Initial trial of bispecific antibody-mediated immunotherapy of CD15-bearing tumors: cytotoxicity of human tumor cells using a bispecific antibody comprised of anti-CD15 (MoAb PM81) and anti-CD64/Fc gamma RI (MoAb 32).

The high-affinity receptor for IgG, Fc gamma RI, expressed on monocytes and interferon-gamma (IFN-gamma)-stimulated neutrophils, is a trigger molecule for cell-mediated cytotoxicity. We have prepared murine monoclonal antibodies (MoAb 22 and MoAb 32) that bind to Fc gamma RI outside the ligand binding site and thus bind to and trigger cytotoxicity that is not competed by other immunoglobulins. Because of these properties, it seemed that these MoAbs would be very useful for the development of bispecific antibodies (BsAb) for targeting normal cellular immune defense mechanisms as a new form of immunotherapy for treatment of cancer. BsAbs incorporate into a single molecule the binding specifities of two different antibodies, and, thus, can be used to target myeloid cells to tumors, ensure activation of cellular cytotoxic mechanisms, and target cell lysis and/or phagocytosis. BsAbs were prepared using anti-Fc gamma RI MoAb and an anti-myeloid cell MoAb, PM81, reactive with the CD15 antigen, for studies of antibody-dependent cellular cytotoxicity. Conjugates were made by cross-linking sulfhydryl groups of Fab fragments of MoAb 32 or 22 (both IgG1) and sulfhydryl groups added to intact PM81 (an IgM) using N-succinimdyl-acetyl-S-thioacetate (SATA). The resulting product was purified by high-performance size-exclusion chromatography. The ability of the BsAbs to mediate attachment of human monocytes to tumor target cells was confirmed in a microtiter well assay of binding of MTT-labeled U937 cells (a human Fc gamma RI-bearing cell line) to SKBR-3 (PM81-reactive breast carcinoma) target cells. The ability of the BsAbs to mediate killing of HL-60 promyelocytic leukemia cells was studied using a 6-hour Chromium-51 release assay. Effector cells were monocytes obtained by cytopheresis and cultured for 18 hours with IFN-gamma. Monocytes alone caused minimal killing (5-20%), monocytes plus BsAb caused moderate killing (20-50%), and monocytes plus BsAb plus human serum resulted in maximal killing (50-80%). Experiments were performed to test the ability of the BsAb to purge bone marrow of small numbers of leukemia cells using bone marrow mononuclear phagocytes treated for 18 hours with IFN-gamma prior to adding target cells. Without the addition of human serum as a source of complement, a 90% depletion of clonogenic HL-60 cells could be demonstrated. With human complement, up to 95% depletion was seen. Thus, this BsAb possessed the ability to lyse tumor cell targets by two different mechanisms, complement and cell-mediated lysis.(ABSTRACT TRUNCATED AT 400 WORDS)

Fc receptors for IgG (Fc gamma Rs) on human monocytes and macrophages are not infectivity receptors for human immunodeficiency virus type 1 (HIV-1): studies using bispecific antibodies to target HIV-1 to various myeloid cell surface molecules, including the Fc gamma R.

Fc gamma Rs (Fc gamma RI, Fc gamma RII, and Fc gamma RIII) are highly expressed on human mononuclear phagocytes and function in the clearance of immune complexes and opsonized pathogens. We have examined the role of Fc gamma R in mediating antibody-dependent clearance of HIV-1 by human monocytes and monocyte-derived macrophages by using bispecific antibodies (BsAbs) to independently target the virus to Fc gamma RI, Fc gamma RII, or Fc gamma RIII. Virus production was markedly reduced in monocytes cultured with strain HIV-1IIIB opsonized with BsAbs that target the virus to either Fc gamma RI or Fc gamma RII compared to monocytes cultured with virus in the absence of BsAbs or in the presence of BsAbs that target the virus to non-Fc gamma R surface antigens (CD33 and HLA-A,B,C). These results were confirmed using the monotropic isolate HIV-1JRFL. Interaction of HIV-1JRFL with Fc gamma RI or Fc gamma RII on human monocytes and Fc gamma RI, Fc gamma RII, or Fc gamma RIII on monocyte-derived macrophages resulted in markedly reduced levels of virus production in these cultures. Moreover, HIV-1 infection of monocytes and monocyte-derived macrophages was completely blocked by anti-CD4 monoclonal antibodies, indicating that interaction with CD4 is required for infectivity even under conditions of antibody-mediated binding of HIV-1 to Fc gamma R. Thus, we propose that highly opsonized HIV-1 initiates high-affinity multivalent interactions with Fc gamma R that trigger endocytosis and intracellular degradation of the antibody-virus complex. At lower levels of antibody opsonization, there are two few interactions with Fc gamma R to initiate endocytosis and intracellular degradation of the antibody-virus complex, but there are enough interactions to stabilize the virus at the cell surface, allowing antibody-dependent enhancement of HIV-1 infection through high-affinity CD4 interactions. However, our results suggest that interaction of highly opsonized HIV-1 with Fc gamma Rs through BsAbs may reduce viral infectivity through Fc gamma R-mediated cytotoxic mechanisms and, therefore, that BsAbs offer promise as therapeutic reagents in HIV-1 infections.

Encapsidation and expression of the herpes thymidine kinase gene in polyoma virus.

A recombinant DNA of 5,150 base pairs was prepared containing the intact early region of polyoma virus, including the viral origin of replication and the structural sequences of the herpes simplex virus type 1 thymidine kinase gene. Although no thymidine kinase activity was detected when herpes structural sequences alone were transfected into cells, activity was produced when the structural gene followed the polyoma early region. The recombinant DNA was encapsidated into polyoma virions when cotransfected into mouse 3T6 cells with helper DNA from an early polyoma virus mutant. Herpes thymidine kinase activity was detected by a rapid in situ autoradiographic assay in which [125]iododeoxycytidine was utilized as a substrate for the viral but not the cellular enzyme.

Viable polyoma virus with four BamHI decanucleotide linkers inserted at the 26-minute HincII site.

BamHI decanucleotide linkers (5' CCGGATCCGG 3') were ligated to full-length linear polyoma DNA partially cleaved with HincII, and the recombinant DNA was transfected into mouse 3T6 cells. A viable virus (PYNB5) was isolated which contains four linkers at the 26-min HincII site. PYNB5 is encapsidated with a larger major viral capsid protein (VP1) as predicted from the DNA sequence. PYNB5 appears to have the same growth and physical properties as the wild-type virus with the exception of greater inactivation upon dilution of virus stocks with water. When PYNB5 DNA is cleaved with BamHI, the larger of the two resulting fragments (PY66) contains an intact early region of the virus, including the origin of DNA replication. PY66 complements a polyoma early region mutant for growth and may be useful as a cloning vector for foreign DNA.