Chemistry: molecular biology and microbiology – Animal cell – per se ; composition thereof; process of... – Animal cell – per se – expressing immunoglobulin – antibody – or...
Reexamination Certificate
2000-09-12
2002-11-12
Park, Hankyel T. (Department: 1648)
Chemistry: molecular biology and microbiology
Animal cell, per se ; composition thereof; process of...
Animal cell, per se, expressing immunoglobulin, antibody, or...
C435S007100, C435S069700, C530S388350
Reexamination Certificate
active
06479284
ABSTRACT:
FIELD OF THE INVENTION
The present invention is directed to a humanized single chain antibody having a framework motif, preferably a motif containing no murine amino acids, that result in the humanized antibody having activity comparable to the corresponding murine antibody. Preferably, the antibody is a Tat antibody.
BACKGROUND OF THE INVENTION
Human immunodeficiency viruses type 1 and type 2 (HIV-1 and HIV-2) are the etiologic agents of acquired immunodeficiency syndrome (AIDS) in humans (Barre-Sinoussi et aL., 1984). AIDS results from the depletion of CD
4-
positive T lymphocytes in HIV-infected individuals (Fauci et al., 1984).
HIV-1 infects T lymphocytes, monocytes/macrophage, dendritic cells and, in the central nervous system, microglia (Gartner et al., 1986; Koenig et al., 1986; Pope et al., 1994; Weissman et al., 1995). All of these cells express the CD4 glycoprotein, which serves as the receptor for HIV-1 and HIV-2 (Dalgleish et al., 1984; Klatzman et al., 1984; Maddon et al., 1986). Efficient entry of HIV-1 into target cells is dependent upon binding of the viral exterior envelope glycoprotein, gp120, to the CD4-amino-terminal domain (McDougal et al., 1986; Helseth et al., 1990). After virus binding, the HIV-1 envelope glycoproteins mediate the fusion of viral and host cell membranes to complete the entry process (Kowalski et al., 1987; Stein et al., 1987; Helseth et al., 1990). Membrane fusion directed by HIV-1 envelope glycoproteins expressed on the infected cell surface leads to fusion with uninfected CD4-positive cells, resulting in syncytia (Lifson et al., 1986; Sodroski et al., 1986). HIV-1 and HIV-2 contain numerous regulatory proteins including tat, rev, nef, vpu/vpx and vpr in addition to pol, gag and the envelope glycoproteins.
Tat, a 16 kD regulatory protein, is expressed early in the viral life cycle and is absolutely required for viral replications
1,2
. Tat acts as a potent transcriptional activator of viral gene expression through its binding to a RNA stem-loop structure called the transactivation response element (TAR) that is located 40 bp downstream from the site of initiation of transcription in the 5′ long terminal repeat (LTR). Tat functions primarily to stimulate transcription initiation and increase transcriptional elongation
3,4,5
. However, new evidence suggests that Tat may also be required for efficient HIV-1 reverse transcription
6,7
.
Apart from its role in viral replication, Tat protein also has an effect on cellular genes that may aid in the dissemination of virus infection. For example, Tat has been implicated in several immunosuppressive effects including increasing the expression of the potent immunosuppressive cytokine transforming growth factor &bgr;1 (TGF-&bgr;1)
8
, suppressing antigen-induced proliferation of T cells
9
and decreasing the activity of an MHC class I gene promoter, thereby providing a mechanism whereby HIV-1-infected cells may be able to avoid immune surveillance and recognition of specific cytotoxic T lymphocytes
10
. Other cellular genes such as those involved in G1 checkpoint control, p53 and in cellular defense against oxidative stress, Mn-superoxide dismutase are also downregulated by Tat
11,12
.
Tat has additional functions in the pathogenesis of AIDS, in part because of its ability to be released from HIV-1-infected cells through a non-classical secretory pathway and to enter the nuclei of both infected and uninfected cells. Tat uptake not only enhances HIV-1 transcription in infected cells, it also affects a range of host cellular genes in both infected and uninfected cells. This includes activation of cellular genes such as tumor necrosis factor (TNF) &agr; and &bgr;
13,14,15
and IL-6
16,17
, which in turn may activate HIV-1 gene expression and replication leading to further spread of HIV-1
18,19,20,21
. Tat has also been shown to upregulate IL2 secretion in ctivated T cells
22
and to recapitulate the phenotype of increased IL-2 secretion in response to costimulation with CD3 plus CD28 that is seen in HIV-1-infected primary T-cells that are stimulated via CD3 and CD28 receptors
23
. Extracellular Tat has been shown to activate uninfected quiescent T cells in vitro and in vivo, thereby causing uninfected cells to become highly permissive to productive HIV-1-infection
24
. In this way, Tat protein is unique among the HIV-1 proteins in not only being critical for ,iral transcriptional activation but also for its role in evolvinga self-perpetuating mechanism to actively generate cells permissive to productive and cytopathic infection
24,25
.
Consequently Tat is likely to have both direct and indirect effects in the pathogenesis of AIDS through its multiple roles in the HIV-1 life cycle and on the immune system. It would be desirable to have more efficient means for disrupting Tat interactions. Disruption of Tat protein interaction with TAR RNA or the cellular factors that bind Tat protein, and of Tat protein release from HIV-1-infected cells, thus represents an important target for pharmacologically and genetically based therapeutic interventions to combat HIV-1 infection. While clinical results with the Tat antagonist Ro24-7429 showed no evidence of anti-viral activity
26
despite prolonged inhibition of HIV-1 replication in vitro
27
, the results of a number of Tat directed in vitro gene therapy studies have been encouraging
28,29,30,31,32,33,34
, particularly when combined with pharmacologic inhibitors of NF-kB
35,36
.
A murine anti-tat sFv antibody, which is directed intracellularly against the proline-rich N-terminal activation domain of HIV-1 Tat and hence sometimes referred to as an intrabody, is a potent inhibitor of Tat-mediated LTR transactivation and HIV-1 infection
36,37,38
. However, murine antibodies can produce undesired immune responses which can reduce or totally abolish the effectiveness of the antibody. The immune response can also cause undesired side effects. In order to minimize evoking an immune response against the murine anti-tat sFv or transgene encoding it in a clinical setting
39
, CDR grafting experiments were performed to completely humanize the murine anti-tat sFv. Unfortunately, “humanizing” an antibody is not as efficient a process as sometimes presented. Compatible human framework regions must be chosen from heavy chain and light chain sequences of over 1000 human sequences each. However, the resulting antibody despite having the same variable region as the murine antibody frequently does not have the same effectiveness as the original murine antibody. Frequently the “humanized” antibody will retain some “murine” amino acid residues. It would be desirable to have a framework motif that produces an antibody having a protective efficiency comparable to the murine antibody.
SUMMARY OF THE INVENTION
We have now discovered a framework motif that produces a humanized antibody such as an anti-tat sFv intrabody that demonstrated a level of activity, e.g., anti-HIV-1 activity that was comparable to that of the parental murine sFv.
The preferred sequence was completely human, retaining none of the murine amino acids. The comparable human heavy chain and light chain are selected. One preferred framework motif is based upon the human V
H
gene K5B8 and V
L
gene TR1.6.
While the sequence is preferably completely humanized, some murine amino acid residues can be retained. The amino acid sequences encoded by these genes are aligned against the murine sequence to determine where the amino acids differ. One humanized antibody retains at least one of the murine amino acid residues at the FRM2/CDR2 border and the FRM3/CDR3 border of the heavy chain. In another embodiment, at least one murine amino acid within the FRM3 sequence of the light chain is also retained. Preferred positions are the first murine amino acid within FRM3 after the CDR2 border that differs from the human sequence and the last such amino acid within FRM3 before the CDR border. In yet another embodiment, at least three of the four murine positions described above are maintained. For example, all four of these murine
Eberhardt Bridget
Lavecchio Joyce
Marasco Wayne A.
Mhashilkar Abner
Porter-Brooks Julie
Dana-Farber Cancer Institute Inc.
Nixon & Peabody LLP
Park Hankyel T.
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