Yashwant M. Deo

Clinical significance of IgG Fc receptors and FcγR-directed immunotherapies

Abstract Fc receptors for IgG (FcγRs) can trigger the inflammatory, cytotoxic and hypersensitivity functions of immune effector cells. Activation or deactivation of effector cells via FcγRs can be exploited to develop novel therapies for cancer, infectious diseases and autoimmune disorders. Initial results of clinical trials for several FcγR-directed immunotherapies show the potential promise of this approach.

Phase I clinical trial of the bispecific antibody MDX-H210 (anti-FcγRI x anti-HER-2/neu) in combination with Filgrastim (G-CSF) for treatment of advanced breast cancer

A phase I study of the bispecific antibody MDX-H210 in combination with granulocyte colony-stimulating factor (G-CSF) was performed in stage IV breast carcinoma patients, overexpressing HER-2/neu. MDX-H210, constructed by crosslinking antigen binding fragments (F(ab′) fragments) of monoclonal antibody (mAb) H22 to Fc gamma receptor I (FcγRI), and mAb 520C9 to HER-2/neu, respectively, mediates the lysis of tumour cells in vitro, and in human FcγRI transgenic mouse models. The proto-oncogene HER-2/neu is overexpressed in approximately 30% of breast cancer patients, and represents a promising target for antibody-based immunotherapy. Fc gamma receptor I (CD64) is an effective trigger molecule, which is expressed on monocytes/macrophages, immature dendritic cells, and G-CSF-primed polymorphonuclear cells (PMN). Patients received G-CSF (Filgrastim) for 8 consecutive days, and cohorts of three patients were treated on day 4 with escalating, single doses of MDX-H210. A total of 30 patients were included, and treatment was generally well tolerated, without reaching dose-limiting toxicity. Side effects consisted mainly of fever and short periods of chills, which were timely related to elevated plasma levels of interleukin 6 and tumour necrosis factor alpha. In the last two cohorts, MDX-H210 plasma levels exceeded 1 μg ml−1, and on circulating myeloid cells >50% saturation of FcγRI was found until day 4. These effector cells were highly effective in antibody-dependent cell-mediated cytotoxicity. Immunohistochemical analyses of tumour biopsies in individual patients documented infiltration of monocytes and PMN after MDX-H210 infusion. Although the clinical course of the disease was not altered by the single dose of MDX-H210, a favourable toxicity profile – even at high doses – and remarkable biological effects were seen when combined with G-CSF. Therefore, the combination of G-CSF and MDX-H210 should be evaluated in further immunotherapeutical strategies.

Differential Effect of Cytokine Treatment on Fcα Receptor I- and Fcγ Receptor I-Mediated Tumor Cytotoxicity by Monocyte-Derived Macrophages

Macrophages represent an important effector cell for Ab-mediated tumor therapy. Previous studies have documented that cytokines can influence Fc receptor (FcR) expression and function. In this study we examined the tumoricidal activities of the type I receptors for IgG (FcγRI) and the IgA FcR (FcαRI) on monocyte-derived macrophages (MDM) cultured in the presence of IFN-γ, M-CSF, or GM-CSF. Bispecific Abs were used to target a Her2/neu breast carcinoma cell line, SKBR-3, to FcαRI or FcγRI on MDM. Although FcαRI and FcγRI share a common signaling pathway contingent on association with the γ-chain (FcRγ subunit), a marked difference in their efficiency in mediating tumoricidal functions was seen in response to specific cytokines. M-CSF- and GM-CSF-treated MDM mediated efficient phagocytosis of SKBR-3 cells by FcαRI and FcγRI; however, IFN-γ-treated MDM phagocytosed tumor cells only with the FcγRI-directed bispecific Abs. Similarly, IFN-γ-cultured MDM lysed tumor cells more efficiently via FcγRI then by FcαRI as measured in Ab-dependent cellular cytotoxicity assays. Conversely, GM-CSF-treated MDM mediated more efficient lysis of tumor cells via FcαRI than FcγRI, while M-CSF-cultured MDM were relatively less efficient in mediating Ab-dependent cellular cytotoxicity through either receptor. With the exception of IFN-γ-mediated enhancement of FcγRI expression and FcγRI γ-chain complexes, the regulation of FcγRI- or FcαRI-mediated activity occurred without significant change in either receptor expression or total complexes with γ subunit. These data suggest that the efficiency of Ab-mediated tumor therapy, which depends on FcR effector cell functions, may be modified by the use of specific cytokines.

Targeting Weak Antigens to CD64 Elicits Potent Humoral Responses in Human CD64 Transgenic Mice

Previous studies have documented that targeting foreign Ags to IgG FcγR leads to enhanced Ag-specific responses in vitro and in vivo. However, the ability to overcome immunologic nonresponsiveness by targeting poorly immunogenic Ags to FcγR has not been investigated. To address this question in a simple model, we immunized transgenic mice expressing human CD64 (FcγRI) and their nontransgenic littermates with Fab′ derived from the murine anti-human CD64 mAb m22. The m22 Fab′ served as both the targeting molecule and the Ag. We found that only CD64-expressing mice developed anti-Id titers to m22. Furthermore, chemically linked multimers of m22 Fab′, which mediated efficient internalization of the human CD64, were significantly more potent than monomeric m22 F(ab′)2 at inducing anti-Id responses. In all cases, the humoral responses were specific for m22 Id and did not react with other murine IgG1 Fab′ fragments. Chemical addition of a second murine Fab′ (520C9 anti-human HER2/neu) to m22 Fab′ multimers demonstrated that IgG1 and IgG2a anti-Id titers could be generated to 520C9 only in the CD64-expressing mice. These results show that targeting to CD64 can overcome immunological nonresponsiveness to a weak immunogen. Therefore, targeting to CD64 may be an effective method to enhance the activity of nonimmunogenic tumor vaccines.

A phase I study of a HER2/neu bispecific antibody with granulocyte-colony-stimulating factor in patients with metastatic breast cancer that overexpresses HER2/neu.

Abstract A phase I study of escalating doses of humanized bispecific antibody (bsAb) MDX-H210 with granulocyte-colony-stimulating factor (G-CSF) was conducted in patients with metastatic breast cancer that overexpressed HER2/neu. The main objectives of the study were to define the maximal tolerated dose (MTD) of MDX-H210 when combined with G-CSF, to measure the pharmacokinetics of MDX-H210 when administered with G-CSF, and to determine the toxicity, biological effects and possible therapeutic effect of MDX-H210 with G-CSF. MDX-H210 is a F(ab)′ × F(ab)′ humanized bispecific murine antibody that binds to both HER2/neu and the FcγR1 receptor (CD64), and was administered intravenously weekly for three doses followed by a 2-week break and then three more weekly doses. A total of 23 patients were treated, and doses were escalated from 1 mg/m2 to 40 mg/m2 with no MTD reached. The toxicity of the bsAb + G-CSF combination was modest, with no dose-limiting toxicity noted: 19 patients had fevers, 7 patients had diarrhea, and 3 patients had allergic reactions that did not limit therapy. The β-elimination half-life varied from 4 h to 8 h at doses up to 20 mg/m2. Significant release of cytokines interleukin-6, G-CSF, and tumor necrosis factor α was observed after administration of bsAb. Circulating monocytes disappeared within 1 h of bsAb infusion, which correlated with binding of bsAb, noted by flow-cytometric analysis. Significant levels of human anti-(bispecific antibody) were measured in the plasma of most patients by the third infusion. No objective clinical responses were seen in this group of heavily pre-treated patients.

Antibody dependent cellular phagocytosis (ADCP) and antibody dependent cellular cytotoxicity (ADCC) of breast cancer cells mediated by bispecific antibody, MDX-210.

Background: MDX-210 is a bispecific antibody (BsAb) with specificity for both the proto-oncogene product of HER-2/neu (c-erbB-2) and FcγRI (CD64). HER-2/neu is overexpressed in malignant tissue of approximately 30% of patients with breast cancer, and FcγRI is expressed on human monocytes, macrophages, and IFN-γ activated granulocytes. We investigated phagocytosis and cytolysis of cultured human breast cancer cells by human monocyte-derived macrophages (MDM) mediated by BsAb MDX-210, its partially humanized derivative (MDX-H210), and its parent MoAb 520C9 (anti-HER-2/neu) under various conditions.Materials and Methods: Purified monocytes were cultured with GM-CSF, M-CSF, or no cytokine for five or six days. Antibody dependent cellular phagocytosis (ADCP) and cytolysis (ADCC) assays were performed with the MDM and HER-2/neu positive target cells (SK-BR-3). ADCP was measured by two-color fluorescence flow cytometry using PKH2 (green fluorescent dye) and phycoerythrin-conjugated (red) monoclonal antibodies (MoAb) against human CD14 and CD11b. ADCC was measured with a non-radioactive LDH detection kit.Results: Both BsAb MDX-210 (via FcγRI) and MoAb 520C9 (mouse IgG1, via FcγRII) mediated similar levels of ADCP and ADCC. ADCP mediated by BsAb MDX-H210 was identical to that mediated by BsAb MDX-210. Confocal microscopy demonstrated that dual-labeled cells represented true phagocytosis. Both ADCP and ADCC were higher when MDM were pre-incubated with GM-CSF than when incubated with M-CSF.Conclusions: BsAb MDX-210 is as active in vitro as the parent MoAb 520C9 in inducing both phagocytosis and cytolysis of MDM. MDX-210 and its partially humanized derivative, MDX-H210, mediated similar levels of ADCP. GM-CSF appears to superior to M-CSF in inducing MDM-mediated ADCC and ADCP. These studies support the ongoing clinical investigations of BsAb MDX-210 and its partially humanized derivative.

Human monocyte‐derived dendritic cells produce macrophage colony‐stimulating factor: enhancement of c‐fms expression by interleukin‐10

Human monocyte‐derived dendritic cells (DC) generated with granulocyte‐macrophage colony‐stimulating factor (GM‐CSF) and IL‐4 express c‐fms (CD115), the receptor for macrophage‐CSF (M‐CSF). Expression of c‐fms on monocyte‐derived DC has been interpreted as the susceptibility of these cells to M‐CSF‐induced macrophage development. We show here that homogenous cultures of CD14− DC constitutively produced large amounts of M‐CSF. However, presence of M‐CSF neither induced macrophage development nor did it prevent terminal maturation into CD83+ DC. M‐CSF production by DC was driven by GM‐CSF and inhibited by the specific phosphatidylinositol 3‐kinase inhibitor wortmannin. M‐CSF synthesis was rapidly induced during the first 24 h of DC culture and then declined during the 5‐day culture period. Replating of the cells, which was associated by a transient adherence, always induced a strong up‐regulation of M‐CSF synthesis. Addition of recombinant IL‐10 to DC cultures enhanced c‐fms expression and induced macrophage development as measured by the strong up‐regulation of CD14 expression as well as by enhanced expression of the Fcγ receptors I, II, and III (CD64, CD32, CD16). Our data demonstrate that immature monocyte‐derived DC produce M‐CSF which does not induce macrophage development, despite the surface expression of c‐fms on DC. IL‐10 appears to induce macrophage development by up‐regulating c‐fms and, thereby, enhancing the sensitivity of the cells to endogenously produced M‐CSF.

Efficient carcinoma cell killing by activated polymorphonuclear neutrophils targeted with an Ep-CAM×CD64 (HEA125×197) bispecific antibody

Abstract. Bispecific antibodies (bsAb) have attracted much attention over the past several years as a mean to improve immunotherapy of cancer. Due to their dual specificity, bsAb are able to redirect effector cells against tumor targets. In this study, the development and preclinical testing of a new quadroma-derived bsAb, HEA125×197, recognizing the tumor-associated Ep-CAM antigen and the high affinity Fc receptor for IgG, CD64, is reported. Using granulocyte-colony stimulating factor (G-CSF) and interferon-gamma (IFN-γ)-stimulated polymorphonuclear neutrophils to induce CD64 expression, bsAb HEA125×197 elicited strong cytotoxic activity towards allogeneic and autologous ovarian carcinoma cells. The cytolytic efficiency of this antibody was comparable to that of a previously described bsAb, HEA125×OKT3, targeting preactivated T lymphocytes against Ep-CAM-carrying tumor cells. Based on the pan-carcinoma specificity and the stable expression of Ep-CAM, bsAb HEA125×197 may broaden the spectrum of bispecific reagents for the treatment of epithelial malignancies.

Development of T-cell lines expressing functional HIV-1 envelope glycoproteins for evaluation of immune responses in HIV-infected individuals.

The human T-lymphoid cell line, CEM, was transfected with gp 160 cDNA of human immunodeficiency virus type 1 (HIV-1)pm213. Three clones expressing the envelope glycoproteins (env), designated CEM-213env1, -env4, and -env7, were isolated. These clones expressed high levels of surface gp41 and gp120, as demonstrated by flow cytometry with anti-HIV env monoclonal antibodies. Processing and function of env was shown by induction of syncytia with CD4-expressing HeLa cells and by immunoblot analysis. The env expression resulted in specific down-regulation of surface CD4 levels, supporting the role of HIV env in CD4 modulation. Furthermore, serum samples from nine of nine HIV-1-infected individuals bound specifically to the env-expressing transfectants, substantiating the presence of conserved antigenic determinants. These sera also mediated antibody-dependent cellular cytotoxicity (ADCC) of the env-expressing cell lines. The env-expressing cell lines provide a relevant, safe, and practical model for qualitative and quantitative analysis of humoral and cellular immune responses and their role in HIV-1 pathogenesis and therapy.

Targeting tumor cell destruction with CD64-directed bispecific fusion proteins.

The human immunoglobulin G Fc receptor type I (FcgRI), or CD64, is a member of the immunoglobulin supergene family, and is the only FcgR with high affinity for monomeric IgG [5]. CD64 is expressed on a variety of leukocytes including monocytes, macrophages, dendritic cells, and CD34+ myeloid progenitor cells. In addition, polymorphonuclear cells can be induced to express FcgRI with granulocyte-colony-stimulating factor and interferon g (IFNg) treatment, both in vitro and in vivo [23, 27]. CD64 mediates antibody-dependent cellular cytotoxicity (ADCC), phagocytosis, and release of inflammatory mediators [5]. The discrete expression of CD64 (confined primarily to cells with cytotoxic effector functions), and its potent immune triggering functions make this receptor an attractive target for redirected cytotoxicity. Several antibodies have been developed that are specific for human CD64 [1, 12]. The monoclonal antibody (mAb) 22 binds to CD64 at a site distinct from the Fc ligandbinding domain, allowing binding of mAb 22 in the presence physiological concentrations of IgG [12]. mAb22 has been humanized to H22 through the process of complementarity determinant region grafting, to increase the potential use of this targeting antibody for clinical applications [10]. Several bispecific antibodies (bsAb) comprising mAb 22 (both the murine, M22, and humanized versions) and anti-target mAb have been constructed. The ADCC, phagocytosis, and superoxide generation mediated by these bsAb demonstrate that M22/H22 can trigger effector cell functions through CD64 [5, 10, 12, 25]. Several members of the epidermal growth factor receptor (EGF-R) family are associated with over-expression in specific malignancies, and are appropriate targets for tumor immunotherapy. In particular, EGF-R is over-expressed in almost all head and neck, kidney and lung tumors, and in about one-third of breast and ovarian tumors [20] and can be over-expressed in cancers of the prostate, bladder, brain, pancreas, and gastrointestinal system. The HER2/neu or erbB-2 protein is over-expressed in breast, ovarian, and other cancers [18, 24, 26]. The other members of the EGF-R family, HER3 (erbB-3) and HER4 (erbB-4) are less well characterized, but their expression has also been associated with carcinomas [15, 21]. Previous studies have shown that growth-factor-receptor-specific mAb can be successfully utilized for making chemically linked bispecific antibodies (bsAb) that target HER2/neu-expressing tumor cells to CD64-expressing effector cells [27, T. Keler et al. manuscript submitted]. We have developed genetically linked CD64-targeted bispecific fusion proteins employing the natural ligands of members of the EGF-R family [8]. CD3-directed cytotoxic fusion proteins with ligands for tumor-associated receptors as tumor-targeting reagents have been described previously [6, 17]. The natural ligand fusion proteins may be more advantageous than chemically linked bsAb by being easier to manufacture and having reduced immunogenicity. Furthermore, such fusion proteins may have direct growth-modulatory effects through interaction with the growth factor receptor. For example, the growth of tumor cell lines that over-express the EGF-R is inhibited by EGF [3, 14, 19]. This report summarizes our current work with two CD64-targeted fusion proteins, H22-EGF and H22heregulin b (H22-HRG). The H22-EGF fusion protein binds specifically to cells that over-express EGF-R, while the H22-HRG is targeted to cells that overexpress HER2, HER3, and/or HER4. Cancer Immunol Immunother (1997) 45: 124 ±127 Ó Springer-Verlag 1997