Antigen sensitivity of CD22-specific chimeric TCR is modulated by target epitope distance from the cell membrane

Abstract

We have targeted CD22 as a novel tumor-associated Ag for recognition by human CTL genetically modified to express chimeric TCR (cTCR) recognizing this surface molecule. CD22-specific cTCR targeting different epitopes of the CD22 molecule promoted efficient lysis of target cells expressing high levels of CD22 with a maximum lytic potential that appeared to decrease as the distance of the target epitope from the target cell membrane increased. Targeting membrane-distal CD22 epitopes with cTCR(+) CTL revealed defects in both degranulation and lytic granule targeting. CD22-specific cTCR(+) CTL exhibited lower levels of maximum lysis and lower Ag sensitivity than CTL targeting CD20, which has a shorter extracellular domain than CD22. This diminished sensitivity was not a result of reduced avidity of Ag engagement, but instead reflected weaker signaling per triggered cTCR molecule when targeting membrane-distal epitopes of CD22. Both of these parameters were restored by targeting a ligand expressing the same epitope, but constructed as a truncated CD22 molecule to approximate the length of a TCR:peptide-MHC complex. The reduced sensitivity of CD22-specific cTCR(+) CTL for Ag-induced triggering of effector functions has potential therapeutic applications, because such cells selectively lysed B cell lymphoma lines expressing high levels of CD22, but demonstrated minimal activity against autologous normal B cells, which express lower levels of CD22. Thus, our results demonstrate that cTCR signal strength, and consequently Ag sensitivity, can be modulated by differential choice of target epitopes with respect to distance from the cell membrane, allowing discrimination between targets with disparate Ag density.

Figure 1. Maximum lytic efficiency of cTCR⁺ CTL correlates with the distance of the target antigen epitope from target cell membrane

(A) Schematic of cTCR structure. scFv: single chain antibody, IgG1 Fc: CH1 hinge and constant heavy domains 2 and 3 of IgG1, CD4TM: CD4 transmembrane. (B) Epitope mapping of HD39 and RFB4 scFv binding sites of CD22. CD20 is shown to scale. (C) Flow cytometric measurement of cTCR expression. cTCR or vector transduced CTL were stained with PE-labeled goat anti-human Fcγ-specific polyclonal IgG. Black solid: empty vector, Black line: Leu16, Grey line: RFB4, and Dashed line: HD39. (D) Target lysis assay using ⁵¹Cr-labeled CD20⁺CD22⁺ Daudi cell line as targets. The data are representative of 4 experiments. Points represent individual samples. * p < 0.05, *** p < 0.0001: paired T-test, two tailed. Dashed line: Leu16 vs. RFB4 (*), Solid black line: RFB4 vs. HD39 (***), Grey solid line: Leu16 vs. HD39 (***).

Figure 2. Lysis defect of CD22-specific cTCR is not due to insufficient target antigen density, insufficient cTCR expression density, or low affinity of scFv

(A) Flow cytometry of transduced CD22⁺ Jurkat clones with varying surface density. Cells were stained with PE-H-SCL-1 anti-CD22 mAb. Six clones out of 12 to 15 total per antigen type are shown. Similar results were obtained for CD20⁺ Jurkat clones. (B and C) Antigen sensitivity of target lysis and cTCR down-modulation. 7-AAD lysis assay and cTCR down-modulation assay were performed as described in Materials and Methods. The expression density of the RFB4 cTCR is five fold greater on the CTL line used in figure 2C compared with the RFB4 cTCR⁺ CTL used in 2B.2. Points represent the average of duplicate samples in this and all following dose-response experiments. (D) Relationship between antigen density and cTCR down-modulation. Numbers of cTCR expressed per T cell were estimated as described in Materials and Methods. Data from cTCR down-modulation assay were converted from percentages to absolute numbers of down-modulated cTCR. Numbers of cTCR down-modulated were plotted vs. numbers of CD20 or CD22 ABS (estimated as described in Materials and Methods) for antigen densities below the cTCR down-modulation inflection point. Data are representative of at least 3 experiments.

Figure 3. Targeting membrane distal epitopes produces both a degranulation and granule targeting defect

Degranulation of cTCR⁺ CTL in response to Jurkat clones expressing varying densities of CD20 or CD22. Degranulation was assessed by BLT-esterase activity as described in Materials and Methods. Data from three experiments are overlaid.

Figure 4. Targeting membrane distal epitopes results in diminished signaling per triggered cTCR

cTCR down-modulation was determined in simultaneous experiments in which target lysis and degranulation were also measured. The number of cTCR expressed per CTL was estimated as described in Materials and Methods and converted to the number of cTCR down-modulated. Percent degranulation (A) or percent target lysis (B) in response to a particular antigen density was plotted against the number of cTCR down-modulated in response to the same antigen density. One representative experiment of three is shown.

Figure 5. Targeting a truncated CD22-based ligand restores sensitivity and efficiency of degranulation and target lysis

(A) RFB4 or HD39 cTCR⁺ or empty vector transduced CTL were incubated on goat anti-human Fcγ-specific IgG-coated plates (200 μg/mL) for two hours in 100 μL RPMI. Supernatants were obtained and degranulation was determined in a BLT esterase assay. Average and standard deviation of duplicate samples are shown and are representative results from one of three experiments. (B) Schematic of truncated CD22-based ligand Ig1-2. (C) Degranulation in response to unsorted transduced Jurkat cells expressing similar, intermediate densities of WT CD22 or Ig1-2. Average and standard deviation of duplicate samples are shown and are representative of two experiments. (D) Degranulation (BLT assay) or target lysis (7-AAD assay) by HD39 cTCR⁺ CTL in response to Jurkat clones expressing varying densities of WT CD22 or Ig1-2. Representative results from one of three experiments are shown. (E) Overlay of data from three experiments in which target lysis by Leu16 or HD39 cTCR⁺ CTL was analyzed in response to Jurkat clones expressing varying antigen densities of CD20 or CD22 WT and Ig1-2, respectively.

Figure 6. Membrane distal target epitopes are not inhibitory in the presence of membrane proximal ones

(A) Lysis of CD22 WT^Hi Ig1-2^Hi or Ig1-2^Hi Jurkat cell lines by RFB4, HD39, or empty vector transduced CTL. Lysis was analyzed by the 7-AAD lysis assay. (B) Schematic of WT CD22, Ig1-2, and rearranged Ig3-7;1-2 CD22-based ligand. (C) Target lysis of Jurkat cell lines expressing equivalent densities of WT CD22, Ig1-2, and Ig3-7;1-2 ligands by RFB4, HD39, or Leu16 cTCR⁺ CTL. Lysis was analyzed by the 7-AAD lysis assay. Data are representative results from one of three experiments. Average and standard deviation of duplicate samples are shown for (A) and (C).

Figure 7. Diminished lytic sensitivity of CD22-specific cTCR targeting WT CD22 permits selective lysis of tumor cell lines and weak responses to normal, autologous B cells

(A) Target lysis of the B cell lymphoma lines Raji or Ramos and of CD19 positively selected autologous B cells by Leu16, RFB4, and HD39 cTCR⁺ CTL or empty vector transduced CTL was assessed by the 7-AAD assay. Average and standard deviation of triplicate samples shown and are representative of four experiments. (B) cTCR down-modulation in response to tumor cell lines or autologous B cells was assessed by the cTCR down-modulation assay. For comparison, the cTCR down-modulation dose-response curve generated by cTCR⁺ CTL responding to Jurkat clones expressing varying CD20 or CD22 expression densities is superimposed. Data are representative of two experiments.