Characterization of a humanized IgG4 anti-HLA-DR monoclonal antibody that lacks effector cell functions but retains direct antilymphoma activity and increases the potency of rituximab

Abstract

HLA-DR is under investigation as a target for monoclonal antibody (mAb) therapy of malignancies. Here we describe a humanized IgG4 form of the anti-HLA-DR mAb L243, hL243gamma4P (IMMU-114), generated to provide an agent with selectivity toward neoplastic cells that can kill without complement-dependent cytotoxicity (CDC) or antibody-dependent cellular-cytotoxicity (ADCC), so as to reduce reliance on intact immunologic systems in the patient and effector mechanism-related toxicity. In vitro studies show that replacing the Fc region of hL243gamma1, a humanized IgG1 anti-HLA-DR mAb, with the IgG4 isotype abrogates the effector cell functions of the antibody (ADCC and CDC) while retaining its antigen-binding properties, antiproliferative capacity (in vitro and in vivo), and the ability to induce apoptosis concurrent with activation of the AKT survival pathway. Growth inhibition was evaluated compared with and in combination with the anti-CD20 mAb rituximab, with the combination being more effective than rituximab alone in inhibiting proliferation. Thus, hL243gamma4P is indistinguishable from hL243gamma1 and the parental murine mAb in assays dependent on antigen recognition. The abrogation of ADCC and CDC, which are believed to play a major role in side effects of mAb therapy, may make this antibody an attractive clinical agent. In addition, combination of hL243gamma4P with rituximab offers the prospect for improved patient outcome.

Figure 1.

Binding characteristics of hL243γ4P and hL243γ1 relative to the parental murine L243. (A) Binding of hL243γ1 to Raji cells was measured using PE-conjugated second antibody (goat anti-human IgG, Fc fragment specific) and counting in a Guava PCA system. (B) Competitive binding assay. A cell-surface competitive binding assay was performed to compare the binding activity of hL243γ4P with the parental murine L243. Varying concentrations of mL243 (▪) or hL243γ4P (▴) were mixed with a constant amount of ¹²⁵I-hL243γ4P and incubated with Raji (left) or Daudi cells (right). The cells were washed to remove unbound mAbs and counted for the bound residual radioactivity. (C) Direct cell-surface saturation binding and Scatchard plot analysis on Daudi cells. mL243, ▪; hL243γ4P, ▴.

Figure 2.

Assessment of CDC and ADCC. (A) Daudi cells were treated with hL243γ4P or control mAbs, as indicated, at the concentrations shown in the presence of human complement. Cell viability was measured using resazurin and reported as percentage of viable population relative to cells treated with complement only (no mAb). (B) ⁵¹Cr-labeled NHL cell lines were incubated with anti-B-cell mAbs in the presence of human complement. Following a 3-hour incubation at 37°C, supernatants were collected and counted. Percentage of specific lysis of 3 cell lines is shown. (C) Calcein-AM cytotoxicity release assay for measurement of ADCC. Labeled NHL cell lines were incubated with anti-B-cell mAbs in the presence of human mononuclear cells. Following a 4-hour incubation at 37°C, supernatants were harvested and transferred to new plates. Samples were measured using a Spectromax Gemini dual-scanning microplate spectrofluorimeter (Molecular Devices, Sunnyvale, CA); excitation filter, 485 nm; bandpass filter, 530 nm. Percentage of specific lysis of 3 cell lines is shown. ▪, with PBMCs; □, without PBMCs. Error bars represent SD.

Figure 3.

Antiproliferative effects of hL243γ4P alone and in combination with rituximab. Effects of mAbs on proliferation of NHL cells lines were determined by MTS assays (A-B) and ³H-thymidine uptake assays. (C) Effect of combining hL243γ4P and rituximab on proliferation of cell lines. Cells were cultured with the mAbs with or without a second antibody for cross-linking. Error bars represent SD of triplicates. On the x-axis, hL243 refers to the γ4P form.

Figure 4.

Apoptotic effect of mAbs on NHL cell lines. (A) Induction of apoptosis was evaluated by flow cytometry determination of haploid DNA on the cell line panel with and without a second antibody for cross-linking, followed by staining with propidium iodide. Error bars represent SD. (B) Apoptosis was quantified in Daudi using annexin V/7-AAD staining. Percentage of apoptotic cells refers to the annexin V-positive, 7-AAD-negative cells; percentage of dead cells refers to the annexin V-positive, 7-AAD-positive population. Cells were 97% viable prior to treatment. (C) Changes in mitochondrial membrane potential were measured by flow cytometry using the JC-1 reagent following antibody incubation in the presence or absence of second antibody. GAM indicates F(ab′)₂ goat anti-mouse IgG, Fcγ specific; GAH, F(ab′)₂ goat anti-human IgG, Fcγ-specific antibody.

Figure 5.

Time course studies in Daudi. (A) Cleaved caspase-3 and (B-C) P-AKT.

Figure 6.

Therapeutic efficacy of hL243γ4P and murine L243 in Raji-bearing SCID mice. SCID mice (10 per group) were given injections of 2.5 × 10⁶ Raji cells. Treatments were initiated 1 day after injection of cells and continued twice weekly for 4 weeks. Control animals were treated with saline. MST indicates median survival time.