Karuna Sundarapandiyan

Triggering FCα-Receptor I (CD89) Recruits Neutrophils as Effector Cells for CD20-Directed Antibody Therapy

CD20 Abs induce clinical responses in lymphoma patients, but there are considerable differences between individual patients. In 51Cr release assays with whole blood as effector source, RAJI cells were effectively killed by a mouse/human chimeric IgG1 construct of CD20 Ab 1F5, whereas ARH-77 proved resistant to killing by this Ab. When whole blood was fractionated into plasma, mononuclear cells, or granulocytic effector cells, RAJI cells were effectively killed in the presence of complement-containing plasma, whereas the mature B cell line ARH-77 proved complement resistant. However, with a bispecific Ab (BsAb) against the myeloid receptor for IgA (CD89; FcαRI) and CD20, a broad range of B cell lines were effectively killed. FcαRI is expressed on monocytes/macrophages, neutrophils, and eosinophils. As the numbers of these effector cells and their functional activity can be enhanced by application of G-CSF or GM-CSF, lysis via (FcαRI × CD20) BsAb was significantly enhanced in blood from patients during therapy with these myeloid growth factors. Interestingly, the major effector cell population for this BsAb were polymorphonuclear neutrophils, which proved ineffective in killing malignant B cells with murine, chimeric IgG1, or FcγRI- or FcγRIII-directed BsAbs against CD20. Experiments with blood from human FcαRI/FcγRI double-transgenic mice showed corresponding results, allowing the establishment of relevant syngenic animal models in these mice. In conclusion, the combination of myeloid growth factors and an (FcαRI × CD20) BsAb may represent a promising approach to improve effector cell recruitment for CD20-directed lymphoma therapy.

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.

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

Development of a Novel Antibody-drug Conjugate for the Potential Treatment of Ovarian, Lung and Renal Cell Carcinoma Expressing TIM-1

T-cell immunoglobulin and mucin domain 1 (TIM-1) is a type I transmembrane protein that was originally described as kidney injury molecule 1 (KIM-1) due to its elevated expression in kidney and urine after renal injury. TIM-1 expression is also upregulated in several human cancers, most notably in renal and ovarian carcinomas, but has very restricted expression in healthy tissues, thus representing a promising target for antibody-mediated therapy. To this end, we have developed a fully human monoclonal IgG1 antibody specific for the extracellular domain of TIM-1. This antibody was shown to bind purified recombinant chimeric TIM-1-Fc protein and TIM-1 expressed on a variety of transformed cell lines, including Caki-1 (human renal clear cell carcinoma), IGROV-1 (human ovarian adenocarcinoma), and A549 (human lung carcinoma). Internalization studies using confocal microscopy revealed the antibody was rapidly internalized by cells in vitro, and internalization was confirmed by quantitative imaging flow cytometry. An antibody–drug conjugate (ADC) was produced with the anti-TIM-1 antibody covalently linked to the potent cytotoxin, monomethyl auristatin E (MMAE), and designated CDX-014. The ADC was shown to exhibit in vitro cytostatic or cytotoxic activity against a variety of TIM-1–expressing cell lines, but not on TIM-1–negative cell lines. Using the Caki-1, IGROV-1, and A549 xenograft mouse models, CDX-014 showed significant antitumor activity in a clinically relevant dose range. Safety evaluation in nonhuman primates has demonstrated a good profile and led to the initiation of clinical studies of CDX-014 in renal cell carcinoma and potentially other TIM-1–expressing tumors. Mol Cancer Ther; 15(12); 2946–54. ©2016 AACR.