Bispecific antibody simultaneously targeting PD1 and HER2 inhibits tumor growth via direct tumor cell killing in combination with PD1/PDL1 blockade and HER2 inhibition

Abstract

Immune checkpoint blockade has shown significant clinical benefit in multiple cancer indications, but many patients are either refractory or become resistant to the treatment over time. HER2/neu oncogene overexpressed in invasive breast cancer patients associates with more aggressive diseases and poor prognosis. Anti-HER2 mAbs, such as trastuzumab, are currently the standard of care for HER2-overexpressing cancers, but the response rates are below 30% and patients generally suffer relapse within a year. In this study we developed a bispecific antibody (BsAb) simultaneously targeting both PD1 and HER2 in an attempt to combine HER2-targeted therapy with immune checkpoint blockade for treating HER2-positive solid tumors. The BsAb was constructed by fusing scFvs (anti-PD1) with the effector-functional Fc of an IgG (trastuzumab) via a flexible peptide linker. We showed that the BsAb bound to human HER2 and PD1 with high affinities (EC₅₀ values were 0.2 and 0.14 nM, respectively), and exhibited potent antitumor activities in vitro and in vivo. Furthermore, we demonstrated that the BsAb exhibited both HER2 and PD1 blockade activities and was effective in killing HER2-positive tumor cells via antibody-dependent cellular cytotoxicity. In addition, the BsAb could crosslink HER2-positive tumor cells with T cells to form PD1 immunological synapses that directed tumor cell killing without the need of antigen presentation. Thus, the BsAb is a new promising approach for treating late-stage metastatic HER2-positive cancers.

Keywords: HER2; PD1 blockade; PD1 immunological synapse; Trastuzumab; antibody-dependent cellular cytotoxicity (ADCC); bispecific antibody.

Fig. 1. The structure and properties of the anti-HER2×PD1 BsAb.

a Schematics of the anti-HER2×PD1 BsAb structure. b SDS-PAGE showing nonreduced and reduced anti-HER2×PD1 BsAb. Lane 1: nonreduced BsAb; Lane 2: reduced BsAb; Lane 3: nonreduced trastuzumab; Lane 4: reduced trastuzumab; M: Molecular weight markers. c SEC chromatogram showing that the BsAb purified by a single-step protein A affinity column had over 95% monomeric species. d Differential scanning calorimetry (DSC) of the anti-HER2×PD1 BsAb showing that the antibody has a T_onset (the temperature at onset of melting) of 52.5 °C and T_m1/2/3 (melting temperatures) of 59.2 °C/68.4 °C/ 83.5 °C, respectively.

Fig. 2. The anti-HER2×PD1 BsAb simultaneously bound to PD-1 and HER2.

a The binding affinity of the BsAb for HER2 was measured by ELISA. Trastuzumab was used as the positive control. b The binding affinity of the BsAb for PD1 was measured by ELISA and compared to that of the parental anti-PD1 mAb, 609A. c The ability of the BsAb to bind to BT474, a HER2-overexpressing cancer cell line, was measured by FACS and compared to that of trastuzumab. d The ability of the BsAb to bind to PD1-overexpressing CHO cells was measured by FACS and compared to that of the parental anti-PD1 mAb, 609A. e A bridging ELISA was set up such that PD1 proteins were coated on the plates followed by the sequential addition of the indicated antibodies and His-tagged HER2 proteins. Anti-6×HisTag monoclonal antibody-HRP was added to visualize the positive binders. The results confirm that the BsAb is capable of simultaneously crosslinking its two targets, HER2 and PD1.

Fig. 3. The anti-HER2×PD1 BsAb inhibited the proliferation of HER2-overexpressing tumor cells and blocked the PD1/PDL1 interaction in cell-based bioassays.

a The BsAb inhibited the proliferation of HER2-overexpressing BT474 cancer cells in a dose-dependent manner similar to trastuzumab. b The ability of the BsAb to block PD1/PDL1 signaling was measured and compared to that of the parental anti-PD1 mAb, 609A, using a PD1/PDL1 blockade cell-based assay, in which the expression of luciferases were monitored under the control of nuclear factor of activated T cells (NFAT) response elements in response to blockade of PD1/PDL1 signaling (Promega Cat#J1250). A nonspecific IgG1 was used as the negative control.

Fig. 4. The anti-HER2×PD1 BsAb retained ADCC toward tumor cells but not T cells.

a The BsAb exhibited a potency in lysing tumor cells similar to that of trastuzumab in the ADCC assay. b The BsAb and trastuzumab failed to mediate ADCC toward T cells, whereas an anti-MHC1 IgG1 antibody showed strong potency in lysing T cells in a dose-dependent manner [44].

Fig. 5. The anti-HER2×PD1 BsAb exhibited synergistic killing effects on cancer cells in a PDL1 expression-independent manner ex vivo.

a Human PBMCs were mixed with PDL1-overexpressing N87 tumor cells (N87-PDL1) in the presence of 2, 10, and 50 nM indicated antibodies (the N87-PDL1/PBMC pair). b Activated Jurkat T cells were mixed with parental N87 tumor cells in the presence of 2 and 9 nM indicated antibodies (the N87/T cell pair). With respect to the combination treatment comprising trastuzumab and 609A, equal molar amounts of the two mAbs (2, 10, and 50 nM each) were combined and added to the cells. The number of viable cells was measured as relative luminescence units (RLUs). The different treatment groups were as follows: N87-PDL1 (N87), N87-PDL1 (N87) tumor cells only; N87-PDL1+PBMCs (N87+T cells), N87-PDL1 cells mixed with PBMCs (N87 cells mixed with T cells) in the absence of antibodies; Anti-PD1 mAb+PBMCs (Anti-PD1 mAb+T cells), N87-PDL1 cells mixed with PBMCs (N87 cells mixed with T cells), and the anti-PD1 mAb, 609A; Trastuzumab+PBMCs (Trastuzumab+T cells), N87-PDL1 cells mixed with PBMCs (N87 cells mixed with T cells) and trastuzumab; Trastuzumab+anti-PD1 mAb+PBMCs (Trastuzumab+anti-PD1 mAb+T cells), combination of trastuzumab and 609A added to N87-PDL1 cells in the presence of PBMCs (N87 cells in the presence of T cells); and Anti-HER2×PD1 BsAb+PBMCs (Anti-HER2×PD1 BsAb+T cells), the BsAb added to N87-PDL1 in the presence of PBMCs (N87 cells in the presence of T cells). *P < 0.05, **P < 0.01, and ****P < 0.0001 by two-way ANOVA. c PD1-overexpressing Jurkat T cells prelabeled with an Alexa Fluor 647-conjugated anti-PD1 mAb (Sinobiological, Cat#MM18) were mixed with N87 cells prelabelled with an Alexa Fluor 546-conjugated anti-HER2 mAb (19H6-Hu) in the presence of the BsAb (left), the mixture of trastuzumab and 609A (center), the BsAb plus 609A (right). The group in red (pseudo-color) (upper right corner) shows cells that have dual emission signals (MFI > 1 × 10⁴ for FITC-A and MFI > 3 × 10⁴ for APC-A), meaning that the two types of cells were associated together. The group in green (pseudo-color) (upper left corner) shows T cells labeled with the Alexa Fluor 647-conjugated anti-PD1 mAb (MFI > 3 × 10⁴ for APC-A). The group in purple (pseudo-color) (lower right corner) shows N87 cells labeled with the Alexa Fluor 546-conjugated anti-HER2 mAb (MFI > 1 × 10⁴ for FITC-A). d Immunofluorescence microscopy showing costaining of PD1 (green) on T cells and HER2 (red) on N87 tumor cells in samples treated with the BsAb or a mixture of the anti-PD1 mAb (609A) plus trastuzumab. Note that one tumor cell was ligated with two T cells, while a single T cell was in contact with two tumor cells in the presence of the BsAb. The combination of 609A and trastuzumab failed to induce PD1 synapse formation with T cells. The white arrows denote T cells (DIC channel), for which PD1 staining (green channel) were barely seen owing to the rearrangement of PD1 during synapse formation.

Fig. 6. The anti-HER2×PD1 BsAb exhibited potent antitumor effects in vivo.

a A control (black circle), the BsAb (blue square and pink triangle), and trastuzumab (green triangle) were i.p. injected into mice bearing NCI-N87 tumors at the indicated doses. Tumor volumes (mm³) were measured at the indicated time points. b A control (black circle), the BsAb (green triangle and diamond), and the anti-PD1 mAb, 609A (red square and triangle) were i.p. injected into human PD1 transgenic mice bearing mouse MC38 tumors at the indicated doses. c An isotype control (blue square), 609A (orange triangle), 609A plus trastuzumab (green diamond, 20 mg/kg each), and the BsAb (red circle) were injected into M-NSG mice bearing JIMT-1 tumors in the presence of human PBMCs at the indicated doses. WT mice (black dot) were used as control. Tumor volumes (mm³) were measured at the indicated time points. Mean ± SEM. *P < 0.05 by two-way ANOVA (the BsAb/PBMC vs Vehicle/PBMC) and ****P < 0.0001 for the comparison of all indicated groups with the control group.