JNJ-78306358, a first-in-class bispecific T cell engaging antibody targeting CD3 and HLA-G

Abstract

T cell-redirecting bispecific antibodies (bsAbs) to treat advanced stage solid tumors are gaining interest after recent clinical successes. The immune checkpoint human leukocyte antigen G (HLA-G) is expressed in several tumor types while in normal tissues expression is limited. Here, we describe JNJ-78306358, a T cell-redirecting bispecific antibody (bsAb) to treat advanced stage solid tumors. JNJ-78306358 binds with high affinity to the α3 subunit of HLA-G on cancer cells and with purposely engineered weaker affinity to CD3ε on T cells. JNJ-78306358 induced potent T cell-mediated cytotoxicity of HLA-G-expressing solid tumors in vitro and in vivo. JNJ-78306358 also blocked the interaction of HLA-G with its receptors in vitro, indicating that immune checkpoint blocking may contribute to its anti-tumor activity. These results suggest that T cell-redirection against HLA-G could be a potent and effective treatment for a wide range of solid tumor indications.

Keywords: Biological sciences; Cancer systems biology; Health sciences; Immunology; Medical specialty; Medicine; Natural sciences; Oncology; Pharmacology.

Graphical abstract

Figure 1

Level of HLA-G expression in human cancer and normal tissue samples by IHC IHC was performed on FFPE tumor (A and B) and normal (C–F) tissues using 4H84 at 1 μg/mL and 2 μg/mL dilution, respectively. Detection and counterstaining of tumor tissues was achieved as per manufacturer’s instructions using Bond Polymer Refine Detection kit (Leica), while secondary HRP-conjugated OmniMap anti-mouse antibody, followed by chromogenic detection with DAB was performed for normal tissues. (A) Quantification of HLA-G expression on large tissue samples from renal (n = 77), ovarian (n = 87) and colon (n = 28) cancers demonstrated overall positivity of 75%, 61% and 64%, respectively. Tumor HLA-G levels are represented as staining intensity (0, 1+, 2+ and −3+) and percent positivity for H-score >0. (B) Representative IHC images (20x magnification) for each indication under respective graph. (C and D) HLA-G expression by IHC in human placenta (C, bar = 1 mm) with positive staining of extravillous trophoblast (EVT) (D, bar = 100 μm). (E and F) HLA-G expression by IHC in pituitary gland (E, bar = 1 mm) with positive staining of neuroendocrine cells (F, bar = 100 μm) in anterior pituitary.

Figure 2

JNJ-78306358 binds to a membrane-proximal region of the α3-subunit of HLA-G and competes with hILT2 and hILT4 binding to HLA-G (A) Hydrogen-deuterium exchange-based mass spectrometry (HDX-MS) was used to identify HLA-G regions bound by JNJ-78980577, human ILT2, or human ILT4. Binding epitopes for each agent (JNJ-78980577, ILT2 and ILT4) are highlighted in red on the crystal structure of HLA-G/β2m (PDB ID 1YDP). JNJ-78980577, ILT2, and ILT4 share a partial binding epitope comprising the AB loop within the HLA-G α3 subunit. (B) Sequence alignment of the α3 domain AB loop of HLA-G and related homologs highlight F219 as unique to human HLA-G (Mafa-AG: cynomolgus monkey homolog, Mamu-AG: rhesus macaque homolog, Qa-2 putative mouse homolog). (C) Dose dependent inhibition of HLA-G binding to hILT2/4-expressing HEK cells was determined by monitoring the changes in fluorescence. The inverse of the normalized fluorescence is reported as % inhibition in HLA-G:ILT2/hILT4 binding. Antibodies and target cell lines tested are indicated in the legend.

Figure 3

JNJ-78306358 binds specifically to K562 cells expressing HLA-G and Jurkat cells expressing CD3, and induces T cell mediated killing of HLA-G-expressing cell lines (A) JNJ-78306358 was designed as a heterodimeric “bipod” type bsAb. The antibody features mutations in the Fc to abolish interaction with Fcγ receptors. The anti-CD3 binding region formatted as a Fab binds to CD3ε with K_D = 22 nM. The anti-HLA-G binding region is formatted as a single-chain fragment variable (scFv) binding to HLA-G with K_D = 13 pM. (B and C) Dose dependent binding of JNJ-78306358 to cells (K562 and CHO) expressing HLA-G or other class I MHC molecules (B), and Jurkat cells negative (CD3^Neg) or positive (CD3^Pos) for CD3 (C). The y axis represents fold-increase in mean fluorescence intensity over background. MHC Class I-transfected cells expressed HLA-A/B/C/E/G from Uniprot IDs P01892/P18464/P30508/P13747/P17693, respectively. (D) JNJ-78306358-induced T cell activation and T cell-mediated killing of K562 cells expressing HLA-G. JNJ-78306358 was incubated for 3 days with K562 and human T cells (E:T ratio 8:1). Percent cytotoxicity and % CD25 expression on T cells were measured by flow cytometry. Error bars represent mean ± SEM.

Figure 4

JNJ-78306358 demonstrates potent T cell mediated killing of HLA-G expressing cancer cells (A) Level of HLA-G expression, ratio of geoMFI, on indicated cell lines was assessed by flow cytometry with anti-HLA-G JNJ-78980577-PE antibody and compared to that of isotype control IgG1-PE antibody. Note, JNJ-78980577 is a mAb consisting of the same anti-HLA-G variable region as JNJ-78306358. BICR6 and HuP-T3 cell lines demonstrated biphasic staining for membrane HLA-G expression. The percentage of subpopulation with high HLA-G expression is indicated for these cell lines in parentheses. Graphs are normalized to mode. (B) Dose response of JNJ-78306358 in healthy donor T cell-mediated killing of indicated cancer cell lines was assessed at 72 h with E:T ratio of 3:1 and 1:1. Duplicate samples were tested for each condition, and data are graphed as the mean ± SD. The experiment was performed with several donors for each cell line; results for a single donor are shown. Error bars represent mean ± SEM. (C) JNJ-78306358 50% effective concentrations (EC₅₀) for T cell activation (CD25), cytotoxicity (at 96 h) and secretion of measured cytokines (at 48 h). Cytotoxicity (% killed RERF-LC-Ad1 target cells) was measured by xCELLigence real-time cell analyzer and percentage was calculated as described in STAR Methods. T cell activation (% CD25⁺ T cells) was measured on T cells by FACS analysis (CD25-BV421, Biolegend 302630, 1:125 dilution). Each donor (n = 6) is presented with a different color. Individual curves are reported in Figure S6. (D) Correlation between level of HLA-G expression by cancer cells with secretion of INF-γ by T-cells and sensitivity to JNJ-78306358 mediated T cell killing. Quantification by MSD of in vitro IFN-γ release from 6 renal cell carcinoma DTC samples upon JNJ-78306358 treatment. HLA-G expression levels were determined by flow cytometry (receptor density, RD and % of HLA-G⁺ cells) and Wes (% area of HLA-G of total protein). IFN-γ arbitrary threshold based on dichotomic maximum induction (<100 pg/mL and >200 pg/mL) is indicated by red dotted line and correlates with HLA-G expression, where higher HLA-G expression leads to increased JNJ-78306358 induced INF-γ production. (E) Extrapolation of sensitivity to JNJ-78306358 mediated T cell killing based on HLA-G quantification and IFN-γ secretion threshold (defined in D). Level of HLA-G expression by capillary Wes for indicated normal and tumor tissue samples, including renal DTC samples (analyzed in D). Values represent total area under the HLA-G^Pos peak normalized to area of total protein peak. HLA-G levels detected in HuP-T3 and RER-FL-CAd1 cell lines are shown as positive controls and correlate with their sensitivity to JNJ-78306358 mediated T cell killing.

Figure 5

Levels of human serum HLA-G Soluble HLA-G (sHLA-G) levels in serum from healthy (normal, pregnant) donors and cancer (lung, kidney, ovary and colorectal) patients were measured by MSD assay with JNJ-78306358 as a capture antibody and 4H84 as a detection antibody. Dash line indicates the limit of quantification (LLOQ) of 39 ng/mL. Statistically significant differences were determined using t-test. Asterisks indicate P-value <0.001. Bars indicate median values.

Figure 6

HLA-G expression and JNJ-78306358 activity against human cell line- and patient-derived xenografts (A) Efficacy of JNJ-78306358 on SC HuP-T3 pancreatic tumor cell line-derived xenografts (CDX) in T cell-humanized NSG mice. Group tumor volumes are graphed as the mean ± SEM (n = 10/group). Tumor cells were implanted on Day 0, T cells were administered IP on Day 8, and antibody treatment occurred on Days 9, 13, 16, 20, 23, 27, 30, 34, 37, and 41 (dosing period indicated by the bar below the X axis). ∗ Denotes significant difference (p ≤ 0.05) of treatment groups versus control group on Day 41. Error bars represent mean ± SEM. (B) JNJ-78306358 induced T cell infiltration in HuP-T3 CDX in T cell-humanized mice. Tumor cells were implanted on Day 0, T cells were implanted on Day 8, followed by treatment with JNJ-78306358 or nullxCD3 antibodies at indicated doses on Days 9 and 13. Representative IHC micrographs of HuP-T3 CDX stained for the presence of intratumoral CD8⁺ and CD4⁺ T-cells are shown. Magnification bar denotes 200 μm. (C) Study scheme of JNJ-78306358 in vivo anti-tumor efficacy against PDX tumor models from different cancer types in T cell humanized NSG mice. Tumors were implanted SC and after randomization (group mean tumor volumes 131–182 mm³) mice received 2 × 10⁷ human CD3⁺ T cells IP (allogeneic T cell transfer, ACT). JNJ-78306358 (0.03 and 0.3 mg/kg) treatment was initiated one day after ACT on Days 1, 5, 8,12,15 and 19. Tumors were collected from an additional untreated cohort (4 mice) on Day 14 and assessed for HLA-G expression by IHC (Table S5; Figure S7). (D) Association between HLA-G expression in PDX (n = 10) and their sensitivity to JNJ-78306358. HLA-G IHC staining was performed with 4H84 and JNJ-78655317. Level of HLA-G expression is presented as H-score. Antitumor efficacy of JNJ-78306358 at 0.3 mg/kg was assessed at the first tumor measurement after the end of therapy, Day 20 or Day 21 depending on the tumor model, using the vehicle control group as a reference. Antitumor efficacy for respective tumor models is annotated on either as complete responses (CR), as the %ΔTGI>0, or %ΔTGI ≤0. HLA-G expression is denoted by different colors; high (blue), low-medium (gray) and no expression (red). (E) Representative anti-tumor efficacy of JNJ-78306358 against tumor models presented in panel D expressing high (RXF 488; blue), low-medium (BXF 439; gray), and no HLA-G (PAXF 546; red), plotted as mean tumor volumes over time.

Figure 7

JNJ-78306358 exerts T cell-mediated anti-tumor activity in CD34⁺ HSC humanized mice (A) JNJ-78306358 efficacy against PAXF 1657 human pancreatic PDX tumors in CD34⁺-Human Stem Cells (HSC)-humanized NSG-SGM3 mice. HSCs were implanted 91 to 105 days prior to tumor implantation followed by treatment with JNJ-78306358 at indicated doses, HLA-G x null at 1 mg/kg or PBS control on Days 10, 13, 17, 20, 24, 27, 31, and 34 (dosing period indicated by the bar below the X axis). (n = 10–11/group). ∗∗∗Denotes significant difference (p ≤ 0.001) between each JNJ-78306358-treatment group versus PBS vehicle control group on Day 35. Error bars represent mean ± SEM. (B) JNJ-78306358 efficacy against SC LXFA 983 lung PDX tumors in CD34⁺ HSC-humanized NSG-SGM3 mice. HSCs were implanted 84 to 91 days prior to tumor implantation, followed by treatment with JNJ-78306358 at 0.3 mg/kg or null x CD3 at 1 mg/kg on Days 18, 21, 25, 28, 32, 35, 39, and 42. (n = 8–9/group). ∗Denotes significant difference (p ≤ 0.05) between the JNJ-78306358 0.3 mg/kg group versus control (null x CD3) group at Day 46. (C) Increased number of T cells (CD3⁺/CD56^neg, CD8⁺ and CD4⁺) and activation (increased CD25 expression) of CD8⁺ T cells upon treatment with JNJ-78306358 (0.3 mg/kg) of PDX LXFA 983 tumors in human CD34⁺ NOG-EXL mice. HSCs were implanted approximately 91 days prior to tumor implantation, followed by biweekly treatment with JNJ-78306358 at 0.3 mg/kg starting on Day 21. Flow cytometry analysis was performed on leukocytes from each collected tumor on experiment day 5 and total cell count for selected population (X axis) plotted. The horizontal bar in each dataset indicates the mean value. (D) Intratumoral CD3⁺ T cell infiltration of experiment described in B at Day 12, after administration of 4 doses of JNJ-78306358 at 0.3 mg/kg. Tumors were FFPE fixed. Representative IHC micrographs are shown for LXFA 983 tumors treated on Days 1, 4, 8 and 11, followed by tumor harvest 24 h later on Day 12, and stained for CD3.