Expression evaluation of AFP and GPC3 in liver most cancers
To develop efficient and secure CAR-T cell remedy for liver most cancers, we investigated the expression of AFP and GPC3 in hepatocellular carcinoma (HCC) and regular tissues. This evaluation helps predict potential therapeutic results and hostile occasions related to CAR-T merchandise focusing on these antigens.
We utilized the GEPIA2 database (http://gepia2.cancer-pku.cn), which incorporates gene expression knowledge from The Most cancers Genome Atlas (TCGA). Evaluation of pan-cancer samples revealed important expression of AFP and GPC3 primarily in liver most cancers tissues, with minimal expression in regular tissues and different most cancers sorts (Supplementary Fig. 1A, B and Supplementary Tables 1, 2).
Moreover, we analyzed a cohort of 368 tumor tissues and 160 regular liver tissues from sufferers with HCC. This evaluation confirmed important variations within the expression ranges of AFP and GPC3 between liver most cancers tissue and adjoining non-cancerous tissues (Supplementary Fig. 1C and D).
To strengthen the medical relevance of our findings, we carried out a retrospective evaluation on 65 liver most cancers sufferers recognized between 2014 and 2021 at Zhuhai Folks’s Hospital. Immunohistochemical staining of their pathological specimens revealed that fifty.77% of the sufferers have been constructive for AFP, 70.77% have been constructive for GPC3, and 38.46% expressed each antigens (Supplementary Fig. 1E, F, G and Supplementary Desk 3).
These findings strongly counsel that CAR-T cells focusing on AFP and GPC3, liver cancer-specific antigens, maintain promise for improved remedy efficacy and enhanced security.
Improvement of high-affinity AFP-specific antibody
Constructing on the success of our second-generation GPC3 CAR-T with excessive anti-tumor efficacy, we’re initiating the event of an AFP-targeting TCR mimic antibody to discover potential synergistic results together remedy.
Given the excessive prevalence of the HLA-A*02:01 allele (8.15% globally, 12.04% in China), we centered on setting up the hAFP158 − 166/HLA-A*02:01 advanced for optimum antigen presentation (Fig. 1A Up). Following purification of recombinantβ2m and HLA-A*02:01 proteins, we co-complexed them with the hAFP158 − 166 peptide at an outlined ratio. Using Capto Q ImpRes, we purified the complexes to attain 99.09% purity (Fig. 1A Down).
To determine high-affinity antibodies focusing on AFP offered by HLA-A*02:01, we employed a biopanning technique on an artificially synthesized phage show antibody library. After 5 rounds of liquid-phase screening, we recognized 5 candidate VHH antibodies (Fig. 1B, C, D). Monoclonal ELISA was used to pick out 5 candidate antibodies exhibiting low background and important signal-to-noise ratios. These candidates have been sequenced and designated as 1B3, 1C11, 1C4, 1D12, and 2F9. Notably, the Ab61 antibody, encoded by the gene described in patent CN107106671A, was synthesized as a constructive management. Protein A affinity chromatography purified this constructive management. Subsequent one-step Protein A affinity chromatography and SEC-HPLC evaluation confirmed excessive purity for all VHH antibodies (Supplementary Fig. 2A, B and Supplementary Desk 4).
Lastly, affinity assays have been carried out utilizing a ForteBio AHC sensor-conjugated antibody loaded with every candidate antibody and the hAFP158 − 166/HLA-A*02:01 advanced. All candidate antibodies, besides 1D12, demonstrated superior dissociation constants in comparison with the Ab61 constructive management. Notably, the 1B3 antibody exhibited an affinity fixed roughly five-fold larger than Ab61 (Fig. 1E and Supplementary Desk 5).
These outcomes spotlight the profitable improvement of a high-affinity, particular antibody (1B3) focusing on hAFP offered by HLA-A*02:01, a vital part for our dual-targeting CAR-T cell remedy technique.
(A) Up, Building technique of hAFP158-166/HLA-A*02:01 advanced. Down, Purity evaluation of hAFP158-166/HLA-A*02:01 advanced by SEC-HPLC. (B) Antibody screening course of: ①Synthesis of synthetic nanobody phage show library. ② Screening for humanized VHH antibodies. ③ Elution of phages. ④ Evaluation of goal phages. ⑤ Phage amplification and acquisition of candidate VHH plasmid sequences. (C) The acquisition and screening technique of antibodies. (D) 5 rounds of liquid part elution antibody enrichment impact (In every spherical of elution, every column represents a focus gradient of phage inventory answer, which is diluted right into a gradient at a 3-fold ratio from left to proper). (E) Outcomes of affinity assay of various antibodies binding to peptide hAFP158-166/HLA-A*02:01: the affinity of AFP Ab (1B3) antibody reached 0.504nM; the affinity of AFP Ab (1C11) antibody was 1.21nM; the affinity of AFP Ab (1D12) antibody was 3.02nM; the affinity of AFP Ab (1C4) was 0.865nM; the affinity of AFP Ab (2F9) was 2.06nM; the affinity of Bench marker (Ab61) was 2.34nM. The affinity of the three antibodies (1C11, 1D12 and 2F9) was akin to the Bench marker (Ab61) (The six curves in Determine E are the fitted curves for the primary six antigen concentrations)
In vitro practical analysis of the VHH antibodies for AFP and the related CAR-T
To evaluate the specificity of the VHH antibodies, we carried out binding assays utilizing T2 cells (The T2 Cell is a human lymphocyte hybridoma cell that faithfully presents the goal antigenic peptide to the cell floor, thereby focusing on the T cell akin to the goal antibody.), loaded with numerous peptide antigens. The outcomes demonstrated that the antibodies exhibited excessive specificity for the hAFP158 − 166/HLA-A*02:01 advanced, with minimal binding to T2 cells loaded with irrelevant peptides (Fig. 2A). Notably, the 1B3 antibody demonstrated sturdy binding affinity not solely to the HLA-A*02:01 advanced but in addition to the HLA-A*02:07 advanced, suggesting potential applicability in a broader affected person inhabitants (Supplementary Fig. 2C and D).
To guage the cytotoxic potential of the VHH antibodies, we carried out in vitro ADCC assays. The goal cells (HepG2) have been engineered to specific the hAFP158-166/HLA-A*02:01 advanced on their floor (HepG2-MiniG). We co-cultured numerous tumor cell traces HepG2 cell (hAFP+/HLA-A*02:01−), HepG2-MiniG cell (hAFP158 − 166+/HLA-A*02:01+), SK-HEP-1 cell (hAFP−/HLA-A*02:01+), and Raji cell (hAFP−/HLA-A*02:01−) and Daudi cell (hAFP−/HLA-A*02:01−) with PBMCs and completely different VHH antibodies. The outcomes confirmed that every one 5 antibodies exhibited particular cytotoxicity in opposition to hAFP158 − 166+/HLA-A*02:01+ tumor cells, with minimal non-specific killing of different cell traces (Supplementary Fig. 3A, B, C, D and E). It’s significantly noteworthy that the AFP Ab (1B3) and AFP Ab (1C11) antibodies didn’t exhibit non-specific cytotoxicity in opposition to SK-HEP-1 cells at any focus (Supplementary Fig. 3D).
To additional examine the therapeutic potential of those VHH antibodies, we engineered CAR-T cells incorporating the VHH sequences. The goal cells (SK-HEP-1 and HepG2) have been engineered to specific the hAFP158 − 166/HLA-A*02:01 advanced on their floor (named SK-HEP-1-MiniG and HepG2-MiniG), enabling recognition and elimination by CAR-T cells (Fig. 2B).
In vitro repeated stimulation experiments demonstrated that 1B3 CAR-T cells exhibited strong growth and particular cytotoxicity in opposition to goal cells, akin to the constructive management Ab61 CAR-T cells. Furthermore, 1B3 CAR-T cells displayed decrease non-specific cytotoxicity in opposition to hAFP−/HLA-A*02:01− cells, suggesting improved security. Cytokine launch assays revealed that 1B3 CAR-T cells produced comparable ranges of cytotoxic cytokines as Ab61 CAR-T cells, however with decrease baseline cytokine secretion within the absence of goal cells. This implies that 1B3 CAR-T cells might supply a positive stability of efficacy and security (Fig. 2C, D, E, and F).
These findings collectively exhibit the promising potential of the AFP Ab (1B3) antibody and its corresponding CAR-T cells for the focused remedy of liver most cancers, significantly in sufferers expressing HLA-A*02:01 or HLA-A*02:07.
(A) All antibodies confirmed binding exercise to T2 cells loaded with hAFP158-166 peptide, however had non-specific binding to T2 cells loaded with NY-ESO and 19 blended peptides. (B) Illustration of hAFP158-166/HLA-A*02:01 TCR-like CAR-T binding to tumor cells and killing them. (C) In vitro cytotoxicity assay of CAR-T cells constructed with numerous antibodies in opposition to completely different most cancers cells (SK-HEP-1, HepG2, SK-HEP-1-MiniG, HepG2-MiniG). (D) The fold growth of every CAR-T cell within the in vitro repeated stimulation experiment (goal cells: SK-HEP-1-MiniG). (E) The secretion ranges of IFN-γ by CAR-T cells within the cytotoxicity assay at an effector-to-target ratio of 1:1. (F) The secretion ranges of IL-2 by CAR-T cells within the cytotoxicity experiment at an effector-to-target ratio of 1:1
In vivo efficacy of 1B3 CAR-T cells
To guage the in vivo efficacy of 1B3 CAR-T cells, we established a subcutaneous tumor mannequin utilizing SK-HEP-1-MiniG cells in mice. Therapy with 1B3 CAR-T cells considerably inhibited tumor development in comparison with the management group (Fig. 3A, B, and C). Notably, intratumoral administration of CAR-T cells demonstrated enhanced efficacy in comparison with intravenous administration. Importantly, 1B3 CAR-T cells have been well-tolerated, with no important modifications in physique weight noticed (Fig. 3D).
To additional assess the sturdiness of the anti-tumor response, we established a secondary tumor problem mannequin (Fig. 4A and Supplementary Desk 6). Mice have been handled with 1B3 CAR-T cells and, after full tumor regression, have been re-challenged with SK-HEP-1-MiniG cells on the contralateral flank. Remarkably, even at decrease doses, 1B3 CAR-T cells successfully inhibited the expansion of the secondary tumor, demonstrating potent long-lasting anti-tumor exercise (Fig. 4B).
To research the mechanisms underlying the therapeutic efficacy of 1B3 CAR-T cells, we analyzed the persistence and cytokine launch profiles of those cells in vivo. Movement cytometry evaluation revealed that 1B3 CAR-T cells persevered within the tumor microenvironment for an prolonged interval, contributing to sustained tumor management (Fig. 4C). Moreover, 1B3 CAR-T cells exhibited a balanced cytokine launch profile, with elevated ranges of IFN-γ and IL-2, essential for efficient tumor cell killing and T cell proliferation, respectively (Fig. 4D, E).
The security profile of 1B3 CAR-T cells was assessed by monitoring physique weight and peripheral cytokine ranges. No important weight reduction was noticed in any of the remedy teams, indicating good tolerability (Supplementary Fig. 4A). Notably, 1B3 CAR-T cells induced decrease ranges of IL-6, a cytokine related to cytokine launch syndrome (CRS), in comparison with the Ab61 management (Supplementary Fig. 4B). These findings counsel that 1B3 CAR-T cells might have a positive security profile, with a lowered danger of cytokine launch syndrome.
(A) Movement chart of the primary spherical animal experiments (On this examine, the primary CAR-T cell remedy injection was administered on Day 0, SK-HEP-1-MiniG have been inoculated in mice on Day − 9, and the second CAR-T cell remedy injection was administered on Day 14. All through the experimental interval, tumor dimension was measured thrice per week. The proportion of human T cells and cytokine ranges have been assessed by peripheral blood testing). (B) Administration strategies and dosages of CAR-T in every group, tumor quantity of mice at endpoint, and tumor inhibition fee. (C) Tumor development curve of mice in all teams (every group: n = 5). (D) Adjustments in physique weight of mice in every remedy group (every group: n = 5)
(A) Movement chart of the second spherical animal experiments (On this examine, SK-HEP-1-MiniG cells have been inoculated subcutaneously in the fitting flank of mice on Day − 31. When the common tumor quantity reached 151 mm³, CAR-T cells have been administered by way of intratumoral injection. As a result of important tumor regression, tumor cells have been re-inoculated subcutaneously within the left flank on Day 43 to simulate a tumor recurrence mannequin, aiming to judge the sustained anti-tumor efficacy of CAR-T cells). (B) Tumor development curve of mice in all teams, rechallenge: mice with tumor regression have been rechallenged with tumor cells on the contralateral aspect to watch the long-lasting tumor-suppressive results of CAR-T (every group: n = 5). (C) CAR-positive fee of CAR-T cells in peripheral blood of mice on day 7, day 14 and day 21 (every group: n = 5). (D) Secretion degree of IFN-γ within the peripheral blood serum of mice on day 7, day 14 and day 21 (every group: n = 5). (E) Secretion degree of IL-2 within the peripheral blood serum of mice on day 7, day 14 and day 21 (every group: n = 5)
Low-dose 1B3 CAR-T remedy: a promising method for most cancers remedy
To additional examine the therapeutic potential of 1B3 CAR-T cells, we carried out a 3rd spherical of in vivo experiments, specializing in low-dose remedy. Mice bearing subcutaneous SK-HEP-1- MiniG tumors have been handled with intratumoral injections of 1 × 106 or 3 × 105 1B3 or Ab61 CAR-T cells (Fig. 5A). Remarkably, even on the low dose of three × 105 cells, 1B3 CAR-T cells demonstrated superior tumor suppression in comparison with Ab61 CAR-T cells (Fig. 5B and Supplementary Desk 7). To evaluate the long-term efficacy, mice have been re-challenged with tumor cells on the contralateral flank. Each 1B3 and Ab61 CAR-T cells successfully suppressed tumor recurrence, highlighting the sturdiness of the anti-tumor response (Fig. 5B).
In distinction to the earlier experiments with larger preliminary tumor burden and CAR-T cell doses, the height in CAR-T cell growth and cytokine launch occurred later within the low-dose setting. Particularly, a peak in IFN-γ and TNF-α ranges was noticed on day 14, whereas IL-2 ranges remained elevated all through the examine interval (Fig. 5C, D, E and Supplementary Fig. 4D).
Relating to security, no important weight reduction was noticed in any remedy group (Supplementary Fig. 4C). The transient elevation in IL-6 ranges, a cytokine related to cytokine launch syndrome (CRS), was minimal and didn’t persist. These findings counsel that low-dose 1B3 CAR-T remedy provides a positive security profile with minimal hostile results.
Total, these outcomes exhibit the potent anti-tumor exercise and favorable security profile of low-dose 1B3 CAR-T remedy. This method holds important promise for the remedy of liver most cancers with 1B3 antibody focusing on AFP.
(A) Movement chart of the third spherical animal experiments (On this experiment, to problem CAR-T cells with bigger tumors, we inoculated SK-HEP-1-MiniG cells subcutaneously on the fitting flank of mice on Day − 33. When the tumor quantity reached 182 mm³, CAR-T cells have been administered by way of intratumoral injection. Subsequently, fast tumor regression was noticed. On Day 59, mice have been re-challenged with SK-HEP-1-MiniG cells subcutaneously on the left flank to evaluate the long-term antitumor efficacy of CAR-T cells). (B) Tumor development curve of mice in all teams, rechallenge: mice with tumor regression have been rechallenged with tumor cells on the contralateral aspect to watch the long-lasting tumor-suppressive results of CAR-T (every group: n = 5). (C) CAR-positive fee of CAR-T cells in peripheral blood of mice on day 7, day 14 and day 21 (every group: n = 5). (D) Secretion degree of IFN-γ within the peripheral blood serum of mice on day 7, day 14 and day 21 (every group: n = 5). (E) Secretion degree of IL-2 within the peripheral blood serum of mice on day 7, day 14 and day 21 (every group: n = 5)
GPC3 CAR-T cells with built-in AFP-CD3 chew: enhanced anti-tumor exercise
Whereas our earlier technology of GPC3 CAR-T cells demonstrated promising anti-tumor exercise, we sought to additional optimize their efficacy by incorporating extra focusing on methods. The mixing of an optimized AFP-CD3 BiTE into the CAR-T cell design supplies a dual-targeting method, probably enhancing tumor cell killing and overcoming potential resistance mechanisms.
We constructed second-generation GPC3 CAR-T cells incorporating 4-1BB as a co-stimulatory area, built-in an optimized, secreted AFP-CD3 BiTE into the CAR-T cell design. The BiTE molecule, upon secretion, can bind to each AFP-expressing tumor cells and T cells, each endogenous and CAR-T cells, forming an immune synapse and activating bystander T cells (Fig. 6A, B).
To guage the practical exercise of those CAR-T cells, we established a goal cell line (Sk-hep-1-GPC3-AFP) overexpressing each GPC3 and the hAFP158 − 166/HLA-A*02:01 advanced (Fig. 6C). In vitro cytotoxicity assays and repeated stimulation experiments demonstrated that GPC3 CAR-T cells with the built-in AFP-CD3 BiTE (GPC3 CAR-T-AFP-CD3) exhibited superior anti-tumor exercise in comparison with single-target GPC3 CAR-T cells (Fig. 6F, G). Moreover, phenotypic evaluation revealed that GPC3 CAR-T-AFP-CD3 cells displayed a blended phenotype of central reminiscence T cells (TCM) and effector reminiscence T cells (TEM) (Fig. 6D, E). This implies that GPC3 CAR-T-AFP-CD3 cells might possess enhanced persistence and long-term anti-tumor exercise. Notably, GPC3 CAR-T-AFP-CD3 cells constantly demonstrated superior efficiency in each cytotoxicity assays and IFN-γ secretion assays (Fig. 6F, G).
These findings spotlight the potential of our dual-targeting technique to enhance the efficacy of CAR-T cell remedy for liver most cancers. By combining direct tumor cell killing with bystander T cell activation, this method might overcome the challenges related to tumor heterogeneity and immune suppression.
(A) Designed and optimized second-generation GPC3 CAR-T cells that incorporate 4-1BB as a co-stimulatory area and are able to secreting BiTE molecules that bind to AFP-expressing tumor cells and T cells. (B) lllustration of GPC3 CAR-T/AFP-BiTE building. (C) Affirmation of GPC3 and AFP expression on track liver most cancers cell line (SK-HEP-1-GPC3-AFP) by circulation cytometry. (D) Movement cytometry evaluation was carried out to detect the reminiscence/effector phenotype of CAR T cells. (E) Movement cytometry evaluation was carried out to detect the reminiscence/effector phenotype of CAR T cells after repeated stimulation experiment in vitro. (F) Cytotoxicity of CAR-T Cells in vitro cytotoxicity assay. (G) The secretion ranges of IFN-γ in each cytotoxicity assays and autocrine secretion of varied CAR-T cells
Mechanistic insights into enhanced efficacy of GPC3 CAR-T-AFP-CD3 cells
To grasp the mechanisms underlying the superior anti-tumor exercise of GPC3 CAR-T-AFP-CD3 cells, we carried out transcriptome sequencing on these cells after repeated stimulation. Principal part evaluation revealed distinct gene expression profiles between GPC3 CAR-T-AFP-CD3 cells and management teams (Fig. 7A and B). Volcano plots and Venn diagrams recognized differentially expressed genes (DEGs) throughout teams (Figs. 7C-F and Supplementary Tables 8, 9, 10 and 11). Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses revealed enrichment of pathways related to enhanced T cell perform in GPC3 CAR-T-AFP-CD3 cells in comparison with controls (Supplementary Fig. 5A, B, C, D, E and F). Notably, genes like TNF, CD40LG, and CSF2 emerged as key gamers within the practical distinction between GPC3 CAR-T-AFP-CD3 and different CAR-T cell variants (Fig. 7G, H and I). These findings counsel that the incorporation of the AFP-CD3 BiTE into GPC3 CAR-T cells (GPC3 CAR-T-AFP-CD3) modulates gene expression, probably contributing to their superior anti-tumor exercise. Additional investigation of those key genes might present priceless insights for optimizing CAR-T cell remedy for liver most cancers.
(A) The field plot of gene expression degree. (B) The principal part evaluation of the gene expression of samples in every group. (C) The volcano plot of the general distribution of differentially expressed genes when evaluating the GPC3 CAR-T-AFP-CD3 group with the GPC3 CAR-T group; (D) The volcano plot of the general distribution of differentially expressed genes when evaluating the GPC3 CAR-T-AFP-CD3 group with the GPC3 CAR-T-AFP group; (E) The volcano plot of the general distribution of differentially expressed genes when evaluating the GPC3 CAR-T-AFP-CD3 group with the GPC3 CAR-T-CD3 group. (F) The frequent and distinctive differentially expressed genes amongst completely different comparability teams. (G) The PPI community constructed by differentially expressed genes when evaluating the GPC3 CAR-T-AFP-CD3 group with the GPC3 CAR-T group. (H) The PPI community constructed by differentially expressed genes when evaluating the GPC3 CAR-T-AFP-CD3 group with the GPC3 CAR-T-AFP group. (I) The PPI community constructed by differentially expressed genes when evaluating the GPC3 CAR-T-AFP-CD3 group with the GPC3 CAR-T-CD3 group
In vivo efficacy of GPC3 CAR-T with AFP-CD3 chew
To evaluate the in vivo efficacy of GPC3 CAR-T cells with the built-in AFP-CD3 BiTE (GPC3 CAR-T-AFP-CD3), we established a subcutaneous tumor mannequin in mice (Fig. 8A). When tumor volumes reached 80–120 mm³, mice have been intravenously administered with 1 × 106 or 3 × 106 CAR-T cells. The group handled with 3 × 106 GPC3 CAR-T-AFP-CD3 cells exhibited important tumor development inhibition and extended survival in comparison with the management teams (Fig. 8B and Supplementary Desk 12). Importantly, no important weight reduction was noticed in any remedy group, indicating a positive security profile (Fig. 8C). Moreover, elevated ranges of IFN-γ within the peripheral blood and growth of CAR-positive T cells have been noticed in mice handled with GPC3 CAR-T-AFP-CD3 cells (Fig. 8D, E). These findings counsel that the BiTE-based technique enhances the anti-tumor exercise of CAR-T cells, with the potential recruiting and activating endogenous T cells.
In conclusion, our examine demonstrates the potential of GPC3 CAR-T cells with the built-in AFP-CD3 BiTE as a promising therapeutic method for liver most cancers. This dual-targeting technique provides improved anti-tumor efficacy and a positive security profile, making it a promising candidate for future medical translation.
(A) Schematic of the liver most cancers xenograft mannequin used to investicate the in vivo activitof CAR-T cells (On this experiment, SK-HEP-1-GPC3-AFP cells have been inoculated subcutaneously on Day − 15. When tumor quantity reached 80–120 mm³, mice have been randomly grouped. The primary intravenous administration of CAR-T cells was carried out on Day 0, adopted by a second intravenous administration on Day 14). NSG mice have been subcutaneously injected with 3 × 106 goal cells, and CAR-T cells have been injected at specified time factors. Every group consisted of n = 5 mice. (B) Tumor development was assessed thrice per week following CAR-T cell injection. (C) ln vivo toxicity was evaluated by monitoring the mice‘s physique weights all through the remedy. (D–E) Peripheral blood samples have been collected weekly to research the degrees of CD3+ T cells (D) and IFN-γ (E)
