium acetate pH 5.0 as the aqueous phase. The lipid phase was prepared with an N:P ratio of 6:1 by adding the lipid solutions SM-102 (MedChemExpress, HY-134541), DSPC (Avanti, 850365), cholesterol (Sigma, C8667), and PEG2000 PE (Avanti, 880150) at the molar ratio of 50:10:38.5:1.5 into ethanol. Aqueous phase and lipid phase were then transferred into individual syringes at 3:1 ratio and loaded to the pre-washed NanoAssemblr Benchtop Acetone Cartridge (Precision, NIT0058). LNP were formulated by mixing of the aqueous phase and lipid phase at a flow ratio of 3: 1 and a flow speed of 6 ml/min. After formulation, LNP were buffer exchanged to PBS by dialysis and concentrated via ultrafiltration. mRNA encapsulation efficiencies and concentrations were determined with the Quant-iT RiboGreen RNA Assay Kit (Thermo Fisher Scientific, R11490). Diameters of LNP were measured by dynamic light scattering (DLS) using a DynaPro™ NanoStar (Wyatt Technologies) and, finally, LNP were sterilized by filtration and stored at -80°C in PBS with 10% sucrose.

Mouse splenic B cell activation and electroporation. Whole spleens from 9-12 weeks old CD45.1 positive donor mice were pulverized and mechanically crushed on the inner top of 70 pm cell strainers in RPMI 1640 medium (Thermo Fisher Scientific, 61870127) with 2% FBS (Thermo Fisher Scientific, 26140-079). After red blood cell lysis in a NH4CI solution (BD Biosciences, 555899) at room temperature for 3 minutes, B cells were neutralized with Ca2+/Mg2+ free DPBS with 0.5% BSA (Miltenyi Biotec, 130-091-376) and 2 mM EDTA and then isolated using mouse B cell purification kit (Miltenyi Biotec, 130-090-862) and LS columns (Miltenyi Biotec, 130-042-401). Before electroporation, B cells were activated for 36-42 hours in RPMI 1640 medium with 10% FBS, 100 pM Non-Essential Amino Acids (NEAA, Thermo Fisher Scientific, 11140050), 1 mM sodium pyruvate (Thermo Fisher Scientific, 11360070), 10 mM HEPES (Thermo Fisher Scientific, 15630080), 55 pM 2- Mercaptoethanol (Thermo Fisher Scientific, 21985023), 100 units/ml penicillin and 100 pg/ml streptomycin (Thermo Fisher Scientific, 15140163), and 5 pg/ml anti-mouse CD180 antibody (Biolegend, 117708).

After activation, B cells were washed twice with Ca2+/Mg2+ free DPBS at room temperature. For each 100 pl of electroporation reaction using the nucleocuvette vessels (Lonza, V4XP-4024), 5 million cells were suspended in 74 pl of P4 Primary Cell solution (Lonza, VSOP-4096). In parallel, 3.12 pl of PBS, 1.26 pl of IM NaCl, 1.12 pl of 250 pM Mb2Casl2a (produced in house), and 4.5 pl of 100 pM gRNA (see Table 4 for sequence information) were mixed and incubated at room temperature for 15 min for RNP complex formation. The RNP were then incubated with 16 pl of 100 pM single strand DNA enhancer for 3 minutes at room temperature. The above 26 pl mixture was then mixed with the 74 pl of suspended B cells and transferred to the nucleocuvette vessels for electroporation in the Lonza 4D nucleofector under the DI- 100 program. For larger scale electroporation in the 1 ml scale of Nucleocuvette Cartridge (Lonza, V4LN-7002), reactions were scaled up 10-fold for cell suspension, RNP, and donors. After electroporation, cells were rested for 10-15 min in nucleocuvette vessels or cartridges before transferred to preheated activation medium with 20% FBS without penicillin-streptomycin, which was added two hour later. Mouse B cell transplantation, immunization, and blood collection. Approximately 18 hours after electroporation, B cells were washed with prechilled Ca2+/Mg2+ free DPBS for three times and then suspended in prechilled DPBS with Ca2+/Mg2+ (Thermo Fisher Scientific, 14040133) and 5% horse serum (Cytiva, SH3007403HI). After filtration (Falcon, 352235) the number of cells was adjusted, and each mouse received 5 million cells in 100 pl buffer via retro-orbital injection under anesthesia with isoflurane. An aliquot of approximately 2 million cells were further cultured in RPMI 1640 medium with 10% FBS, 100 pM NEAA, 1 mM sodium pyruvate, 55 pM 2-mercaptoethanol, 10 mM HEPES, 100 units/ml penicillin and 100 pg/ml streptomycin, 5 pg/ml LPS, 10 ng/ml mouse IL-4 (PeproTech Inc, 214-14), and 2 pg/ml anti-mouse CD180 antibody for additional 24 hours to validate editing efficiency by flow cytometry. For protein immunization, 5 pg gpl20 was adjuvanted with 10 pg monophosphoryl lipid A (InvivoGen, vac-mpla) and 10 pg saponin (InvivoGen, vac-quil) in PBS, and formulated into 250 pl per mouse. Mice were injected subcutaneously and intramuscularly. For mRNA-LNP, 0.5 pg in 20 pl was injected intramuscularly at each hind leg. Sera were collected one week after each immunization via submandibular bleeding.

Analytical cytometry, cell sorting, and immunoglobulin repertoire sequencing. For flow cytometry and cell-sorting assays, spleens and lymph nodes were homogenized by mechanical disassociation and filtered through a 70-micron cell strainer. Red-blood cells were lysed with NH4CI Buffer (BD Biosciences, 555899). B cells were then isolated with Pan B Cell Isolation Kit II (Miltenyi Biotec, 130-104-443) and LS columns (Miltenyi Biotec, 130-042-401), and resuspended in DPBS with 0.5% BSA and 2 mM EDTA. For cell sorting, antigen- specific B cells were stained by DAPI (BioLegend), CD45.1-FITC (BioLegend, 110706), IgG-PE (Biolegend, 405307), and ConM gpl20-Fc conjugated with APC using the Lightning-Link (R) Fluorescein Antibody Labeling Kit (Novus, 705-0010). The antibody cocktail for GC B-cell staining consisted of CD45.1-FITC (BioLegend, 110706), CD19- BV605 (BioLegend, 115539) CD138-PE-Cy7 (BioLegend, 142513), GL7-PE (BD Biosciences, 561530), and CD38-AF700 (BioLegend, 102741). Cells were incubated for 20 min on ice in dark, then washed and filtered before analysis or sorting on BD FACS LSR II, BD FACS Aria III, BD FACS Aria Fusion, or CytoFLEX S. Data were analyzed using FlowJo. CD45.1 and CD45.2 markers were used to distinguish donor and host B cells.

Antigen- specific B cells were gated as singlet live CD45.1+ IgG+ gpl20+; germinal center B cells were gated as singlet live CD19+ CD138- CD38- GL7+. At least 100,000 events per sample were analyzed.

Sorted B cells were lysed for RNA extraction by the RNeasy Micro Kit (Qiagen, 74004). Primers used for reverse transcription and library amplification are provided in Table 4. First-strand cDNA synthesis was performed on 8 pl of total RNA using 5 pmol of IgM and IgG specific primers in a 20 pl total reaction with SuperScript III (Thermo Fisher, 18080044) according to the manufacturer’s protocol. Second-strand synthesis reactions were performed in 50 pl using HotStarTaq Plus (Qiagen, 203603) and 10 pmol of each primer tagged with unique molecular identifiers (UMIs). P5 and P7 flow cell-adaptor sequences and offset were introduced into dsDNA products with 10 pmol of i5i7 primers in a 50 pl total reaction volume for 20 PCR cycles. Libraries were verified in 1.5% agarose gel (Invitrogen, 16550100) and concentration was determined by NanoDrop. Libraries were optionally amplified for additional 6-12 PCR cycles if concentration was below 5 ng/ pl. Finally, single indices for demultiplexing were added using the NEBNext Multiplex Oligos for Illumina (NEB, E7335S, E7500S, or E7710S) in 6-cycle PCR. All PCR products were purified by ExoSAP-IT (Thermo Fisher, 78201.1. ML) and SPRI beads (Beckman Coulter Genomics, SPRIselect). Bead-purified libraries were quality controlled on Agilent 2100 Bioanalyzer or 4200 TapeStation, and quantified by qPCR (Roche, KR0405). Pooled samples were sequenced using MiSeq 2x300 bp paired end reads (Illumina, MS- 102-3003).

Neutralization assays. Neutralizing activity was measured as the reduction in luciferase (Luc) reporter-gene expression after a single round of infection in TZM-bl cells. HIV-1 pseudoviruses were produced by co-transfecting Env plasmids with an Env-deficient backbone plasmid (pNL4-3 Env) in HEK293T cells grown in DMEM containing 10% FBS in a 1:3 ratio, using polyethylenime “Max” (PEI MAX; Polysciences, 49553-93-7). Plasmids were acquired through the NIH HIV Reagent Program. Cell supernatants were harvested, centrifuged at 4000 g, filtered through 0.45pm filter 48 h after transfection, and stored at - 80°C. Neutralization assays were performed using pre-titrated HIV-1 Env pseudoviruses and TZM-bl cells growing in DMEM containing 10% FBS as previously described⁴⁵. Briefly, 50 pl of titrated pseudoviruses were incubated with 50 pl of serially diluted antibodies/sera for 60 min at 37°C in 96-well flat bottom TC-treated plates (Corning, 353075). Subsequently, 100 pl of TZM-bl cells resuspended at 0.1 million/ml were added to each well and incubated at 37°C. At 48 h post infection, 100 pl supernatant was removed and cells were lysed in wells and subjected to firefly luciferase assays. Luciferase expression was determined using Britelight Plus substrate (PerkinElmer, 6066761). The luminescence signal was acquired with a Victor Nivo plate reader (PerkinElmer) or a GloMax® Plate Reader (Promega). Percent infection was calculated using background-subtracted signals from wells containing virus only as a 100% infection reference, and neutralization curves were fitted by nonlinear regression using a four-parameter hill slope equation. Experiments were done in triplicate and repeated twice. IC50 and ID50 values were determined as the concentration or dilution required to inhibit infection by 50%.

Surface plasmon resonance. Single-cycle kinetics analysis of CD4-Ig binding to the 16055- ConM-v8.1 SOSIP with C-terminal Spy-tag-2 (ST2) was performed at 25°C on a Biacore T100 (GE Healthcare). IX HBS-EP containing 10 mM HEPES, pH 7.4, 150 mM NaCl, 3 mM EDTA, and 0.05% surfactant P-20 (Cytiva, BR100669) with 0.25% BSA and additional 150 mM NaCl was prepared and filtered. Mouse anti-human IgG Fc antibody (Novus, #NBP 1-51523) was immobilized onto all flow cells of a CM5 sensor to ~ 10,000 response units (RU) using the amine-coupling (Cytiva, BR- 1000-50) method according to manufacturer’s instruction. SEC -purified CD4-Ig variants were captured at 5 nM and a flow rate of 5 pl/min for 60 s in sample flow cells. The capture level was kept to 5-25 RU to minimize mass transport effects and steric hindrance. The reference flow cells were left blank. A 2-fold increasing series of SEC-purified SOSIP (50 nM, 100 nM, 200 nM, 400 nM, 800 nM) was then injected into both the reference and sample flow cells at 50 pl/min for 240 s in a single cycle, resulting in a maximum of 30 RU. 360s of dissociation phase was followed by regeneration with 3M MgCh. Sensorgrams were corrected with double reference by subtracting the response over the reference surface and the response of a blank injection from the sample binding responses. The association and dissociation phase data sets were globally fitted with Biacore Insight Evaluation software (v5.0) using a 1:1 Langmuir model, because the capture density and conformation of CD4-Ig prevents intra-protomer crosslinking of the SOSIP protein⁴⁶. These kinetic binding studies were repeated twice on different sensor surfaces for each CD4-Ig variant.

Pharmacokinetics in mice. SCID hFcRn transgenic mice were injected intravenously with

8 mg/kg of CD4-Ig diluted in PBS. Serum samples were drawn from each mouse via the tail vein on days 1, 3, 6, 14, 21 and 30 post-injection. Sera were collected in tubes containing clotting activators and frozen at -8°0C right after processing.

ELISA. To monitor the serum D1D2 concentration in B-cell engrafted mice by ELISA, high- binding 96-well plates (Corning, 3690) were coated overnight at 4°C with a rabbit antihuman CD4 antibody (Cell Signaling Technology, 93518) at a concentration of 2 pg/ml in PBS. To measure the CD4-Ig concentrations in serum samples in pharmacokinetic studies, plates were coated with 2 pg/ml of a mouse anti-human CD4 antibody (BioLegend, 300502) diluted in PBS overnight at 4 °C. Wells were washed two times with 0.05% Tween 20 in PBS and blocked for 2.5 hours at room temperature with 150 pl of 4% BSA (Thermo Scientific, 37525) in PBS. After blocking, wells were loaded with 50 pl of serially diluted mouse sera, or standards of either SEC -purified CD4-OKT3-IgG for 1.5 hour or SEC-purified CD4-Ig for an hour at 37°C in duplicate. After five washes, wells were incubated with 50 pl of 1:2000 diluted horseradish peroxidase-conjugated goat anti-mouse IgG Fc antibody (Jackson ImmunoResearch, 115-035-008) in 4% blocking buffer for 1.5 hours, or horseradish peroxidase-conjugated goat anti-human IgG Fc antibody (Sigma, A0170) diluted 1:20,000 in the blocking buffer for an hour at 37°C. Following eight washes, 50 pl of 1-Step™ Ultra TMB-ELISA substrate (Thermo Scientific, 34028) was added to each well and incubated at room temperature. The reaction was terminated with 50 pl of TMB Stop solution (SeraCare, 5150-0020). Absorbance at 450 nm was measured with a Victor® Nivo™ plate reader (PerkinElmer) or a GloMax® Plate Reader (Promega). The concentrations of the samples were determined by extrapolation from a standard curve made with a four-parameter nonlinear regression model in Prism.

Differential scanning fluorimetry. Samples were prepared by mixing SEC-purified CD4-Ig with 25X GloMelt™ Dye (Biotium, #99843-20uL) to reach a final concentration of 10 nM CD4-Ig and 2X dye. Fluorescence was detected in SYBR channel of Bio-Rad CFX96. Temperature was incremented by 0.5°C per 30 s, from 25°C to 95°C. Melting curves were monitored for homogeneity. Melting temperatures were determined as the thermal transition points by the instrument software.

Immunofluorescence assay on HEp-2 cells. Immunofluorescence assays were performed with ANA HEp-2 Test Kits (Zeus Scientific, FA2400EB) according to manufacturer’s instructions. Briefly, 100 pg/ml antibodies or CD4-Ig variants or manufacturer-provided control serum were incubated on HEp-2 cell slides at RT for 40 min, and then washed 3 times with PBS. FITC-conjugated anti-human IgG antibodies were coated to each well at RT for 25 min and slides were again washed. Slides were viewed using a Leica DMIL LED microscope at a 292ms exposure, and mean pixel intensity was measured by Image J.

Bioinformatic analysis. All fastq files were initially processed with the in-house tool “dsa” (short for deep sequencing analysis). Briefly, reads were first processed by trimming bases from the 3' ends with Phred quality scores falling below 32. Subsequently, low quality reads that did not contain the adaptor sequences were discarded. Reads with shared UMI were merged into single reads. Merged reads had a minimum UMI group size of 2. dsa identifies open reading frames in deep-sequencing datasets and aligns them to CD4-Ig-vO D1D2 nucleotide or amino acid templates using the Needleman-Wunsch algorithm. Following alignment and UMI group consensus formation, reads with undetermined nucleotides marked as N were removed, dsa outputs alignments of the reads to the template, tables of amino acid substitution frequencies, and summaries of coding and non-coding substitution frequencies. It further compiles lists of unique amino acid and nucleotide sequences used for further analysis including clustering and phylogenetic inference. Mutational frequency is calculated from these data as the number of sequences bearing a nucleotide mutation at a given position for each mouse divided by the total number of sequences from that mouse.

Clustering and phylogenetic inference was performed with a custom-made tool called “Dandelions”. Dandelions uses a maximum parsimony approach to generate the N-ary phylogenetic trees. The output represents a consensus of at least 500 minimum spanning trees constructed over the input sequences and a set of inferred ancestral sequences not present in the original data. Each node in the final tree corresponds to a unique amino acid sequence. Colored nodes are “centroids”, so designated based on the total of their non-coding variants and the number of their descendants in the tree. The root path of each centroid shares its color. The 18 largest centroids are rank ordered and labeled. The size of a node is proportional to the number of non-coding variants that share the amino-acid sequence of the node. The tree is rooted on the known ancestor, represented as black circle. The layout of the tree is determined by an interactive physics simulation where nodes are modeled as masses in a viscous medium connected by springs. Quantification and Statistical analysis. Flow cytometry analysis was carried out using FlowJo™ v.10 software. Throughout the study, statistical tests were performed using a nominal type I error rate of p = 0.05 and two-tailed tests where applicable. Mixed effects analysis, one- and two- way ANOVA were performed in GraphPad Prism 10. Flow cytometry data was analyzed using generalized linear mixed models in JMP Pro 17, where distribution was binomial, repeated measures aspect was included and generalized chisq/df value was monitored to make sure close to 1 to avoid overdispersion. Statistical information including n, mean, geometric mean, median, standard error of the mean, and statistical significance values are indicated in the figure legends. Normality and equal variances were examined in residual plot, homoscedasticity plot and QQ plot. Continuous outcome variables exhibiting a skewed distribution were log-transformed, and diagnostic plots were evaluated to assure assumptions are met in the final analysis. Mixed effects model with Geisser- Greenhouse correction was used for repeated measures. H-Sidak test (pairwise comparison) or Dunnett’s test (comparison to control) was performed following ANOVA and mixed effects analysis. Adjusted p-values for multiplicity were used in multiple comparison tests.

Data were considered statistically significant at *p < 0.05, **p < 0.01, ***p < 0.001, and ****p<0.0001.

Table 4. gRNA, ssDNA enhancer and NGS primers.

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EQUIVALENTS AND SCOPE

While several inventive embodiments have been described and illustrated herein, those of ordinary skill in the art will readily envision a variety of other means and/or structures for performing the function and/or obtaining the results and/or one or more of the advantages described herein, and each of such variations and/or modifications is deemed to be within the scope of the inventive embodiments described herein. More generally, those skilled in the art will readily appreciate that all parameters, dimensions, materials, and configurations described herein are meant to be exemplary and that the actual parameters, dimensions, materials, and/or configurations will depend upon the specific application or applications for which the inventive teachings is/are used. Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific inventive embodiments described herein. It is, therefore, to be understood that the foregoing embodiments are presented by way of example only and that, within the scope of the appended claims and equivalents thereto, inventive embodiments may be practiced otherwise than as specifically described and claimed. Inventive embodiments of the present disclosure are directed to each individual feature, system, article, material, kit, and/or method described herein. In addition, any combination of two or more such features, systems, articles, materials, kits, and/or methods, if such features, systems, articles, materials, kits, and/or methods are not mutually inconsistent, is included within the inventive scope of the present disclosure. All definitions, as defined and used herein, should be understood to control over dictionary definitions, definitions in documents incorporated by reference, and/or ordinary meanings of the defined terms.

All references, patents, and patent applications disclosed herein are incorporated by reference with respect to the subject matter for which each is cited, which in some cases may encompass the entirety of the document.

The indefinite articles “a” and “an,” as used herein in the specification and in the claims, unless clearly indicated to the contrary, should be understood to mean “at least one.”

The phrase “and/or,” as used herein in the specification and in the claims, should be understood to mean “either or both” of the elements so conjoined, i.e., elements that are conjunctively present in some cases and disjunctively present in other cases. Multiple elements listed with “and/or” should be construed in the same fashion, i.e., “one or more” of the elements so conjoined. Other elements may optionally be present other than the elements specifically identified by the “and/or” clause, whether related or unrelated to those elements specifically identified. Thus, as a non-limiting example, a reference to “A and/or B”, when used in conjunction with open-ended language such as “comprising” can refer, in one embodiment, to A only (optionally including elements other than B); in another embodiment, to B only (optionally including elements other than A); in yet another embodiment, to both A and B (optionally including other elements); etc.

As used herein in the specification and in the claims, “or” should be understood to have the same meaning as “and/or” as defined above. For example, when separating items in a list, “or” or “and/or” shall be interpreted as being inclusive, i.e., the inclusion of at least one, but also including more than one, of a number or list of elements, and, optionally, additional unlisted items. Only terms clearly indicated to the contrary, such as “only one of’ or “exactly one of,” or, when used in the claims, “consisting of,” will refer to the inclusion of exactly one element of a number or list of elements. In general, the term “or” as used herein shall only be interpreted as indicating exclusive alternatives (i.e. “one or the other but not both”) when preceded by terms of exclusivity, such as “either,” “one of,” “only one of,” or “exactly one of.” “Consisting essentially of,” when used in the claims, shall have its ordinary meaning as used in the field of patent law.

As used herein in the specification and in the claims, the phrase “at least one,” in reference to a list of one or more elements, should be understood to mean at least one element selected from any one or more of the elements in the list of elements, but not necessarily including at least one of each and every element specifically listed within the list of elements and not excluding any combinations of elements in the list of elements. This definition also allows that elements may optionally be present other than the elements specifically identified within the list of elements to which the phrase “at least one” refers, whether related or unrelated to those elements specifically identified. Thus, as a non-limiting example, “at least one of A and B” (or, equivalently, “at least one of A or B,” or, equivalently “at least one of A and/or B”) can refer, in one embodiment, to at least one, optionally including more than one, A, with no B present (and optionally including elements other than B); in another embodiment, to at least one, optionally including more than one, B, with no A present (and optionally including elements other than A); in yet another embodiment, to at least one, optionally including more than one, A, and at least one, optionally including more than one, B (and optionally including other elements); etc.

It should also be understood that, unless clearly indicated to the contrary, in any methods claimed herein that include more than one step or act, the order of the steps or acts of the method is not necessarily limited to the order in which the steps or acts of the method are recited.

In the claims, as well as in the specification above, all transitional phrases such as “comprising,” “including,” “carrying,” “having,” “containing,” “involving,” “holding,” “composed of,” and the like are to be understood to be open-ended, i.e., to mean including but not limited to. Only the transitional phrases “consisting of’ and “consisting essentially of’ shall be closed or semi-closed transitional phrases, respectively, as set forth in the United States Patent Office Manual of Patent Examining Procedures, Section 2111.03. It should be appreciated that embodiments described in this document using an open-ended transitional phrase (e.g., “comprising”) are also contemplated, in alternative embodiments, as “consisting of’ and “consisting essentially of’ the feature described by the open-ended transitional phrase. For example, if the disclosure describes “a composition comprising A and B,” the disclosure also contemplates the alternative embodiments “a composition consisting of A and B” and “a composition consisting essentially of A and B.”

Claims

CLAIMS What is claimed is:

1. A method comprising: introducing a nucleic acid comprising a nucleotide sequence encoding a peptide of interest into a genomic locus encoding the endogenous B cell receptor (BCR) in a B cell, thereby generating an engineered BCR; and administering the B cell comprising the engineered BCR to a mammalian subject, wherein the peptide of interest is not an antibody.

2. The method of claim 1, wherein the peptide of interest is grafted onto the endogenous heavy chain constant region in the engineered BCR.

3. The method of claim 1, wherein the peptide of interest is grafted onto the endogenous light chain constant region in the engineered BCR.

4. The method of any one of claims 1-3, wherein the nucleic acid comprising the nucleotide sequence encoding a peptide of interest further comprises a nucleotide sequence encoding a variable region of a heterologous antibody.

5. The method of any one of claims 1-3, wherein the peptide of interest is inserted between FR3 and FR4 of the variable region in the heavy chain or light chain in the engineered BCR.

6. The method of claim 5, wherein the nucleotide sequence encoding the peptide of interest is inserted into a sequence encoding a variable region of a heterologous antibody between the framework 3 region and framework 4 region, in the nucleic acid being introduced.

7. A method, comprising: contacting a B cell with (i) a homology-directed repair (HDR) template comprising a nucleotide sequence encoding a peptide of interest, (ii) a Cas protein, and (iii) a guide RNA, wherein the B cell comprises heavy and light chain genomic loci encoding a B cell receptor (BCR), and wherein the guide RNA comprises a sequence having complementarity to a region of heavy chain or light chain genomic locus encoding the BCR; whereby the Cas protein introduces a double-strand DNA break immediately adjacent to a target site in the heavy chain or light chain genomic locus, and the target site is replaced with the nucleotide sequence encoding the peptide of interest through HDR, thereby generating an engineered BCR; and administering the B cell comprising the engineered BCR to a mammalian subject, wherein the peptide of interest is not an antibody.

8. The method of claim 7, wherein the target site is in the heavy chain genomic locus.

9. The method of claim 7, wherein the target site is in the light chain genomic locus.

10. The method of claim 8, wherein the peptide of interest is grafted onto the endogenous heavy chain constant region in the engineered BCR.

11. The method of claim 9, wherein the peptide of interest is grafted onto the endogenous light chain constant region in the engineered BCR.

12. The method of any one of claims 7-11, wherein the HDR template further comprises a nucleotide sequence encoding a variable region of a heterologous antibody.

13. The method of claim 12, wherein the HDR template comprises, from 5’ to 3’, nucleotide sequences encoding the peptide of interest, a linker, and the variable region of the heterologous antibody.

14. The method of any one of claims 7-11, wherein the HDR template further comprises a nucleotide sequence encoding a heterologous light chain constant domain.

15. The method of claim 14, wherein the HDR template comprises, from 5’ to 3’, nucleotide sequences encoding the peptide of interest, a first linker, the heterologous light chain constant domain, and a second linker.

16. The method of any one of claims 6-11, wherein the peptide of interest is inserted between FR3 and FR4 of the variable region in the heavy chain or light chain in the engineered BCR.

17. The method of claim 16, wherein the nucleotide sequence encoding the peptide of interest is inserted into a sequence encoding a variable region of a heterologous antibody between the framework 3 region and framework 4 region, in the HDR template.

18. The method of claim 17, wherein the HDR template comprises, from 5’ to 3’, nucleotide sequences encoding FR1 to FR3 of the variable region of the heterologous antibody, a first linker, the peptide of interest, a second linker, and FR4 of the variable region of the heterologous antibody.

19. The method of any one of claims 7-18, wherein the HDR template comprises a 3’ homology arm that is homologous to a region proximal to the 3’ region of a J segment and a 5’ homology arm that is homologous to the 5’ region of a V segment.

20. A method, comprising: contacting a B cell with (i) a homology-directed repair (HDR) template comprising a nucleotide sequence encoding a peptide of interest and a nucleotide sequence encoding a variable region of a heterologous antibody, (ii) a Cas protein, and (iii) a guide RNA, wherein the B cell comprises heavy and light chain genomic loci encoding a B cell receptor (BCR), and wherein the guide RNA comprises a sequence having complementarity to a region of heavy chain or light chain genomic locus encoding the BCR; whereby the Cas protein introduces a double-strand DNA break immediately adjacent to a target site in the heavy chain or light chain genomic locus, and the target site is replaced with the nucleotide sequence encoding the peptide of interest and the nucleotide sequence encoding the variable region of the heterologous antibody through HDR, thereby generating an engineered BCR; and administering the B cell comprising the engineered BCR to a mammalian subject, wherein the peptide of interest is not an antibody.

21. The method of claim 20, wherein the variable region of the heterologous antibody is a heavy chain variable region and the guide RNA comprises a sequence having complementarity to a region of the heavy chain genomic locus.

22. The method of claim 21, wherein the nucleotide sequence encoding the heavy chain variable region of the heterologous antibody comprises recombined germline VDJ segments.

23. The method of claim 20, wherein the variable region of the heterologous antibody is a light chain variable region and the guide RNA comprises a sequence having complementarity to a region of the light chain genomic locus.

24. The method of claim 23, wherein the nucleotide sequence encoding the light chain variable region of the heterologous antibody comprises recombined germline VJ segments.

25. The method of any one of claims 20-24, wherein the peptide of interest is connected to the N-terminus of the variable region of the heterologous antibody.

26. The method of any one of claims 20-25, wherein the peptide of interest is connected to the variable region of the heterologous antibody via a linker.

27. The method of claim 26, wherein the linker is between 5-30 amino acids in length.

28. The method of claim 26 or 27, wherein the linker is a glycine-serine linker.

29. The method of claim 28, wherein the linker is a (G+Sja (SEQ ID NO: 8) linker.

30. The method of any one of claims 7-29, wherein the replacement of the target site with the sequence encoding the peptide of interest does not result in integration of any exogenous genetic regulatory elements into the heavy chain or light chain genomic locus encoding the BCR.

31. The method of any one of the preceding claims, wherein the mammalian subject is a rodent.

32. The method of any one of the preceding claims, wherein the mammalian subject is a wild-type mouse.

33. The method of any one of claims 1-32, wherein the mammalian subject is not a transgenic mouse.

34. The method of any one of claims 7-33, wherein the Cas protein is Cas9, Casl2a or Cas 13.

35. The method of any one of the preceding claims, whereby the method generates an affinity-matured peptide of interest in the subject that is a variant of the peptide of interest.

36. The method of any one of the preceding claims, whereby the method results in somatic hypermutation and affinity maturation of the peptide of interest in the subject.

37. The method of claim 36, whereby the method provides rates of somatic hypermutation of about 0.1%-50% in the peptide of interest.

38. The method of claim 35, wherein the affinity-matured peptide of interest has an enhanced biological property relative to the peptide of interest.

39. The method of claim 38, wherein the biological property is bioavailability.

40. The method of claim 38, wherein the biological property is binding affinity to a target.

41. The method of claim 38, wherein the biological property is inhibition of a target.

42. The method of any one of the preceding claims, wherein the peptide of interest is an

FDA-approved therapeutic peptide.

43. The method of any one of the preceding claims, wherein the peptide of interest binds to a target selected from a soluble protein, a transmembrane protein, and a pathogenic antigen.

44. The method of claim 43, wherein the peptide of interest binds to a viral antigen, optionally, an HIV antigen.

45. The method of claim 44, wherein the peptide of interest is human CD4 domain 1 and 2 (D1D2) or a variant thereof.

46. The method of any one of claims 1-43, wherein the peptide of interest is a blood factor, a chemokine, a cytokine, a soluble receptor, an adhesin, a thrombolytic agent, a serum protein, a hormone, a hematopoietic growth factor, an interferon, an interleukin, or an enzyme, a nanobody, an adnectin, or a DARPin.

47. The method of any one of claims 7-46, wherein the HDR template is comprised within a double-stranded DNA (dsDNA) vector.

48. The method of any one of claims 7-47, wherein the HDR template is comprised within an adeno-associated viral (AAV) vector.

49. The method of claim 48, wherein the AAV vector is encapsidated in an AAV6 or AAV-DJ capsid.

50. The method of any one of claims 7-49, wherein the guide RNA comprises a sequence of between 15 and 200 nucleotides that is complementary to a region of the heavy chain or light chain genomic locus.

51. An affinity-matured variant of the peptide of interest generated using the method of any one of the preceding claims.

52. An engineered mature B cell generated using the method of any one of claims 1-51.

53. A B cell comprising the affinity-matured variant of claim 51.

54. A population of B cells in accordance with claim 53.

55. A method of administering the affinity-matured variant of claim 51, or the B cell or B cells of any one of claims 52-54, to a subject.

56. The method of claim 55, wherein the subject is a human.

57. A system of affinity maturation of a peptide of interest, comprising: a B cell, a nucleic acid comprising a nucleotide sequence encoding a peptide of interest, for generating an engineered BCR in the B cell; and an injection mechanism for administering the B cell comprising the engineered BCR to a mammalian subject, wherein the peptide of interest is not an antibody.

58. A system of affinity maturation of a peptide of interest, comprising: a B cell, wherein the B cell comprises heavy chain and light chain genomic loci encoding a B cell receptor (BCR), a homology-directed repair (HDR) template comprising a nucleotide sequence encoding a peptide of interest, a Cas protein, and a guide RNA, wherein the guide RNA comprises a sequence having complementarity to a region of the heavy chain or light chain genomic locus encoding the BCR, for generating an engineered BCR in the B cell, and an injection mechanism for administering the B cell comprising the engineered BCR to the subject, wherein the peptide of interest is not an antibody.

59. A system of affinity maturation of a peptide of interest, comprising: a B cell, wherein the B cell comprises heavy chain and light chain genomic loci encoding a B cell receptor (BCR), a homology-directed repair (HDR) template comprising a nucleotide sequence encoding a peptide of interest and nucleotide sequence encoding a variable region of an antibody, a Cas protein, and a guide RNA, wherein the guide RNA comprises a sequence having complementarity to a region of the heavy chain or light chain genomic locus encoding the BCR, for generating an engineered BCR in the B cell, and an injection mechanism for administering the B cell comprising the engineered BCR to the subject, wherein the peptide of interest is not an antibody.

60. The system of any one of claims 57-59, wherein the injection mechanism is adapted for subcutaneous, intravenous, intramuscular, or intraperitoneal injection.

61. A nucleic acid molecule encoding an engineered murine B cell receptor (BCR) comprising a peptide of interest, wherein the peptide of interest is not an antibody, and wherein the nucleic acid molecule comprises endogenous murine BCR regulatory elements.

62. A nucleic acid molecule encoding an engineered murine B cell receptor (BCR) comprising a peptide of interest and a variable region of a heterologous antibody, wherein the peptide of interest is not an antibody, and wherein the nucleic acid molecule comprises endogenous murine BCR regulatory elements.

63. A pharmaceutical composition comprising the affinity-matured variant of claim 51, or the B cell of any one of claims 52-54.