Engineered B cells expressing an anti-HIV antibody enable memory retention, isotype switching and clonal expansion

Abstract

HIV viremia can be controlled by chronic antiretroviral therapy. As a potentially single-shot alternative, B cells engineered by CRISPR/Cas9 to express anti-HIV broadly neutralizing antibodies (bNAbs) are capable of secreting high antibody titers. Here, we show that, upon immunization of mice, adoptively transferred engineered B cells home to germinal centers (GC) where they predominate over the endogenous response and differentiate into memory and plasma cells while undergoing class switch recombination (CSR). Immunization with a high affinity antigen increases accumulation in GCs and CSR rates. Boost immunization increases the rate of engineered B cells in GCs and antibody secretion, indicating memory retention. Finally, antibody sequences of engineered B cells in the spleen show patterns of clonal selection. Therefore, B cells can be engineered into what could be a living and evolving drug.

Fig. 1. Engineering B cells to express an anti-HIV bNAb.

a Targeting scheme. An rAAV-delivered cassette is targeted to the J-C intron of the IgH locus using CRISPR/Cas9. The bicistronic cassette encodes the light and heavy chains of the 3BNC117 anti-HIV bNAb, under the control of an enhancer dependent (ED) promoter. Splicing with endogenous constant segments allows the expression of a BCR and differentiation into memory B cells and Ig secreting plasma cells upon subsequent antigen-induced activation and alternative polyadenylation (Alt. PolyA). Targeting the J-C intron upstream of the intronic enhancer (iEμ) and switch region further facilitates CSR and SHM. See also Supplementary Fig. 1. b Activation and engineering scheme. Human B cells are collected from blood samples, activated using an anti-RP105 (TLR4 homolog) antibody, electroporated by CRISPR/Cas9 RNP and transduced using rAAV-6. Splenic B cells are activated using the TLR4 agonist LPS electroporated by CRISPR/Cas9 RNP and transduced using rAAV-DJ. c Flow cytometry plots measuring binding of the HIV gp120 antigen by the 3BNC117 BCR following activation and engineering of primary cells. Cells transduced with the donor rAAV and without gRNA serve as a negative control, gating on live, singlets. d, e Quantification of C for mouse (n = 5 for –gRNA and n = 8 for +gRNA, each dot represents a biologically independent sample, data represented as mean values +/− SD) d and human (n = 3, each dot represents a biologically independent sample, data represented as mean values +/− SD) e primary cells. ****p < 0.0001, **p = 0.0040; two-tailed t-test. f Flow cytometry plots demonstrating ERK phosphorylation in primary mouse or human B cells engineered with 3BNC117 and in vitro activated with the gp120 antigen of the YU2.DG HIV strain, gating on singlets.

Fig. 2. Engineering B cells with a reduced risk for the mispairing of transgenic and endogenous chains.

a Flow cytometry of gRNA-dependent IgK ablation, gating on live, singlets for human cells and live, singlets, CD19⁺ for mouse cells. b Scheme of 4 alternative rAAV donor vectors where the 3BNC117 bNAb is coded as two chains separated by a furin cleavage site and a sequence coding for a 2A peptide (bottom) or where the bNAb is coded as a single chain using a linker (up) and is preceded by an ED promoter (left) or a splice acceptor sequence (right). c Representative analysis by flow cytometry of 3BNC117 expression in primary mouse splenic lymphocytes monitored two days following transduction and detected by anti-idiotypic antibody. Gating on live, singlets. d Quantification of c for ED promoter driven cassettes, two-tailed t-test (n = 7 for the two chain and n = 3 for the single chain construct, each dot represents a biologically independent sample, data represented as mean values +/− SD).

Fig. 3. Adoptively transferred engineered B cells can undergo antigen-induced activation in-vivo.

a Experimental scheme of the in vivo assays. Splenic B cells, from C57BL/6 CD45.1 mice, were engineered as in Fig. 1 and infused to otherwise syngeneic CD45.2 recipient mice. Different mice groups were immunized on the following day with gp120 antigens from either the THRO4156.18 (THRO) or the YU2.DG (YU2) HIV strains. When boosted by an additional injection, the mice received the same antigen as in the prime injection. Different mice groups were sacrificed 8 days following injections for spleen collection or terminally bled 14 days after injection for serum collection. b Representative analysis by flow cytometry of the accumulation of engineered cells in the GCs of mice immunized with either the YU2.DG or the THRO4156.18 gp120 antigens, 8 days following a prime antigen injection. Gating on live, singlets, B220⁺, GL-7⁺. c Quantification of b. Each dot represents a different mouse. Error bars represent SD (n = 3, represents amouse, data represented as mean values +/− SD). d Representative analysis by flow cytometry of CD45.1 expression among gp120 binding GC cells. Pre-gating on singlets, live, B220⁺, GL-7⁺. e Quantification of d. Each dot represents a different mouse. Error bars represent SD (n = 3, each dot represents a mouse, data represented as mean values +/− SD). For gating strategy see Supplementary Fig. 12.

Fig. 4. Adoptively transferred engineered B cells enable memory retention upon immunization.

a ELISA of sera collected 14 days following either prime or boost immunization, quantified using an anti-idiotypic antibody to 3BNC117. ####pv < 0.0001, #pv = 0.0465 for two-way ANOVA and **pv = 0.0024, ***pv = 0.0003 for Tukey’s multiple comparison (n = 3, each dot represents a mouse). b Analysis by flow cytometry of CD45.1 expression and gp120 binding in the GCs of mice following prime or boost immunizations, gating on live, singlets, B220⁺, GL-7⁺. c Quantitation of b. ####pv < 0.0001, ###pv = 0.0003 for two-way ANOVA and ***pv = 0.0008 and **upper = pv = 0.0095 and **lower = pv = 0.0067 for Tukey’s multiple comparison (n = 6, each dot represents a mouse). d, e Analysis by flow cytometry of CD38 or CD138 expression among donor derived cells in the spleens of recipient mice after prime or boost immunizations by the gp120 antigens from either the THRO4156.18 (THRO, Red) or the YU2.DG (YU2, Blue) HIV strains, gated on live, singlets, CD45.1⁺. ###pv = 0.0003, ##pv = 0.0044, #(D) = pv = 0.0338, #(E) = pv = 0.0125, for two-way ANOVA and **pv = 0.0012, *(D) = pv = 0.0222, *(E) = pv = 0.0143, Tukey’s multiple comparison (n = 3, each dot represents a mouse). For gating strategy see Supplementary Fig. 12.

Fig. 5. Adoptively transferred engineered B cells can undergo CSR and clonal expansion upon immunization.

a Isotype specific anti-idiotypic ELISA measuring 3BNC117 isotypes in mice sera collected after boost immunizations. # left = pv = 0.0278, # right = pv = 0.0309, ##pv = 0.0014 for Dunnett’s multiple comparisons and ***pv = 0.0003 and * left = pv = 0.0343 and * right = pv = 0.0461 for two-tailed t-test. Comparisons performed to sera of mice receiving adoptive transfer of non-engineered B cells and boost immunized with the YU2.DG gp120 antigen. b Analysis by flow cytometry of IgA and CD45.1 expression among GC cells after prime and boost immunizations, gating on live, lymphocytes, GL-7⁺, B220⁺. ## upper = pv = 0.0087 and ##pv = 0.0041 for two-way ANOVA, *pv = 0.0074 for two-tailed t-test and indicated p value is for one-sample t-test c Ratio of non-synonymous to synonymous mutations in the different samples. #pv = 0.0143 for two-way ANOVA between the prime and boost cohorts. *pv = 0.0169, **pv = 0.0088, ***pv = 0.0002 for two-tailed t-test and indicated p value is for one-sample t-test. d Quantitation of the clonal expansion by measuring polarity: the relative cumulative share of the ten most abundant clones. # pv = 0.0496; two-way ANOVA. e Pie charts of mice immunized with the YU2.DG gp120 antigen and having at least one clone representing more than >10% of the mutant repertoire. The most abundant clones in each mouse were colored. Shades of red indicate clones that were not found in the ten most abundant clones of other mice (Supplementary Fig. 10). Shades of blue indicate shared clones. Indicated clones are the K46R, A89T, and D97Y. f Quantitation of amino acid (AA) substitutions per clone in mice immunized once or twice with the YU2.DG or THRO4156.18 gp120 antigens. ##pv = 0.005, #pv = 0.0177 for two-way ANOVA and **pv = 0.0043, *pv = 0.0167 for Tukey’s multiple comparison. For c, d, f n = 3 for all except THRO Boost samples in which n = 2, each dot represents a mouse.