CA2000989A1 - Conjugates of soluble t4 proteins and toxins and methods for treating or preventing aids, arc and hiv infection - Google Patents

Abstract

ABSTRACT
This invention relates to compositions and methods useful for the treatment and prevention of acquired immunodeficiency syndromes (AIDS), AIDS related complex (ARC), and human immunodeficiency virus (HIV) infection. More particularly, this invention relates to immunotoxins formed by conjugating a toxin to a soluble T4 protein, an HIV virus receptor that binds to the HIV virus or to target cell carrying the gp120/160 HIV marker. This invention also relates to methods for secreting soluble T4-toxin fusion protein conjugates through the cell membrane of a host. The compositions and methods of this invention advantageously provide a highly specific, site-directed delivery system for delivery of toxin to HIV infected cells and to circu-lating HIV virus.

Description

21~00~89 TOXINS AND METHODS FOR TREATING
OR PREVENTING AIDS. ARC AND HIV INFECTION

TECHNICAL FIELD OF INVENTION
This invention relate~ to compositions and methods useful for the treatment and prevention of acgulred immunode~iciency syndrome (AIDS), AIDS related complex (ARC), and human immunode~iciency virus (HIV) infection. More particularly, this invention relates to immunotoxins formed by con~ugating a toxin to a soluble T4 protein, an HIV virus receptor that binds to the HIV virus or to target cells carrying the gpl20/160 HIV marker. This invention also relates to methods for lS secreting soluble T4-toxin fusion protein con~ugates through the cell membrane of a host. The compositions and methods of this invention advantageously provide a highly specific, site-directed system for delivery of toxin to HIV infected cells and to circulating HIV
, . , ,1 ' ' . ! ' ' virus.
BACKGROUND OF THE INVENTION
In immunocompetent individuals, T4 lymphocytes interact with other specialized cell types of the immune system to confer immunity to or to defend against infection [E. L. Reinherz and S. F. Schlossman, "The Differentiation Function Of Human T-Cells", Cell, 19, pp. 821-27 (1980)]. More specifically, T4 ~ .

lymphocytes stimulate the production of growth factors which are critical to a functioning immune system. For example, they act to stimulate B cells, the descendants of hemopoietic stem cells, which promote the production o~ defensive antibodies. They also activate macrophages ("killer cells") to attack infected or otherwise abnormal host cells, and they induce monocyte~ ("scavenger cells") to encompass and destroy invading microbes.
The T4 surface protein of T4 lymphocytes is the primary target of certain infective agents, such as viruses and retroviruses. When T4 lymphocytes are exposed to ~uch viruses, they are functionally impaired. As a re~ult, the host 1 5 complex immune defense system is suppressed, and the host becomes su~ceptible to a wid~ range of opportunistic in-fections.
Such immunosuppression i8 seen in patients suffering from AIDS. AIDS is a disease characterized by severe or complete immunosuppre~sion and attendant host susceptibility to a wide range of opportunistic infections and malignancies. In some cases, AIDS
infection is accompanied by central nervous system di~order~. Complete clinical manifestation of AIDS is usually preceded by ARC, a syndrome accompanied by symptoms such a persistent generalized lymph-adeno~athy, fever and weight loss. The HIV retrovirus is thought to be the etiological agent responsible for AIDS in~ection and its precursor, ARC tM.G. Sargadharan et al., "Detection, Isolation And Continuous Production Of Cytopathic Retroviruses (HTL~-III) From Patients 200~)989 .
' With AIDS And Pre-AIDS", Science, 224, pp. 497-508 (1984)].*
The genome of retroviruses, such as HIV, contains three regions encoding structural proteins.
The g~g region encodes the core proteins of the virion.
The EQl. region encodes the virion RNA-dependent DNA
polymerase ~reveree transcriptase). The env region encodes the ma~or glycoprotein found in both the membrane envelope o~ the virus and the cytoplasmic membrane of infected cells. The capacity of the virus to attach to target cell receptors and to cause fusion of cell membranes ~syncytia) are two HIV properties controlled by the çnv gene. These properties are ;~
believed to play a fundamental role in the pathogenesis of the virus.
The HIV env proteins arise from a precursor polypeptide -- gpl20/160 -- that, in mature for~, i5 cleaved into a large, heavily glycosylated exterior membrane protein of about 481 amino acid~ -- gpl20 --, and a smaller transmembrane protein of about 345 amino acids, which may be glycosylated -- gp41 -- [L. Ratner et al., "Complete Nucleotide Sequence Of The AIDS
Virus, HTLV-III", Nature, 313, pp. 277-84 (1985)~. For convenience, in this applicati.on, the HIV exterior mem-brane protein will be referred to in terms of theprecursor polypeptide, as -- gpl20/160 --.
The host range of HIV is associated with cells which bear the T4 surface glycoprotein. Such * In this application, human immunodeficiency virus ("HIV"), the generic term adopted by the Human Retro-virus Subcommittee of the International Committee On Taxonomy Of Viruses to refer to independent isolates from AIDS patients, including human T cell lymphotropic virus -type III ("HTLV-III"), lymphadenopathy- associated virus ("LAV"), human immunodeficiency virus type 1 ("HIV-l") and AIDS-associated retrovirus ("ARV") will be used.

cells include T4 lymphocytes and brain cells tP. J. Maddon et al., "The T4 Gene Encodes The AIDS
Virus Receptor And Is Expressed In The Immune System And The Brain", Cell, 47, pp. 333-48 (1986)]. The infection o~ a host by the HIV virus results in the depletion of functional T4 lymphocytes. Accordingly, the progression of AIDS/ARC syndromes can be corre-lated with the depletion of T4 lymphocytes which display the T4 surface glycoprotein (T4- lymphocytes).
This T-cell depletion, with ensuing immunological compromise, may be attributable both to recurrent cycles of infection and to lytic growth, from cell-mediated ~pread of the virus. In addition, clinical observations sugge~t that HIV is directly responsible for the central nervous system disorders seen in many AIDS patient~. The tropism of HIV for these cells is believed to be attributable to the role of the T4 protein as a membrane-anchored virus receptor.
Becau~e the T4 protein behaves as the HIV
receptor, it~ extrac~llular sequence probably plays a direct role in binding HIV. More specifically, it is believed that the gpl20/160 HIV envelope protein 6electively binds to the T4 epitope(s), using this ;~;
interaction to initiate entry into the host cell ;~
~A. G. Dalgelish et al., "The CD4 (T4) Antigen Is An Essential Component Of The Receptor For The AIDS
Retrovirus", Nature, 312, pp. 763-67 (1984); ;~
D. Klatzmann et al., "T-Lymphocyte T4 Molecule Behaves As The Receptor For Human Retrovirus LAV", Nature, 312, pp. 767-68 (1984)]. Accordingly, cellular expression of the T4 protein is believed to be sufficient for HIV
binding, with the T4 protein serving as a receptor for HIV.
This T4 tropism of HIV has been demonstrated 35 in vitro. When HIV isolated from AIDS patients is ~;

Z0~:)989 cultured together with T helper lymphocytes pre-selected for T4 protein presentation, the lymphocytes are efficiently infected, display cytopathic effects, including multinuclear syncytia formation, and are killed by lytic growth [D. Klatzmann et al., "Selective Tropism Of Lymphadenopathy Associated Virus (LAV) For Helper-Inducer T Lymphocytes", Science, ~, pp. 59-63 (1984); F. Wong-Staal and R. C. Gallo, "Human T-Lymphotropic Retroviruses", Nature, 317, pp. 395-403 (1985)]. It has also been demonstrated that a cloned cDNA version of a human T4 protein, when expressed on the surface of transfected cells from non-T cell lineages, including murine and fibroblastoid cells, endows those cells with the ability to bind HIV
~P. J. Maddon et al., "The T4 Gene Encodes The AIDS
Virus Receptor And Is Expre3sed In The Immune System And The Brain", ÇÇ11, 47, pp. 333-48 (1986)].
Therapeutic uses of soluble T4 proteins have been proposed for the prevention and treatment of the HIV-related infections AIDS and ARC. The nucleotide sequence and a deduced amino acid sequence for a DNA
that purportedly encodes the full length human T4 protein have been reported tP. J. Maddon et al., "The Isolation And Nucleotide Sequenae Of A cDNA Encoding The T Cell Surface Protein T4: A New Member Of The Immunoglobulin Gene Family", Cell, 42, pp. 93-104 (1985)]. Based upon its deduced primary structure, the T4 protein can be functionally divided as follows:
,~
. " :

2000~389 Amino Acid Structure/Pro~osed Location Coordinates Hydrophobic/Secretory Signal -23 to -1 Homology to V-Regions/ +1 to +94 Extracellular Homology to J-Regions/ +95 to +109 Extracellular Glycosylated Region/ +110 to +374 Extracellular Hydrophobic/Transmembrane +375 to +395 Sequence Very Hydrophilic/ +396 to +435 Intracytoplasmic Soluble T4 proteins have been constructed by truncatlng the full length T4 protein at amino acid 375, to ellminate the transmembrane and cytoplasmic domains. Such proteins have been produced by recombinant DNA techniques ~R. A. Pisher et al., "HIV
Infection Is Blocked In Vitro By Recombinant Soluble CD4", Nature, ~1, pp. 76-78 (1988)]. These soluble T4 proteins advantageously interfere with the T4~
lymphocyte/HIV interaction by blocking or competitive binding mechanisms which inhibit HIV infection of cells expressing the T4 protein. By acting as soluble virus receptors, soluble T4 proteins are useful as antiviral therapeutics to inhibit HIV binding to T4- lymphocytes and virally induced syncytia formation.
Other methods for treating AIDS and ARC have included the administration of antiviral drugs, such as HPA-23, phosphonoformate, suramin, ribavirin, azidothymidine (~AZT") and dideoxycytidine, which apparently interfere with replication of HIV through reverse transcriptase inhibition. Although each of these drugs exhibits activity against HIV n vitro, only AZT has demonstrated potential benefits in clin-,'," ".

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200098~

ical trials. AZT administration in effective amounts,however, has been accompanied by undesirable and debilitating side effects, such as bone marrow depression. It is likely, therefore, that hematologic toxicity will be a major limiting factor in the long term use of AZT.
Other proposed methods for treating AIDS have focu~ed on the development of agents having activity again~t steps in the viral replicative cycle other than reverse transcription. Such methods include the administration of interferons or the application of hybridoma technology. Most of these treatment strategies are expected to reguire the co-admin-istration of immunomodulators, such as interleukin-2.
To date, therefore, the need exist~ ~or the development of immunotherapeutic agents, methods and ~trategies for the treatment or prevention of AIDS, ARC, HIV infection and other immunodeficiencies caused by T-lymphocyte depletion or abnormalities.
~I~CLOSURE ~F THE INVENTION
The present invention 601ves the problems re~srred to above by providing compositions and methods for the treatment or prevention of AIDS, ARC and HIV
infections. The compositions and methods of this invention are characterized by cytotoxic soluble T4 protein-toxin conjugates or "immunotoxins" comprising toxins conjugated to soluble T4 proteins. These immunotoxins utilize soluble T4 proteins as soluble ~-virus receptors for the HIV envelope protein gpl20/160. -~
This results in a toxin delivery system characterized by a high affinity for HIV and cells expressing the HIV
envelope protein. ~dvantageously, the immunotoxins of this invention inhibit HIV binding to T4- lymphocytes by ~ -virtue of their competitive binding characteristics, , ";:

;~000989 and they act$vely destroy HIV and HIV infected cells expressing the gpl20/160 protein and producing the HIV
virus.
More specifically, the soluble T4 protein portion of the immunotoxins of this invention binds to HIV or an HIV infected cell which presents the gpl20/160 surface marker, and the immunotoxin, or a portion thereof, is internalized or endocytosed into the cell. The toxin then intoxicates and kills the infected cell, thus eliminating the source or reser-voir of the virus. The high taxget specificity of the soluble T4 protein portion of the immunotoxin results in delivery of the toxin directly to the target HIV or HIV-infected cells, enabling a high kill ratio with minimal tGxin dosage, while minimizing any damage to non-HIV infected cells.
According to one embodiment of this inven-tion, recombinant DNA techniques are employed to construct i~munotoxins which are fusion protein con-~ugates comprising a toxin and a soluble T4 protein("soluble T4-toxin fusion protein conjugates").
Another embodiment of this invention provides a method for secreting these soluble T4-toxin fusion protein con~ugates through the cell membrane of a host. In a further embodiment of this invention, a soluble T4 protein and a toxin are chemically fused to form an immunotoxin. ~;~
The compositions and methods of this invention are useful in a variety of immunotherapeutic compositions and methods for treating immunodeficient patients suffering from diseases caused by infective ;:
agents whose primary targets are T4- lymphocytes, particularly humans having AIDS, ARC, HIV infection or antibodies to HIV. These compositions and methods may also be used for treating AIDS-like diseases caused by 20~09~

g retroviruses, such as simian immunodeficiency viruses, in mammals, including humans.
BRIEF DESCRIPTION OF THE DRAWINGS
Figure 1 depicts the nucleotide sequence and the derived amino acid sequence of the T4 cDNA of plasmid pl70.2.
Figure 2 depicts the nucleotide sequence and the derived amino acid sequence of plasmid pBG391. The amino acid sequence listed at line a is the reading frame which can be transcribed to produce a soluble T4 protein or portion thereof.
Figure 3 depicts the nucleotide sequence of the entire plasmid defined by pl99.7 (PL mutet.rsT4) and its rsT4.2 insert and the amino acid sequence deduced from the rsT4 sequence. This includes the ClaI-Çl~I
caissette which defines the Met perfect rsT4.2 coding sequence.
In Figure 1, the T4 protein translation start (AA-23) i9 located at the methionine at nucleotides 1199-1201 and the mature N-terminus is located at the lysine (AA3) at nucleiotides 1274-1276.
In Figure 2, the T4 protein translation start (AA-23) is located at the methionine at nucleoides ~;;
1207-1209 and the mature N-terminus is located at the lysine (AA3) at nucleotides 1282-1285. ~`
In Figure ~, the T4 protein translation start (AA-l) is located at the methionine at nucleotides 64- , 66. ;
Figure 4 is a schematic outline of the construction of plasmids pEX2 and pEX3.
Figure 5 is a schematic outline of the ~-construction of plasmids pEX15 and pEX27.
Figure 6 is a schematic outline of the construction of plasmids pEX5 and pEXll. -200~)9~

Figure 7 is a schematic outline of the construction of plasmid pEX39.
Figure 8 depicts the nucleotide sequence of a portion of pSA307 and the amino acid sequence of the regions of that portion coding for a streptavidin-like polypeptide. The amino acid sequence listed at line c is the reading frame which can be transcribed to produce a streptavidin-like polypeptide.
Figures 9A and 9B are schematic outlines of the construction of plasmids pEX45, pEX46, and pEX56.
Figure 10 illustrates the competition for gpl20/160 binding and cell killing activity among the competitor, rsT4.3, and the toxin, P~eudomonas Exotoxin A, or the immunotoxins, PEx45 and PEx46.
lS Figure 11 depicts the amino acid sequence of the immunotoxin PEx45.
Figure 12 depicts the amino acid sequence of the immunotoxin PEx46.
Figure 13 depicts the amino acid sequence of P~eudomonas Exotoxin A.
Figure 14 illustrates the effects of contacting the immunotoxins of the present invention with '~C-NAD and elongation factor 2 (EF-2).
Figure 15 depicts selective killing of a cell ~ ;~
line expressing gpl20/160 on its surface by Pseudomonas ;
Exotoxin A, or the immunotoxins, PEx45 and PEx46. The viability of the cells is then measured by the incorporation of 3H-labelled thymidine. ;
Figure 16 depicts the amino acid sequence of angiogenin.
Figures 17A and 17B are schematic outlines of the production of plasmids pTANGll and pTANG12.
Figure 18 depicts the amino acid sequence of TANGll, a fusion protein comprising a soluble T4 protein (having the formula AA3-AA183 of Figure 2), a ; ZO(~9~9 portion of Pseudomonas Exotoxin A comprisinq the translocation domain, a portion of the ADP ribosylation domain and angiogenin.
Figure 19 depicts the amino acid sequence of 5 TANG12, a fusion protein comprising a soluble T4 protein (having the formula AA3-AA183 of Figure 2), a portion of ~eudomonas Exotoxin A comprising the translocation domain, a portion of the ADP ribosylation domain, the angiogenin signal sequence, and angiogenin.
Figure 20 is a echematic outline oî the con~truction of the plasmids pTANG13 and pTANG14.
Figure 21 depicts the amino acid sequence of TANG13, a fusion protein comprising a soluble T4 protein (having a formula AA3-AA183 of` Figure 2) and 15 mature angiogenin.
Figure 22 depicts the amino acid sequence of TANG14, a fusion protein comprising a soluble T4 protein (having a formula AA3-AA183 of Figure 2), the angiogenin signal sequence, and angiogenin.
Figure 23 illustrates the spatial ~ s relationship of the domains of ~3domonas Exotoxin A
and various immunotoxins according to this invention.
Figure 24 illustrates the plating efficiency -~
cell assay.
Figure 25 illustrates the gpl20 cell binding assay.
In the Figures, the amino acids are represented by single letter codes as follows:
Phe: F Leu: L Ile: I Met: M
S~al: V Ser: S Pro: P Thr: T ;
Ala: A Tyr: Y His: H Gln: Q
Asn: N Lys: K Asp: D Glu: E
Cys: C Trp: W Arg: R Gly: G
* = position at which a stop codon is present. ;

. , ., - , , :

DETAILED DESCRIPTION OF THE INVENTION
This invention relates to immunotoxins comprising soluble T4 proteins conjugated to toxins, use~ul in methods and compositions for treating AIDS, ARC and HIV infections. The affinity of the soluble T4 protein component of the immunotoxins of this invention f~r the gpl20/160 HIV surface marker advantageously permits highly specific targeting of the toxin to HIV
and to HIV infected cells.
The soluble T4 proteins useful in producing the immunotoxins of this invention include all proteins, polypeptldes and peptides which are natural or recomblnant ~oluble T4 proteins, or portions thereo~, and which are characterized by the immuno-therapeutic (antiretroviral) or immunogenic activity of soluble T4 proteins. They include soluble T4-like compounds from a variety of Cources~ such as soluble T4 protein derived from natural sources, recombinant ~ -soluble T4 protein and synthetic or semi-~ynthetic soluble T4 protein. Such soluble T4-like compounds advantageously interfere with the T4~HIV interaction by blocking or competitive binding mechanisms which inhibit HIV infection of cells expressing the T4 sur~ace protein. The soluble T4 proteins used in the immunotoxins of this invention are all characterized by an affinity for the gpl20/160 HIV surface markers. ! ' " ' Soluble T4 proteins include polypeptides selected from the group consisting of a polypeptide of -23 AA362 f Figure 2, a polypeptide of AAl AA362 f Figure 2, a polypeptide of the formula Met-AAl_36~ of Figure 2, a polypeptide of the formula AA-23-AA374 of Figure 2, a polypeptide of the formula AA1-AA374 of Figure 2, a polypeptide of the Met AA1-374 f Figure 2, a polypeptide of the formula AA1-AA377 of Figure 2, a polypeptide of the X~)01)98!3 formula Met AAl_377 of Figure 2, a polypeptide of the formula AA_23-AA377 of Figure 2, or portions thereof.
Other soluble T4 proteins include poly-peptides selected from the group consisting of a poly-peptide of the formula AA_23-AA182 of Figure 2, a polypeptide of the formula Met-AA1_182 of Figure 2~ a polypeptide of the formula AAl-AA182 of Figure 2, a polypeptide o~ the formula AA3-AA183 of Figure 2, a polypeptide of the formula AA_23-AA182 of Figure 2, followed by the amino acids asparagine-leucine-glut-amlne-histidine-serine-leucine, a polypeptide of the formula AA1 AA182 of Figure 2, followed by the amino acids asparagine-leucine-glutamine-histidine-serine- ~, leucine, a polypeptide of the formula Met-AA1_182 of ;
15 Figure 2, followed by the amino acids asparagine- ;
leucine-glutamine-histidine-serine-leucine, a poly-peptide of the formula AA_23-AA111 of Figure 2~ a polypeptide of the formula AA1-AA111 of Figure 2, a - .;~
polypeptide of the formula Met-AA1 111 of Figure 2, a :;
polypeptide of the formula A~_23-AA113 of Figure 2, a :
polypeptide of the formula AA1-AA113 of Figure 2, a polypeptide of the formula Met-AA1_113 of Figure 2, a polypeptide of the formula AA_23-AA131 of Figure 2, a .
polypeptide of the formula AA1-AA131 of Figure 2, ~
polypeptide of the formula Met-AA1_131 of Figure 2, a polypeptide of formula AA_23-AA145 of Figure 2, a ;-polypeptide of the formula AA1-AA145 of Figure 2, a polypeptide of the formula Met-AA1_145 of Figure 2, polypeptide of the formula AA_23-AA166 of Figure 2, a polypeptide of the formula AA1-AA166 of Figure 2, a polypeptide of the formula Met-AAl_166 of Figure 2, or portions thereof.
Additionally, soluble T4 proteins include polypeptides selected from the group consisting of a polypeptide of the formula AA1-AA362 of mature T4 20~09~39 protein, a polypeptide of the formula Met-AAl 362 f mature T4 protein, a polypeptide of the formula AA1-AA374 of mature T4 protein, a polypeptide of the formula Met-AAl_374 of mature T4 protein, a polypep-tide of the formula AA1-AA377 of mature T4 protein, a polypeptide of the formula Met-AAl_377 of mature T4 protein, a polypeptide of the formula AA-23-AA374 of mature T4 protein, a polypeptide of the formula AA 23-AA377 of mature T4 protein, or portions thereof.
Soluble T4 proteins also include polypeptides selected from the group consisting of a polypeptide of :.
the ~ormula AA-23-AA182 f mature T4 protein~ a polypeptide of the formula AA1-AA182 f mature T4 :
protein, a polypeptide of the formula Met-AAl_l82 of 15 mature T4 protein, a polypeptide of the formula :
AA-23-AA182 of mature T4 protein, followed by the amino acids asparagine-leucine-glutamine-histidine-serine- : ;;.;~
leucine, a polypeptide of the formula AA1-AA182 f mature T4 protein, followed by the amino acids asparagine-leucine-glutamine-histidine-serins-leucine, a polypeptide of the formula Met-AAl_l82 of mature T4 protein, followed by the amino acids asparagine- ;.
leucine-glutamine-histidine-serine-leucine, a polypeptide of the formula AA-23 AA113 protein, a polypeptide of the formula AA1-AA113 f mature T4 protein, a polypeptide of the formula Met-AAl_ll3 of mature T4 protein, a polypeptide of the formula AA-23-AAlll of mature T4 protein, a polypeptide of the formula AAl-AAl of mature T4 protein, a polypeptide of the formula Met-AAl 111 f mature T4 protein, a polypeptide of the formula AA-23-AA131 of mature T4 protein, a polypeptide of the formula AA1-AA131 f mature T4 protein, a polypeptide of the formula Met-AAl 131 of mature T4 protein, a polypeptide of the formula AA-23 AA1 , ,; ,, :," "

200098g protein, a polypeptide of the formula AA1-AAl45 of mature T4 protein, a polypeptide of the formula Met-AAl_l45 of mature T4 protein, a polypeptide of the formula AA_23-AA166 f mature T4 protein, a polypeptide of the formula AAl-AA166 of mature T4 protein, a polypeptide of the formula Met-AA1_166 of mature T4 - protein, or portions thereof.
The amino-terminal amino acid of mature T4 protein isolated from T cells begins at lysine, the third amino acid of the sequence depicted in Figure 2.
Accordingly, soluble T4 proteins also include polypeptides of the formula AA3-AA377 of Figure 2, or portions thereof. Such polypeptides include polypeptides selected from the group consisting of a polypeptide of the formula AA3-AA362 of Figure 2, a polypeptide o~ the formula AA3-AA374 of Figure 2, a polypeptide of the formula AA3-AA183 of Figure 2, a polypeptide o~ the formula AA3-AA182 of Figure 2, a polypeptide of the formula AA3-AA113 of Figure 2, a polypeptide of the formula AA3-AA131 of Figure 2, a polypeptide oi the formula AA3-M 145 f Figure 2, a polypeptide of the formula AA3-AA166 of Figure 2, and a polypeptide of the formula AA3-AA111 of Figure 2-Soluble T4 proteins also include the above-recited polypeptides preceded by an N-terminal methionine group.
Soluble T4 proteins useful in the immuno-toxins and methods of this invention may be produced in a variety of ways. We have depicted in Figure 1 the nucleotide se~uence of full-length T4 cDNA obtained from pl70.2 and the amino acid sequence deduced therefrom. The T4 cDNA of pl70.2 is almost identical to the approximately 1,700 bp sequence reported by Maddon et al., supra. The T4 cDNA of pl70.2, however, contains three-nucleotide substitutions which, in the .,;
.:
,. . - .

Z0009~9 translation product of this cDNA, produce a protein containing three different amino acids compared to the sequence reported by Maddon et al. These differences are at amino acid position 3, where the asparagine of Maddon et al. i8 replaced with lysine; position 64, where the tryptophan of Maddon et al. is replaced with arginine and at position 231, where the phenylalanine of Mad~on et al. is replaced with serine. The asparagine reported at positlon 3 in Maddon et al.
instead of lysine was the result of a DNA sequencing error [D. R. Littman et al., "Corrected CD4 Sequence", Ç~ll, 55, p. 541 (1988)].
Soluble T4 protein constructs or DNA inserts may be produeed by truncating the full length T4 protein sequence at various positions to remove the eoding regions for the transmembrane and intracyto-plasmie domains, while retaining the extracellular region believed to be responsible for HIV binding.
More partieularly, soluble T4 proteins may be produeed by eonventional techniques of oligonucleotide directed mutagenesis and restriction digestion, followed by insertion of linkers, or by chewing baek full-length T4 protein with enzymes.
Prior to sueh eonstructions, the cDNA coding sequenee of a full length T4 elone, such as pl70.2, may be modified in sequential steps of site-directed mutagenesis and rest~iction fragment substitution to modify the amino acids at positions 64 and 231. For example, one may employ oligonucleotide-dlrected mutagenesis to modify amino acid 64. Subsequently, restriction fragment substitution with a fragment including the serine 231 codon of a partial T4 cDNA
isolated from a T4 positive lymphoeyte eell line [0.
Aeuto et al., Cell, 34, pp. 717-26 (1983)] library in ., -;. 1 ''' . . ~

20~09~9 - - 17 - `

~gt 11 may be used to modify the amino acid at position 231 [R. A. Fisher et al., ~ature, suDra].
DNA sequences coding for 601uble T4 proteins may be used to trans~orm eukaryotic and prokaryotic host cells by conventional recombinant DNA techniques to produce recombinant soluble T4 (rsT4) proteins in clinically and commercially use~ul amounts. Such soluble T4 proteins include those produced according to the processes set ~orth in copending, commonly assigned United States patent applications Serial No. 094,322, ~iled September 4, 1987 and Serial No. 141,649, filed January 7, 1988, and PCT patent application Serial No. PCT/US88/02940, ~iled September 1, 1988, the disclosures o~ which are hereby incorporated by reference.
Mlcroorganisms and recombinant DNA molecules characterized by DNA sequences coding for soluble T4 proteins ar~ exempli~ied by cultures deposited in the In Vitro International, Inc. culture collection, in 20 Linthicum, Maryland, on September 2, 1987 and identified as:
EC100: E.coli JM83/pEC100 - IVI 10146 BG377: E.coli MC1061/pBG377 - IVI 10147 BG380: E.coli MC1061/pBG380 - IVI 10148 BG381: E.coli MC1061/pBG381 - IVI 10149.
Such microorganisms and recombinant DNA molecules are also exempli~ied by cultures deposited in the In Vitro International, Inc. culture collection on January 6, 1988 and identified as:
BG-391: E.coli MC1061/pBG391 - IVI 10151 BG-392: E.coli MC1061/pBG392 - IVI 10152 ~
BG-393: E.coli MC1061/pBG393 - IVI 10153 `
BG-394: E.coli MC1061/pBG394 - IVI 10154 BG-396: E.coli MC1061/pBG396 - IVI 10155 203-5 : E.coli SG936/p203-5 - IVI 10156.
.~ , 200(~9~g Additionally, such microorganisms and recombinant DNA molecules are exemplified by cultures deposited in the In Vitro International, Inc. culture collection on August 24, 1988 and identified as:
211-11: ~.coli A89/pBG211-11 - IVI 10183 214-10: E.coli A89/pBG214-10 - IVI 10184 215-7 : E.coli A89/pBG215-7 - IVI 10185.
Alternatively, soluble T4 proteins may be chemically synthesized by conventional peptide syn-thesis techniques, such as solid phase synthesis.tR. B. Merrifield, "Solid Phase Peptide Synthesis. I.
The Synthesis Of A Tetrapeptide", J. Am. Chem. Soc., 83, pp. 2149-54 (1963)].
By coupling that portion of a DNA sequence coding for a toxin with a DNA sequence coding for a soluble T4 protein, immunotoxins which primarily affect target HIV or HIV-infected cells may be produced in accordance with this invention. The term "toxin" as used in this application includes any molecule which kills or down regulates cells either upon contact or when introduced intracellularly. Toxins useful in this in~ention include, but are not limited to, proteins such as ricin, abrin, angiogenin, ~seudomonas Exo-toxin A, pokeweed antiviral protein, saporin, gelonin and diptheria toxin, or toxic portions thereof.
Many toxins exert their cytotoxic effects via a three step mechanism which includes: 1) binding of the toxin to the cellular membrane, 2) translocation of the toxin through the cellular membrane into the cytosol*, and 3) target cell inactivation. Such toxins * Like other molecules that bind to receptors on the cell surface, these toxins are internalized by the pathway of receptor mediated endocytosis. In the first step of this process, a ligand binds to its receptor.
The receptor-ligand complex then diffuses laterally within the cell membrane until it encounters a clathrin ;~
,:

200as~s - 19 - . :

each comprise several different functional regions or "domains", which mediate one of these steps.
Typically, one domain contains information for cell recognition and binding, a second domain translocates the toxin across the cell membrane to the cytosol --where the targets of most toxins reside, and a third domain catalyzes the reaction responsible for cell killing [I. Pastan et al., "Immunotoxins", ÇÇll, 47, pp. 641-648 (1986)]. Although the third domain is the portion of the toxin which actually inactivates the target cell, the other two domains are normally critical to overall toxicity, because the toxin usually must be internalized into the cytosol to have a high activlty.
When such toxins are used to produce the immunotoxins of this invention, the translocation and cell inactivating domains o~ the toxin are kept substantially intact, while the binding domain is inactivated or deleted, being replaced with a soluble T4 protein which will specifically bind to HIV or an HIV-infected cell. In this application, the translocation and cell inactivating domains of the toxin are the "toxic portion" of the toxin.
Ricin, abrin, saporin and gelonin are plant ribosome-inactivating proteins tI. Pastan et al., Cell, supra]; angiogenin is a human blood vessel inducing ;
protein which is also ribosome inactivating [D. K. St. Clair et al., "Angiogenin Abolishes Cell-Free Protein Synthesis By Specific Ribonucleolytic Inactivation 0~ Ribosomes", Proc. Natl. Acad. ~ci. USA, 84, pp. 8330-34 (1987)]; while diphtheria toxin and -: . .
coated pit. The coated pit then gives rise to an endocytic veæicle, which moves away from the cell surface by saltatory motion. Once in the cytosol, the toxin then exerts its lethal effect.

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;
p~eudomonas Exotoxin A are bacterial toxins that inhibit protein synthesis by catalyzing the transfer of the ADP ribosyl moiety of oxidized NAD onto elongation factor-2 [G. Gray et al., "Cloning, Nucleotide Sequence, And Expression In Escherichia Coli Of The Exotoxin A Structural Gene Of Pseudomonas Aeruainosa", Proc. Natl. Acad. Sci. USA, ~1, pp. 2645-49 (1984)].
Con~ugates of toxins and cell-reactive antibodies dlrected against determinants on microorganisms, neoplastic cells and virally infected cells have been used to ~orm cell-specific immunotoxins [E. S. Vitetta, "Redesigning Nature's Poisons To Create Anti-Tumor Re-agents", & ience, ~, pp. 1098-1104 (1987); I. Pastan et al., ~ll, suDra]. However, only recently have advances in recombinant DNA technology enabled the production of immunotoxins characterized by highly speci~ic binding to a target cell antigen.
A preferred toxin useful in the compositions and methods of the present invention to produce soluble T4-toxin fusion protein con~ugates is Fseudomonas Exotoxin A, and portions thereof. Pseudomonas Exotoxin A (PE) is an extremely active, monomeric protein (molecular weight 66 kd) secreted by Pseudomonas aeruainosa. It comprises a single polypeptide chain of 613 amino acids [Allured et al., Proc. Natl. Acad. Sci.
USA, 83, pp. 1320-324 (1986)]. Pseudomonas Exotoxin A
inhibits protein synthesis in eukaryotic cells through the inactivation of elongation factor-2 ~EF-2), by catalyzing the transfer of the ADP ribosyl moiety of oxidized NAD onto EF-2.
The specific intoxication process caused by PE is believed to proceed by the following steps.
First, PE binds to cells through a specific receptor on the cell surface. Next, the PE-receptor complex is 35 internalized into the cell. Finally, PE is transferred ;

~o~s~

to the cytosol, where it enzymatically inhibits protein synthesis. Because cellular intoxication is prevented by weak bases, such as NH4~, which raise the pH in acidic vesicles, PE is believed to be translocated across the cell membrane when it reaches an acidic environment. Upon exposure to acidic conditions, the hydrophobic domain of PE enters into the cell membrane, resulting in the formation of a channel through which the enzymatic domain, in extended form, passes across the membrane o~ the endocytic vesicle into the cytosol.
The DNA sequence coding for the PE protein has been mapped and cloned [G. Gray et al., Proc. Natl.
A~ad. Sci. USA, su~ra]. The PE gene i~ characterized by three structurally distinct functional domains which mediate the toxic effects o~ PE ~J~ Hwang et al., "Functional Domains Of Pseudomonas Exotoxin Identif$ed By Deletion Analysis Of The Gene Expressed In E.coli", Ç~Ll, 48, pp. 129-36 (1987)]:
-- domain I, or the binding domain (B)) which mediates binding of the toxin to the cell membrane. The binding domain is characterized by its ability to bind to the mammalian cell surface.
This domain comprises AA1-AA252 of PE (domain Ia) and AA365-AA404 of PE (domain Ib);
-- domain II, or the translocation domain (T), which mediates translocation of the toxin through the cell membrane. The translocation domain is responsible for processing the exotoxin out of the endosome through the cell membrane into the cytosol. This domain comprises AA2s3~AA364 of PE; and -- domain III, or the ADP ribosylation domain (A), which mediates killing of the cell by inactivating elongation factor 2. The ADP
ribosylation domain comprises AA405-AA613 of PE.

Z~0~9S9 A boundary region has also been identified on the Pseudomonas Exotoxin A peptide backbone. This region corresponds to a 26 amino acid residue fragment extending adjacent from the tranclocation domain into the binding domain. The amino acid sequence of p~udomonas Exotoxin A is depicted in Figure 13 and the spatial relationships of the particular domains of PE
are illustrated in Figure 23.
By deleting portions of the PE gene sequence, it is possible to identify the domains responsible for each function. This procedure is also useful to develop shortened versions of the PE gene sequence which no longer code for the binding function of domain I, but still retain the functions of domain II
and III. Such shortened versions of the PE gene sequence which code for the toxic portion of PE, may be conjugated to a soluble T4 protein to form the immuno-toxins of this invention. The inclusion o~ the boundary region of PE in several soluble T4-toxin fusion proteins of this invention leads to an unexpected increase in the cytotoxic effects of these proteins. "~.
We employed shortened versions of the PE gene sequence to express immunotoxins including portions of ~ ~
25 PE without the binding region. One such PE fragment, ~;
identified as PE40, contains only the translocation domain and the ADP ribosylation cell inactivating domain of PE. In addition, we prepared various immunotoxins having a PE fragment, known as PE43, which includes the boundary region located at the juncture of the binding and translocation domains, together with the translocation domain and the ADP ribosylation cell inactivating domain. The boundary region comprises a 26 amino acid fragment located between the binding ,. ,, ",.,,,",...
, . j"

;~000~89 domain and the translocation domain. These immunotoxins are depicted in Figure 23.
Another preferred toxin useful in the compositions and methods of the present invention is S angiogenin, and toxic portions thereof, used alone or in combination with Pseudomonas Exotoxin A. Angiogenin is a human serum protein compri~ing 125 amino acids, without the signal peptide (see Figure 16). The ability o~ angiogenin to inhibit or inactivate protein ~ynthesis [D. St. Clair et al., "Angiogenin Abolishes Cell-~ree Protein Synthesi~ by Specific Ribonucleolytic Inactivation of 40S Ribosomes", ~iochem., 27, pp. 7263-68 (1988)~ constitutes an activity in common with all toxins known to date. AB previously stated, angiogenin i~ a human protein; whereas all toxins known to be useful as immunotoxins have been derived from plants or~i ;
bacteria. Thus, angiogenin is likely to exhibit less of an immunogenic response than its plant or bacterial~;~
counterparts. Moreover, soluble T4 fu~ion proteins comprising angiogenin should be capable of demonstrating efficacy for prolonged periods of treatment.
In addition, angiogenin is resistant to acidic conditions (low pH) as well as to proteolytic degradation. It is known that toxins are typically internalized into the cell in an acidic environment.
Although angiogenin has not typically been considered a "toxin", we believe that its ability to inhibit or inactivate protein synthesis in a target cell or to down regulate a cell renders it useful in the fusion protein conjugates of thi~ invention in place of conventional toxins to achieve comparable, if not enhanced, toxic effects. Accordingly, the use of the term "toxin" to describe angiogenin and soluble T4 protein-angiogenin fusion proteins, described :, 2~9~9 herein, will also be referred to as immunotoxins.
Further, the term "immunotoxin", as used herein, is meant to include all toxins that act through an immunological pathway or a directed biochemical pathway to induce their toxic response.
The immunotoxins of this invention are useful in a variety of immunotherapeutic compositions and methods. Advantageously, the immunotoxins of this invention inhibit HIV binding to T4+ lymphocytes by v~rtue o~ their competitive binding characteristics, and they actively destroy HIV in~ected cells express-ing the gpl20/160 protein and producing HIV. Accord-ingly, the immunotoxins o~ this invention may be used in pharmaceutical compositions and methods to treat humans having AIDS, ARC, HIV infection, or antibodies to HIV. In addition, these immunotoxins and methods may be used ~or treating AIDS-like diseases caused by retroviruses, such as ~imian immunode~iciency viruses, in mammals, including humans.
The immunotoxins o~ this invention may be produced by con~ugating a soluble T4 protein to a toxin. According to one embodiment o~ this invention, toxin~ may be con~ugated to soluble T4 proteins using a chemical coupling method. In this method, the toxin is chemically coupled directly to the soluble T4 protein using, for example, techniques which have been used to -couple toxins to antibody molecules via either disul~ide or thioether linkages. More specifically, disul~ide bonds may be created by introducing sul~hydryl groups into the toxin's amine groups by treating the toxin with iminothiolane ~D.J.P. ~ ~
Fitzgerald, "Construction of Immunotoxins Using !; ' Pseudomonas Exotoxin A", Methods EnzYmol., 151, pp. 139-45 (1987)], or by using N-succinimidyl-3-(2-pyridyldithio)-propionate [J. A. Cumber et al., 2000g~9 "Preparation Of Antibody-Toxin Conjugates", Methods Enzvmol., 112, pp. 207-25 (1985)]. Alternatively, thioether bonds may be created by introducing thiol groups into the protein and an alkylating function into the toxin [M. J. Bjorn et al., "Antibody-Pseudomonas Exotoxin A Conjugates Cytotoxic To Human Breast Cancer Cells In Vitro", Cancer Res., 46, pp. 3262-67 (1986)].
Example 4 illustrates the production of a soluble T4 protein-toxin conjugate by chemical coupling using disulfide linkages.
In another embodiment of thi~ invention, toxins may be con~ugated to soluble T4 proteins using a genetic fusion method. In this method, a hybrid DNA
~equence is constructed from a DNA sequence coding for the toxin and a DNA sequence coding for a soluble T4 protein using recombinant DNA techniques. Expression of the hybrid DNA sequence in an appropriate host re~ults in a fusion protein comprising the ~oluble T4 protein and the toxin ("a soluble T4-toxin fusion protein con~ugate") [Lorberboum-Galski et al., "Cytotoxic Activity Of An Interleukin 2-Fseudomonas Exotoxin Chimeric Protein Produced In E~çherichia ÇQLi", Proc. ~atl. Acad. Sci. USA, 85, pp. 1922-26 ~1988); Hwang et al., Ç~ll, su~ra].
According to a preferred embodiment of the present invention, various soluble T4 protein-P~eudomona~ ExotoxinlA immunotoxins which are toxic to HIV or HIV-infected cells may be prepared. The soluble T4 portion of these fusion proteins may comprise any soluble T4 polypeptide capable of binding HIV cells.
The preferred soluble T4 polypeptide is characterized by the formula AA-23-AA377 of Figure 2, or frag~ents thereof. Particularly preferred are soluble T4 polypeptides represented by the formula AA 23-AA182 or AAl-AA182 of Figure 2. Most preferred is a soluble T4 . . .
' : :

20009~3~

polypeptide represented by the formula AA3-AA183 of Figure 2.
The Pseudomonas Exotoxin A portion of the immunotoxins of this invention preferably comprises the entire protein toxin, less a substantial portion of the binding domain (Ia). A particularly preferred immunotoxin of this invention comprises a fragment of Rseudomonas Exotoxin A having the translocation domain (II) and the ADP ribosylation cell inactivating domain ~III) fused to the soluble T4 protein. Another particularly preferred immunotoxin is a fragment of Pseudomonas Exotoxin A having the boundary region, the translocation domain and the ADP ribosylation cell inactivating domain substantially intact and fused to a soluble T4 protein. Most preferred is an immunotoxin comprising a fragment of Pseudomonas Exotoxin A
comprising the boundary region, the translocation domain, and the ADP ribosylation cell inactivating domain fused to a soluble T4 polypeptide of the formula M 3-AA183 of Figure 2.
Another particularly preferred embodiment of the present invention includes soluble T4 protein-angiogenin immunotoxins which exhibit toxic effects on HIV or HIV-infected cells. These immunotoxins may be produced by conjugation using chemical coupling procedures suitable for linking toxins to antibodies.
These procedures include, but are not limited to, the creation of disulfide bonds by introducing sulfhydryl side chains into an amine functionality of angiogenin through iminothiolane treatment [Fitzaerald, supra].
Another mode of chemical construction employs thioether linkage incorporation through the introduction of sulfhydryl functionalities to the angiogenin followed by alkylation with a suitable alkylating reagent [Biorn, supra].

z~

Alternatively, genetic fusion techniques may be utilized to produce the soluble T4 protein-angiogenin immunotoxins of the present invention. We have expressed such proteins from E.coli using a hybrid DNA sequence constructed from the DNA sequence coding ~or angiogenin and a DNA sequence coding for a soluble T4 polypeptide using recombinant DNA technology. The hybrid DNA sequence, when expre6sed in an appropriate host, encodes an immunotoxin comprising a soluble T4 polypeptide and angiogenin.
Further, an immunotoxin protein comprising a soluble T4 protein capable of binding HIV cells fused to the translocation domain -- AA253-AA364 -- of Pseudomonas Exotoxin A and angiogenin may also be prepared by genetic ~usion techniques. In such immunotoxins, the soluble T4 portion of the protein preferably comprises a soluble T4 polypeptide Or the formula AA3-AA183 of Figure 2. A hybrid DNA sequence comprising a DNA sequence coding for angiogenin with or without its signal-sequence, a DNA sequence coding for the soluble T4 polypeptide o~ the formula AA3-AA183 of Figure 2, and a DNA sequence coding for the translocation domain -- AA253-AA364 o Exotoxin A may be prepared by genetic fusion techniques. Alternatively, such immunotoxins may also comprise the ADP ribosylation domain of Pseudomonas Exotoxin A.
Similarly, a hybrid DNA sequence comprising a DNA sequence coding for angiogenin with or without its signal sequence and a DNA sequence coding for the soluble T4 polypeptide of the formula AA3-AA183 of Figure 2 may be prepared by recombinant techniques.
This invention also relates to immunotoxins, as described above, in which any soluble receptor, rather than soluble T4 protein, may be coupled to a z~og~9 :

toxin. In addition, antibodies, or portions thereof, such as Fab' fragments, variable regions, hypervariable regions, and the like, may also be fused or conjugated to toxins to form immunotoxins according to this invention.
As i5 well known in the art, for expression of the DNA sequences of thi~ invention, the DNA
sequence should be operatively linked to an expression control sequence in an appropriate expression vector and employed in that expression vector to transform an appropriate unicellular host.
Such operative linklng of a DNA sequence of this invention to an expression control seguence, of course, include~ the provision of a translation start signal in the correct reading frame upstream of the DNA
sequence. If a particular DNA sequence being expressed does not begin w~th a methionine, the start signal will result in an additional amino acid -- methionine --being located at the N-terminus o~ the product. While such methionyl-containing product may be emplsyed directly in the compositions and methods of this ;
invention, it is usually more desirable to remove the methionine before use. Methods are available in the art to remove such N-terminal methionines from polypeptides expressed with them. For example, certain hosts and fermentation conditions permit removal of~
substantially all of the N-terminal methionine ~ vivo.
Other hosts require n vitro removal of the N-terminal methionine. However, such i~ vivo and La vitro methods are well known in the art.
A wide variety of host~expression vector combinations may be employed in expressing the DNA
sequences of this invention. Useful expression vectors, for example, may consist of segments of chromosomal, non-chromosomal and synthetic DNA

,, , , . , , ", ,. , ", . . . .. . .. . . ..

200'09~
~.

.

sequences, such as various known derivatives of SV40 and known bacterial plasmids, e.g., plasmids from .coli including colEl, pCRl, pBR322, pMB9 and their derivatives, wider host range plasmids, e.g., RP4, phage DNAs, e.g., the numerous derivatives of phage ~, e.g., NM989, and other DNA phages, e.g., M13 and filamenteous single stranded DNA phages, yeast plas-mids, such as the 2~ plasmid or derivatives thereof, and vectors derived from combinations of plasmids and phage DNAs, such as plasmids which have been modified to employ phage DNA or other expression control sequences.
In addition, any of a wide variety of expres-~ion control sequences -- sequences that control the expression o~ a DNA sequence when operatively linked to it -- may be used in these vectors to express the DNA
~equence of this invention. Such useful expression control ~equence~, include, for example, the early and late promoters of SV40 or the adenovirug, the 1~E
6ystem, the tr~ system, the Ia_ or TRC system, the ma~or operator and promoter regions of phage A, the control regions of fd coat protein, the promoter for 3-phosphoglycerate kinase or other glycolytic enzymes, the promoters of acid phosphatase, e.g., Pho5, the pro- ;
moters of the yeast a-mating factors, the polyhedron promoter of the baculovirus system and other sequences known to control the expression of genes of prokaryotic or eukaryotic cells or their viruses, and various combinations thereof.
A wide variety of unicellular host cells are also useful in expressing the DNA sequences of this invention. These hosts may include well known eukaryotic and prokaryotic hosts, such as strains of E.coli, Pseudomonas, Bacillus, Streptomvces, fungi, -~-~
35 such as yeasts, and animal cells, such as CH0 and mouse ~ ;

' ' , 20a 09~ ~

cells, African green monkey cells, such as COS 1, COS 7, BSC 1, BSC 40, and BMT 10, insect cells, and human cells and plant cells in tissue culture. For animal cell expression, we prefer CH0 cells and COS 7 S cells.
It should of course be understood that not all vectors and expression control sequences will function equally well to express the DNA sequences of khis invention. Neither will all hosts function equally well with the same expression system. How-ever, one of skill in the art may make a selection among these vectors, expression control sequences, and hosts without undue experimentation and without departing from the scope of this invention. For lS example, in selecting a vector, the host must be considered because the vector must replicate in it. ~
The vector~s copy number, the ability to control that ~i copy number, and the expression of any other proteins encoded by the vector, such as antibiotic markers, should also be considered.
In selecting an expres~ion control sequence, a variety of factors should also be considered. These include, for example, the relative strength of the system, its controllability, and its compatibility with the particular DNA sequence of this invention, parti-cularly as regards potential secondary s~ructures.
Unicellular hosts should be selected by consideration of their compatibility with the chosen vector, the toxicity of the product coded for on expression by the DNA sequences of this invention to them, their secretion characteristics, their ability to fold proteins correctly, their fermentation requirements, and the ease of purification of the products coded on expression by the DNA sequences of this invention.

~' "

~9~

- 31 - ~
-Within these parameters, one of skill in the art may select various vector/expression control system/host combinations that will express the DNA
sequences of this invention on fermentation or in large scale animal culture, e.g., CHO cells or COS 7 cells.
The polypeptides produced on expression of the DNA sequences of this invention may be isolated from the fermentation or animal cell cultures and purlfied using any of a variety of conventional methods. One of skill in the art may select the most appropriate isolation and puri~ication techniques without departing from the scope of this invention.
Examples 1-3 and 5-8 of this application illustrate the production of immunotoxins of this invention using genetic fusion techniques.
According to another embodiment of this invèntion, a hybrid DNA sequence comprising a DNA
~equence coding for a toxin and a DNA ~equence coding for solubl~ T4 protein may further comprise a signal ~equence coding for a streptavidin-like polypeptide.
Expression of this hybrid DNA sequence in an appro-priate host results in the secretion of the soluble T4-toxin fusion protein conjugate through the membrane of the cell. This greatly facilitates the production and purification of the soluble T4-toxin fusion protein conjugate, because the secreted soluble T4-toxin fusion protein i8 in its biologically active form.
Streptavidin is an antibiotic produced by the ;
bacteria Streptomvces avidinii and other Streptomvces species tE. O. Stapley et al., Antimicrobial Aaents And Chemothera~v 1963, pp. 20-27 (J. C. Sylvester ed.
1964)]. It occurs naturally as a tetramer with a molecular weight of about 60,000 daltons. Streptavidin is characterized by its strong affinity for biotin and ~ ~-biotin derivatives and analogues. Each of the four : :
, :

200~9~g identical subunits of steptavidin has a single biotin binding site [K. Hoffman et al., ~roc. Natl. Acad. Sci.
USA, 77, pp. 4666-68 (1980)].
As used in this application, "streptavidin-like polypeptide" refer~ to a polypeptide which displays the biological or immunological activity of native streptavidin and which is able to bind to biotin or biotin derivatives or analogues. This polypeptide may contain amino acids which are not part of native streptavidin or may contain only a portion of native streptavidin. The polypeptide may also not be iden-tical to native streptavidin because the host in which it is made may lack appropriate enzyme~ which may be required to transform the host-produced polypeptide to the structure of native streptavidin. The DNA and amino acid sequences corresponding to streptavidin-like polypeptides have been published in PCT patent application W0 86/02077, and are depicted in Figure 8 of the present application.
A signal DNA sequence is that portion of a DNA sequence coding for a polypeptide or protein which encodes, as a template for mRNA, a sequence of hydrophobic amino acids at the amino terminus of the polypeptide or protein, i.e., a "signal sequence" or "hydrophobic leader sequence" of the polypeptide or protein. A signal DNA sequence is located in a gene for a polypeptide or protein immediately before the DNA
sequence coding for the mature protein or polypeptide, and after the translational start signal (ATG) of the 30 gene. Expression of the signal DNA sequence together , with the DNA sequence encoding a polypeptide or protein, produces a precursor of the polypeptide or protein.
A precursor of a protein or polypeptide is a polypeptide or protein as synthesized within a host ,, , ~' ' , cell with a signal sequence, e.g., prestreptavidin. A
mature polypeptide or protein iB ~ecreted through a host~s cell membrane with the attendant loss or clipping (i.e., maturation) of the slgnal sequence of its precursor.
It is believed that only a portion of a ~ignal sequence of a precursor of a protein or poly-peptide ls essential for the precursor to be trans-ported through the cell membrane of a host and for the occurrence of proper clipping of the precursor's signal sequence to form the mature protein or polypeptide during secretion. Hence, the term "signal DNA
~equence" means the DNA sequence which codes for the portion of the signal sequence essential to secretion of a precursor of a protein, polypeptide or peptide, produced within a host cell.
A soluble T4-toxin fusion protein con~ugate of this invention may be secreted through the membrane of the host cell, upon expression in a suitable host, of a hybrid DNA sequence comprising a DNA signal sequence coding for a sufficient portion of a pre-streptavidin-like polypeptide to cause the resulting fusion protein to be secreted through the membrane of the host cell, and a DNA sequence coding for the soluble T4-toxin fusion protein con~ugate. When expre~ed, the soluble T4-toxin fusion protein con- ~-~ugate i5 secreted through the membrane of the host cell transformed by the hybrid DNA sequence.
The hybrid DNA sequence6 o~ this invention may additionally code for a sufficient portion of a streptavidin-like polypeptide to allow binding of the resulting soluble T4-toxin fusion protein conjugate to biotin or its derivatives or analogues. Upon expression of this hybrid DNA sequence, the streptavidin moiety of the soluble T4-toxin fusioh protein conjugate may be bound to biotin or to one of its derivatives or analogues. Other secreted proteins or contaminants which do not bind to biotin may then be washed away and the soluble T4-toxin fusion protein con~ugate sluted from the biotin. This embodiment of the present invention advantageously provides a method ~or producing a soluble T4-toxin fusion protein con-~ugate that may be easily purified due to the binding affinity of streptavidin-like polypeptides for biotin and its derivatives or analogues.
Example 3 of this application illustrates the production and subsequent secretion of a soluble T4-toxin fusion protein con~ugate using a streptavidin-like polypeptide as a signal sequence.
The immunotoxins of this invention may also be used in combination with other therapeutics employed ln the treatment o~ AIDS, ARC and HIV infection. For example, these immunotoxins may be used in combination with antiretroviral agents that block reverse tran~criptase, such as AZT, HPA-23, phosphonoformate, suramin, ribavirin and dideoxycytidine. Additionally, these immunotoxins may be used with antiviral agents such as interferons, including alpha interferon, beta interferon and gamma interferon, or glucosidase inhibitors, such as castanospermine. Further, the immunotoxins and fusion proteins of the present ;
invention may be administered with other rsT4-immunotoxins or toxins as described herein, which may result in an additive or synergistic effect. Such combination therapies advantageously utilize lower dosages of those agents, thus avoiding side effects which may be associated with high dosage regimens of those agents alone. ~;~
The pharmaceutical compositions of this invention typically comprise an immunotherapeutically 2~0~

effective amount of an immunotoxin of this invention and a pharmaceutically acceptable carrier. Therapeutic methods of this invention comprise the stQp of treating patients in a pharmaceutically acceptable manner with those compositions. An immunotherapeutically effective amount of an immunotoxin of this invention is that which is sufficient to lessen the immunocompromising effects of HIV infection, to prevent the spread of HIV
infection or to kill HIV or HIV-infected cells.
The pharmaceutical compo6itions of this tnvention may be in a variety of forms. These include, for example, solid, semi-solid and liquid dosage forms, such as tablets, pills, powders, liquid solutions or suspensions, liposomes, suppositories, in~ectable and infusable solutions. The preferred form depends on the intended mode of administration and therapeutic application. The compositions also preferably include conventional pharmaceutically acceptable carriers and ad~uvants which are known to those of skill in the art.
Generally, the pharmaceutical compositions of the present invention may be formulated and administered using methods and compositions similar to those used for pharmaceutically important polypeptides such as, for example, alpha interferon. Thus, the immunotoxins of this invention may be stored in lyophilized form, reconstituted with sterile water just prior to administration, and administered by conventional routes of administration such as parenteral, subcutaneous, intravenous, intramuscular or intralesional routes. An effective dosage may be in the range of about 0.5 to 0.25 mg/kg body weight/day, it being recognized that lower and higher doses may also be useful. ; ~
In order that this invention may be better ;
35 understood, the following examples are set forth. ;
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. ~ ,. : .

These examples are for the purposes of illustration only, and are not to be construed as limiting the scope of the invention in any manner.
Examples 1-3 illustrate the production of soluble T4-toxin fusion protein conjugates using modified Pseudomonas Exotoxin A and recombinant soluble T4 protein (rsT4). Example 3 further illustrates the con~truction and the secretion of a soluble T4 protein-toxin fusion protein conjugate using a streptavidin-like polypeptide as a signal sequence. Example 4illustrates a soluble T4 protein-toxin con~ugate produced via chemical coupling techniques. Example 5 illustrates the production of a soluble T4-toxin fusion protein con~ugate comprising a soluble T4 polypeptide having th~ formula AA3-AA183 f Figure 2 and a portion of ~ omonas Exotoxin A comprising domains II and III. Example 6 illustrates the production of a soluble T4-toxin fusion protein con~ugate comprising a soluble T4 polypeptide having the formula AA3-AA183 f 20 Figure 2, a portion of ~seudomonas Exotoxin A ;
comprising domains II and III, and the boundary region of Pseudomonas Exotoxin A. Example 7 illustrates the production of a soluble T4-toxin fusion protein con~ugate comprising a soluble T4 polypeptide having the formula AA3-AA183 of Figure 2, a portion of Pseudomonas Exotoxin A comprising domains II and III, and the boundary region of Psçudomonas Exotoxin A. ~;
This protein was expressed from a modified nucleotide sequence which facilitated its purification. Example 8 illustrates the expression, isolation, and purification of the immunotoxins, PEx45, PEx46, and PEx56.
Examples 9 and 10 illustrate the in Yitro activities of the immunotoxins of this invention. Example 11 illustrates the expression, isolation, and purification of TANG11 and TANG12. Example 12 illustrates the expression, isolation, and purification of TANG13 and TANG14.
EXAMPLES
In these examples, we conjugated soluble T4 proteins to toxins to form the immunotoxins of this invention. The soluble T4 protein used wa~ recombinant ~oluble T4 protein ("rsT4") supplied by Biogen Research Corp. ~Cambridge, Massachusetts). The rsT4 was derived ~rom a Chinese hamster ovary cell trans~ected with pBG391, which is characterized by DNA coding ~or AA-23 to AA377 o~ T4 protein, a full-length soluble T4 protein, as depicted in Figure 2 [R. A. Fisher et al., ~HIV In~ection Is Blocked In Vitro By Recombinant Soluble CD4", Nature, ~, pp. 76-78 ~1988)].
In the examples that ~ollow, the molecular biology technigue~ employed, such as cloning, cutting with restriction enzymes, isolating DNA ~ragments, treating with Calf Intestinal alkaline Pho~photase (CIP), ligating, and trans~orming E.coli, are standard protocol exempli~ied and further described in T. Maniatis et al., Molecular Clonin, Cold Spring Harbor Laboratory (1982).
Exam~le 1 ~CTERIAL CELL EXPRESSION
As depicted ln Figure 4, we isolated DNA from PA103, a culture o~ the natural producer o~ Exotoxin A, ;
pseudomonas aeruainosa, [American Tissue Culture -Collection (ATCC), accession no. 29,260], using standard procedures. We then constructed a cosmid library by cloning completely digested Pseudomonas DNA
into an EcoRI-digested pHC79 plasmid (Boehringer Mannheim, Cat. No. 567,795).
". :, We then synthesized an oligonucleotide probe (PA85), corresponding to nucleotide6 827-840 of the published sequence of PE [G. Gray et al., p~oc. Natl.
Acad. Sci. USA, 81, pp. 2645-49 (1984)~, using standard procedures and a 380A DNA synthesizer (Applied Bio-sy6tems Inc.). We probed the cosmid library with PA85, and i601ated cosmids which contained a 6Kb EcoRI
fragment. Ba6ed upon the published sequence of PE and a Southern Blot, we determined that the 6Kb EQRI frag-ment contained the complete PE sequence. One of thecosmids which tested positive for the fragment, designated pEXl, wa6 then 6elected for further work.
Baeed upon the publi6hed sequence of the gene coding for PE, we knew that domain II of the gene begin~ ~ust downstrea~ of the ~glII site at 1500bp tJ. Huang et al., ÇÇ11, 48, pp. 129-36 (1987)], and that domain II and the domain for ADP-ribo6ylation codes through the C-terminal portion of the PE
~equence. We al60 knew that the PE structural gene could be subcloned as a 2.7Kb EQRI-PstI fragment.
Therefore, we cut pEXl with EcoRI and PstI, and subcloned the resulting fragment into EcoRI/E~
digested pUCl9 (Bethe6da Research Laboratory, Cat. No.
5364SA), to produce pEX2.
Several expression vectors utilize a Ç
site downs~ream of the promoter for insertion o~ a gene -~
of intere6t. Because thé gene coding for PE may be ~ ;
conveniently employed as a ~glII-Çl~I fragment, a Ç~
6ite was introduced into the EcoRI site of pEX2 using oligonucleotide linkers, and the resulting 1.2Kb BqlII-Çl~I fragment was subcloned into ClaI/~3~ digested pHC79, to yield pEX3.
The 1.2Kb BglII-ClaI fragment of pEX3 contains domains II, Ib and III of the Pseudomonas Exotoxin A gene ~J. Hwang et al., Cell, supra]. That fragment may be linked to DNA sequences coding for soluble T4 proteins to form a hybrid DNA sequence with ~glII sites engineered into the coding sequence. Upon expression, such hybrid DNA sequences code for an immunotoxin of this invention: a soluble T4-toxin ~usion protein con~ugate. ;;
Accordingly, as depicted in Figure S, we constructed a shortened version of rsT4 containing a 124 amino acid segment and a convenient ~1II site as follows. Plasmid pl99.7 tIVI #10191], which contains full length soluble T4 protein, was cut with BstNl, ~len~wed, and a ~g1II linker ~NEB#1036) WaB ligated to the blunt ended DNA. Following inactivation of the ligase at 6SC, the mixture was digested with ÇL~I and ~g1II. Plasmid pHC79 was also cut with Çl~I and ~31II
and the 2.7Kb fragment containing the ampicillin (Amp) marker and Ç~i was electroeluted. This fragment was ligated with the pl99.7 digest and the mixture used to transform E~Qli strain DH5 (Bethesda Research Laboratorie~, Cat. No. 8265SA). Ampicillin resistant colonies were then probed using a T4-6 oligo probe, corresponding to a region of T4 protein (AA94_l03), synthesized from the published sequence (Maddon et al., Ç~ll, supra).
A clone ~pEX15) was identified which carried an amino acid sequence extending 124 amino acids downstream of the initiating Met of the T4 protein sequence (AA_l-AA124, Figure 3). By sequencing analysis, it was established that the BalII site was correctly inserted at the BstNI site at amino acid 124 (Figure 3). The pEX15 plasmid was then digested with ClaI and ~g1II, and the 380 bp fragment containing the 124 amino acid rsT4 (AA-1-AA124~ Figure 3), was electroeluted. pEX3 was cut with ClaI and BalII and the 1.2kb ClaI-BglII fragment was also electroeluted.

zooosas The above-described 124 amino acid rsT4 and the segment containing Pseudomonas Exotoxin A
domains II and III from pEX3 were then ~oined by ligating the ClaI-BalII fragments into the Çl~I site of Çl~I-cut expression vector pl97.12, to yield plasmid pEX27. Portions of pEX27 were sequenced to establish that they contained the soluble T4-toxin fusion protein coding sequence. All junction regions were found to be correct.
We then tested for expression of pEX27 as ~ollows. SG936, an E.coli ~Qn ht~r double mutant [S. Go~f and A. Goldberg, "ATP-Dependent Protein Degradation In E.Coli", Maximizina Gene Exoression, W. Reznikof~ and L. Gold (eds.) (1986)] was transformed with pEX27 by conventional procedures [T. Maniatis et al., Molecular Clonina, p. 187, Cold Spring Harbor Laboratory ~1982)]. Induction showed Western positive material which ran at a molecular weight of about 60 kd, conflrming the presence of the soluble T4-toxin ~usion protein con~ugate.
The selectivity and specificity of the rsT4-PE fusion protein conjugate may be assessed on ~
uninfected and HIV-infected H9 cells as follows. ~ ~;
Twenty-four well cluster plates are seeded with 200,000 non-HIV infected Hg cells per well [M. Popovic et al., "Detection, Isolation And Continuous Production Of Cytopathic Retrovirus (HTLV-IIIj From Patients With AIDS And Pre-AIDS", Science, 224, pp. 497-500 (1984)], various concentrations of rsT4-PE fusion protein con~ugate added, and the cells incubated at 37C
overnight. The cells are then pulse-labelled with (3H)-leucine at 1 microCurie/ml for two hours to evaluate protein synthesis, as assessed by incor-poration of leucine into trichloroacetic acid-precipitable radioactivity. Alternatively, HIV

:: . , : , . ~ .. `, -infected cells are incubated for four days with the rsT4-PE fusion protein conjugate at various concen-trations, (3H)-thymidine is added to 1 microCurie/ml, the cells are then incubated overnight at 37C, and cell viability assessed by incorporation of thymidine into trichloroacetic acid-precipitable radioactivity.
~xam~le 2 ANIMAL CE~L EXPRESSION
rsT4-PE immunotoxins according to this invention may also be produced in eukaryotic tissue culture. Accordingly, as depicted in Figure 6, we linked the 1.2kb PE fragment obtained as described in Example 1 to a shortened 182 amino acid version of soluble T4 protein as follows.
Plasmid pBG391 tIVI #10193], which contains full length soluble rsT4, was cut at the StuI site corresponding to amino acid 184 of the T4 protein sequence (bp 1825, Figurs 2). A ~glII linker (NEB
#1052), which puts the site in the correct reading ~i frame, was ligated to the cut DNA. The resulting plasmid, pEX5, contained the necessary expression machinery for animal cells to direct the expression of a 182 amino acid rsT4 (AA3-AA184).
We then modified pEX3 (as described in Example 1) so that domains II and III of the PE gene were located on a ~g¦II fragment that could be ligated into the pEX5 plasmid. To do this, we changed the Çl~I
site on pEX3 to a ~lII site. The resulting plasmid, pEX4, contained the II, Ib, and III regions of the gene coding for Pæeudomonaæ Exotoxin A on a BqlII fragment.
This 1.2kb fragment was electroeluted and ligated into pEX5 to yield pEXll. This plasmid was used for transient expression of an immunotoxin of this invention in COS 7 cells [derived from African Green '' "' zooosas Monkey cells]. As a positive control for soluble T4 protein, the full length soluble construct, pBG391, was used. pBG393, a plasmid which produces a truncated 182 amino acid version of r~T4, was also used as a positive control ~or soluble T4 protein. This vector was constructed by placing a translational stop signal at the S~ul site at bp 1825 of pBG391 (Figure 2). The truncated rsT4 produced by pBG393 is comparable to full length rsT4 in its ability to bind OKT4A.
Media ~rom transient expression u~ing pBG391, pBG393 and pEXll was harvested after 48 hours. The media was i~munoprecipitated using OKT4A [Ortho Diagnostics #7142]. No 601uble T4 protein speci~ic material was precipitated ~rom pEXll trans~ormants, whereas both the pBG391 and pBG393 transformants produced soluble T4 protein.
Northern analysis of the transient cell lines was carried out to establish RNA levels specific to soluble T4 protein. Both pBG391 and pBG393 tran~ient lines gave strong signals hybridizing to a T4 protein prob~. There was no T4 protein specific signal in the pEXll transients.
The Northern Blot result was negative, because the cells transformed the with immunotoxin construct had died. We believe that the toxin moved into the cytosol during secretion, thus killing the cells. Nonethèless, we believe that expression may be achieved in eukaryotic cell lines that are resistant to the toxin. We also believe that the immunotoxin con-structs of this invention may be expressed in yeastcell cultures.
Example 3 STREPTAVIDIN SIGNAL SEOUENCE

200~98~3 In this example, we used a streptavidin-like polypeptide signal sequence, to secrete a soluble T4-toxin ~usion protein conjugate into the periplasm of E.coli, as well as into the culture media. This greatly facilitated the production and purification of the fusion protein, because the protein was secreted in its mature, correctly folded form. The streptavidin gens has been cloned and used successfully to secrete other heterologous proteins (PCT patent application W0 86/02077). The starting plasmid pSA307 and the coding sequence for streptavidin and streptavidin-like polypeptides are depicted in Figure 8.
As illustrated in Figure 7, the expression plasmid p211.11 [IVI #10190], which contains an amino acid sequence extending 113 amino acids downstream of the initiating Met (AA_1-AA113, Figure 3), was first transformed into the host strain DH5, which methylates the upstream Çl~I site, making it resistant to cutting.
The plasmid was then isolated from the transformed DH5, cut with ÇlaI, at its downstream site, Klenowed and transformed into the methylation negative strain GM2929 [IVI #10187]. The resulting plasmid pSA400 contained only the Çl~I site upstream of the 113aa rsT4 coding sequence. Plas~id pSA400 was then cut with ~laI, followed by limited digestion with Mung Bean Nuclease to remove the 5' CG overhang of the ClaI site.
Meanwhile plasmid pSA307 [IVI #10194] was digested with ~inII and ~l~I and electroeluted. The resulting 429 bp fragment included the HincII site at bp 194, up to the AluI site at bp623 (Figure 8). It contained the promoter, the translational start codon ATG, the signal sequence and 12 amino acids of the mature streptavidin protein.
This 429 bp HincII-AluI fragment was then ligated into the blunted pSA400 ClaI site. The '~ ,: '";~ ' ~oo9s9 resulting plasmid, pSA410, contained the SA signal (Figure 7) genetically linked to the 113aa rsT4 ~AA l-AA113~ Figure 3). Plasmid pSA410 was then cut with ~lMl and HindIII, and the resulting 612 bp frag-ment was ligated into E~lMl and ~in_III digestedplasmid pSA313 [IVI #10195]. The resulting plasmid, pSA415, contained the SA signal sequence linked to the AA-1-AA62 r~T4 segment at the ~ln~ III site (bp 249, Figure 3). The remainder of the 124aa rsT4 coding sequence and the toxin was derived from pEX27.
Plasmid pEX27, obtained according to Example 1, was digested with EÇQRI, Klenowed, and ligated with ~in~III linkers, ~ollowed by digestion with ~lngIII. The resulting 1.45kb fragment containing the soluble T4-toxin ~usion protein con~ugate was electroeluted and ligated into the HindIII site of pSA415. The resulting plasmid, pEX39, contained the lac promoter directing expression of a hybrid protein comprising streptavidin's.signal sequence and 12aa o~
the mature streptavidin protein, fu6ed to a sequence containing a 124aa rsT~ (AA_l-AA124, Fig was fused to domains II and III of the Exotoxin A
segment.
Plasmid pEX39 was then transformed into the E.coli strain HB101 IQ ~IVI #10188]. The transformant HBlOlIQ/pEX39 was grown at 25C in the presence of 5 mM
IPTG. We tested for the expression and secretion of the hybrid protein into the cell periplasm and the extracellular medium using Western Blot and standard osmotic shocX procedures.
We detected hybrid protein both in the periplasm and in the medium, suggesting that the hybrid protein was secreted out of the cell. To determine if the protein was properly folded, OKT4A was used to immunoprecipitate the osmotic shock fraction. Upon analysis, it was shown that the hybrid secreted soluble T4-toxin ~usion protein con~ugate is precipitated by OKT4A, suggesting that the protein is properly ~olded upon secretion. Additionally, both the osmotic shock ~raction and the media containing the immunotoxin tested positive for ADP-ribosylation activity [H. Hwang et al., ÇÇll, supra] further confirming the activity of the immunotoxin.
We believe that angiogenin, or toxic portions thereof, fused independently or in con~unction with other proteins which exhibit toxic effects may also be particularly well-suited to benefit from the use of the streptavidin signal sequence to assist in the secretion of these angiogenin comprising fusion proteins.
EX~m~e 4 ÇHEMICAL CONJUGATION
In this example, a ricin A chain is con-~ugated to a full length amino acid, soluble T4 protein obtained according to R. A. Fisher et al., Nature, 331, pp. 76-78 ~1988). Highly purified ricin A chain is commercially available ~XOMA Corporation, San Francisco]. The purity of the ricin A chain can be verified free of contaminating B chains by a whole cell toxicity assay.
The full length amino acid soluble T4 pro-tein and purified ricin A chain are conjugated with the heterobifunctional crosslinker N-succinimidyl-3-(2-pyridyldithio)-propionate [SPDP, Pharmacia, Piscataway, New Jersey] according to standard protocols [A. J. -30 Cumber et al., "Preparation Of Antibody-Toxin ;
Conjugates", Meth. Enzymol., 112 pp. 207-25 (1985)], as ~
follows. ;
SPDP, dissolved at 6 mg/ml in dry dimethyl-formamide, is added to a solution of the soluble T4 '''. "' ' Z00~)989 protein at 10 mg/ml in Dulbecco's calcium and magnesium-~ree phosphate buffered saline (PBS); 10 microfilters SPDP per ml of the soluble T4 protein.
The mixture is incubated for 30 minutes at room temperature, then applied to a column of Sephadex G25 Qquilibrated with PBS to separate the rsT4-SPDP
con~ugate from unreacted SPDP. ;
Ricin A chain is then fully reduced with dithiothreitol (DTT) as follows. DTT is added to a solution of ricin A at 10 mg/ml in PBS to 0.1 M final concentration. The solution is incubated for one hour at room temperature. The ~ully reduced ricin A chain is then separated from excess DTT by gel filtration on Sephadex G25 in PBS.
The fully reduced ricin A chain is imme-diately incubated with soluble T4-protein-SPDP, for two hours at room temperature, then for two days at 4C., to effect coupling. The resultlng ~oluble T4 protsin ricin A con~ugate i5 separated from uncoupled soluble T4 protein and ricin A chain by seguential chromato-graphy on an affinity column of Cibacron blue F3GA-Sapharose [P. P. Knowles and P. E. Thorpe, ~Purifi-cation Of Immunotoxins Containing Ricin A Chain And Abrin A Chain Using Blue Sepharose CL-68", Analyt~al Biochemistry, 160, pp. 440-43 (1987)], to remove soluble T4 protein; and in an immunoaffinity column containing T4 protein specific antibody [R. A. Fisher et al., "HIV Infection Is Blocked In Vitro By Recom-binant Soluble CD4", Nature, su~ra~, to remove excess ricin A chain.
The conjugation of soluble T4 protein and ricin A is confirmed by sodium dodecyl sulfate~
polyacrylamide gel electrophoresis and by radio- ; ~
immunoassay with the use of rabbit anti-mouse Ig- ;
coated microtiter plates and 125I labeled rabbit anti-200C~989 . "
ricin A chain antiserum [M. Okhuma and J. Poole, "Fluorescence Probe Measurement Of The Intralysomal pH ', In Living Cells And The Perturbation Of PH By Various Agents," ~,oc. Natl. Acad. Sci. USA, ~, p. 3327 (1978~].
Each molecule of soluble T4 protein is thus associated with at least one molecule of ricin A chain.
These con~ugates can be stabilized with carrier protein ~reduced and alkylated human IgG), and stored at -70C.
Exam~le 5 ~XOTOXIN A FUSION PROTEIN ~PEx45~
We then prepared PEx45, a soluble T4-~aç~,d,omonas Exotoxin A fusion protein containing the boundary region of PE linked to the translocation and ADP ribosylation domains of PE. As depicted in Figure 9A, we cut plasmid pEX11 ~as prepared in Example 2) with ~in~III and EcoRI, and electroeluted the resulting restriction fragment encoding containing the soluble T4-~E43 fusion protein. We next cut the T4 expres~ion plasmid p218~ with EcoRI and HindIII and electroeluted the 241 bp fragment encoding the ' N-terminus of T4. We then isolated the vector promoter ,'',;
portion of pEX27 (as prepared in Example 1) by ' , 25 digesting it with EcoRI and electroeluted the vector. , ' We ligated the EcoRI-HindIII fragment carrying the ~ ,~"
N-terminus of T4 and the _in~ EcoRI fragment from ~ -pEXll to the vector promoter fragment isolated from ;~
pEx27 to yield pEX45. ~ ~' The pEX45 plasmid contains DNA encoding a, '~,' polypeptide of the formula AA3-AA183 of Figure 2 linked - : ':,.':' * The expression vector p218 is prepared from ~ pL
DC197 containing a soluble T4 protein of the formula AA
AA182 of Figure 2-ZU~0989 . . .
at AA224 to a portion of Pseudomonas Exotoxin A
comprising AA224-AA613 of that protein (as illustrated in Figures 11 and 13). We transformed the E.coli strain A89 with pEx45. E. coli A89 is a tetracycline sensitive derivative of E. coli SG936. E.coli A89 had been isolated from ~coli SG396 according to the method of S~R. Maloy and W.D. Numm, "Selection For Loss of Tstrachycline Resistance By sçherichia coli", Bact., ~45, pp. 110-12 (1981). We then grew the A89/pEX45 transformant under conditions of temperature shift induction.
Using standard techniques, we renatured and puri~led the PEx45 fusion protein after temperature ~hlft lnduction. We then used the re~ulting fusion protein as an ~mmunotoxin to selectively kill a CHO
cell llne expres~lng gpl20/160 on its surface. The Xllllng level of 50% was determined to be at a concentration of 5 x 10-1 M as further described in Example 8 and depicted in Figure 15.
Figure 11 depicts the amino acid sequence of the lmmunotoxln product of hosts transformed with pEX45.
Ex~m~le 6 , " .
SOLUBLE T4 - ~$EUDOMONAS
EXOTOXIN A FUSION PROTEIN (PEx46 .,,, ~ ,.....
We next prepared PEx46, a soluble T4 ~eudomonas Exotoxin A fusion protein in which all of the binding region of PE was removed and which did not include the boundary region of PE (See Figures 9A, 9B, and 13). As depicted in Figure 9B, we cut pEX3 (a~
prepared in Example 1) with BqlII and AvaI. We then ligated the ~g~ AvaI fragment to an oligonucleotide adapter having the sequence GAT CAT CAT CGA TCT CCA TGG ~ ~
GGA AGA TCT ACC to place a BalII site immediately ~-,:

.

ZO(:~09~39 _ 49 _ upstream of the AvaI site in PE, yielding plasmid pEX31.
We then cut pEX31 with ag1II and ~çQRI, and electroeluted the 1.2 kb fragment encoding PE40. We also cut pEX27 (as prepared in Example 1) with EcoRI
and ~glII and electroeluted the resulting fragment encoding the N-terminus o~ T4. We ligated these two ~ragments into a ~çQRI vector promoter segment which had previously been isolated from pEX27 (as described in Example 5). The resulting plasmid, pEX44, contained DNA encoding soluble T4 protein linked to PE40 which corr25pondg to PE at AA2S3-AA613 (See Figures 13 and 23).
As shown in Figure 9A, we then cut pEX44 with ~~RI and ~glII, and again electroeluted the fragment encoding PE40. We next cut pEX45 (as prepared in Example 5) with EcoRI and ~glII, and electroeluted the 615 bp ~ragment encoding the N-terminua of T4. We then isolated the vector promoter segments by cutting pEX45 wlth EcoRI and isolated the resulting 2.7 kb ~ragment.
The three fragments -- ~çQRI-~g~ rom pEX45, the BalII-EcoRI from pEX44, and the vector promoter ~çQRI
~ragment ~rom pEX45 -- were ligated to produce plasmid pEX46. That plasmid encodes a soluble T4 polypeptide ~ ;
of the formula AA3-AA183 of Figure 2 linked to PE40.
Thls construct was then transformed into the ~.coli strain A89. Following temperature shift induction, we renatured and isolated the protein. We found that the fusion protein selectively killed CH0 cells expressing~i;
gpl20/160 on their surface (See Figure 15). In addition, PEx46 also killed HIV infected H9 cells.
Figure 12 depicts the amino acid sequence of the immunotoxin product of hosts transformed with pEX46. ~
In addition, we also constructed a pEX60 ;
plasmid which contained a soluble T4 protein (having Z001~198~

the formula AA3-AA183 of Figure 2) fused to only the ADP ribosylation cell inactivation domain of PE
(without the translocation domain of PE) (See Figure 23). This fusion protein failed to exert a toxic effect on celle expressing gpl20/160 on their ~urface. Accordingly, we believe that the translocation domain of PE is necessary to permit the ~usion proteins of this invention to enter the cytoplasm and therefore inactivate protein synthesis in target cells.
Exam~le 7 EXQ5~OXIN A FUSION PROTEIN ~PEx56) The DNA sequences surrounding AA96 of T4 permit the initiation of translation o~ a second, shortened version of a soluble T4-Pseudomonas Exotoxin A fusion protein without the binding domain of PE. This shortened fusion protein interferes with the purification of the desired soluble T4-Pseudomonas Exotoxin A fusion protein in which the entire binding domain of PE is removed.
To avoid such interference, we prepared another soluble T4-Pseudomonas Exotoxin A fusion protein in which the entire the binding region of PE
was removed (See Figure 23). Specifically, we mutagenized plasmid pEX46 (see Figure 9A) with oligonucleotide T4-176 to form plasmid pEX56.
Oligonucleotide T4-176 has the following bp sequence:
GGA GGA CCA GAA AGA AGA AGT TCA GCT GCT GGT TTT CGG ATT
GAC T. In the resulting plasmid pEX56, the initiation of protein translation does not occur at AA96 of the T4 portion of this fusion protein. The amino acid sequence of PEx56 is identical to that of PEx46 ~see Figure 12).

~00~9~9 Example 8 ~XPRESSION OF PEx45. PEx46 and PEx56 We expressed each of PEx45, PEx46, and PEx56 in E. coli and achieved induction of the lambda pL
promoter by temperature shift. In each case, we found the expression level of the protein of interest to be approximately 15-20% of total cell protein as d~termined by densitometric scanning of a protein gel.
Renaturation and Immunoaffinity ~urification of PEx45. PEx46 and PEx56 We suspended 10 g of E. coli cell pellet in 40 ml of 50 mM Tris-HCl, ad~usted to about pH 8.0, containing 2 mM EDTA at 0C. We then ruptured the suspended cells in a French press and pelleted the inclusion bodies at 10,000g for a period of about 30 min. at about 4C. We washed the pellet by resuspending it into 50 ml of 50 mM Tris-HCl, adjusted to about pH 8.0, containing 2 mM EDTA and the inclusion bodies were ~ubsequently repelleted at 10,000g ~or a -20 period of about 30 min. at about 4C. ;
We next dissolved the washed pellet in 50 mM
Tris, 7 M guanidine-HCl, 2 mM EDTA, 10 mM DTT, ad~usted to about pH 8.0 with HCl at about 4C. After about 1 -hour of gentle agitation at about 4C, we clarified the ;;
solution by centrifugation at 10,000g for a period of about 40 min. at about 4C. We then renatured the fusion protein by dropwise addition, at the rate of 1 ml/min, of the fusion protein containing sample into a well stirred buffer of 50 mM Tris-HCl, adjusted to about pH 8.0, having a dilution ratio o~ 1:100. We allowed renaturation to continue for a period of about 24 hours at about 4C under rapid stirring. This was performed in an open container, or alternatively, in a 200098~

vessel under the positive influence of air or oxygen to facilitate oxidation.
We clarified the solution by filtering it through a 5 ~m and 0.2 ~m low protein binding filter (Millpore, Inc., Massachusetts). We then pumped the solution at a rate of about 10 ml/hr through a 10 ml immunoa~finity column at about 4C. As the stationary phase o~ the column, we used a monoclonal antibody 6C6 ~a gi~t of Drs. Blake Pepinsky and Werner Meier, Biogen, Inc., Cambridge, Massachusett6) coupled to BrCN
activated Sepharose 4B (Sigma, Inc., St. Louis, Missouri). This antibody is known to interfere with gpl20/160 and OXT4 binding.
After loading the column with the protein 15 containing ~ample, we washed the column with about 10 ;~
times the column volume with 50 mM Tris-HCl, pH 8Ø
We eluted antibody bound proteins with 50 mM Tris, 3M
K8CN, ad~usted to pH 8Ø Immediately after elution, we exchanged the buffer to Dulbecco's PBS, without Ca"
and Mg", by repeated dialysis with stirring at about 4C.
We verified the structure of the purified fusion proteins by one or more of the following identi~ication methods: N-terminal sequence analysis, ~-amino acid analysis, and bloactivity.
Without optimizing our purification condi-tions, we recovered at least about 0.5 - 1.0 mg/g E.coli of the fusion proteins PEx45, PEx46, and PEx56.
We measured the purity o~ each of these fusion proteins to be > 90% by conventional SDS-PAGE (12% polyacryl-amide gel under reducing and non-reducing conditions) and HPLC size-exclusion chromotography (TSK 2000 SW, Toya Soda Corp., Japan).
We determined each of the fusion proteins, PEx45, PEx46, and PEx56, to have a monomeric physical form by using non-reducing SDS-PAGE and HPLC size-exclusion chromotography in phosphate buffer, adjusted to about pH 7Ø
Exam~le 9 IN VITRO ACTIVITY OF TH~
IM~UNOTOXINS OF ~HIS INVENTION
The activities of PEx27, PEx44, PEx45, PEx46 and PEx56 immunotoxins were assessed ln vitro by measuring gpl20 blnding activity by direct competition with rsT4.3 in a solid phase receptor binding asisay.
The toxicity of PEx45 and PEx46 was then determined by an ~ vitro ADP ribosiylation assay using elongation factor 2 as a substrate.
Wlthout being bound by theory, we believe that the rsT4-PE immunotoxin fuision is taken up by endocytosis. Once in the endosome or ly~osome, the fusion protein i8 then expo~ed to low pH conditions thereby induclng a conformational change in its translocation domain. This structural change of the translocation domain iis likely to be the causie of the permeation of the fusion protein or portions thereof into the cytoplasm. Finally, the cell inactivating domain of the fusion protein performs the ADP
ribosylation of elongation factor 2 which results in the blocking of protein translation and eventuating the death of the invaded cell. Only cells béaring gpl20/160 on their surface should be affected by this immunotoxin cell incorporation process. ;
apl20 Bindina Activity ;
We coated Immulon II ELISA plates (Dynatec) with purified recombinant gpl20 by incubating each well with a solution of 50 ~1 of gpl20 (OD280 = 0.005).
After we removed the gpl20 by washing the plate with nulbecco's PBS, without Ca2 and Mg~-, we incubated the ~ ,; .. . . . - . . ~ : -.. ... .. , . :

X001~9~9 gpl20-coated plate with samples (25 ~l/well) of fusion proteins PEx27, PEx44, PEx45, PEx46 or PEx56 at dilutions ranging from about 1000 ~M to 0.5 ~M. We diluted the constructs in Dulbecco's PBS in the presence of milk proteins in order to block non-~pecific binding. The negative control used was rsT4.3, the soluble T4 polypeptide expression product of pBG381 (IVI 10149). After a period of at least about 1 hour, we added horseradish peroxidase - - ;
conjugated to rsT4.3 (HRP-rsT4) at a concentration of about 60 ng/ml (25 ~l/well) as a competitor.
We continued incubation for at least about 2 hours. After we had washed the plates with Dulbecco's PBS, without Ca2+ and Mg~, containing about 0.05% Tween 20 (Sigma, Inc., St. Louis, Missouri), we detected the gpl20-HRP-rST4 complex by reacting 0-phenylenediamine (OPD, Sigma, Inc., St. Louis, Missouri) and H202 with the horseradish peroxidase substrate and then scanned -~
the ELISA plate at 490 nm (as depicted in Figure 25).
Fusion proteins containing 124 a~ino acids of T4 (PEx27 and PEx44) demonstrated an affinity to gpl20, while the proteins comprising 181 amino acids of T4 (PEx45 and PEx46) demonstrated a higher binding affinity to gpl20.
The ribosylation activity was determined to be about the same for PE40 (PEx44 and PEx46) and PE43 (PEx27 and PEx45) containing proteins.
ADP Ribosylation of EF-2 The toxicity of Pseudomonas Exotoxin A is based on its enzymatic activity which carries out ADP
ribosylation of elongation factor 2 (EF-2) thereby inhibiting protein translation of a target cell. This ADP ribosylation activity may be assayed by measuring the transfer of radioactively labeled substrate ( 14C- . ' NAD-) to EF-2. The fusion protein becomes bound with unpurified EF-2 on one domain and onto another domain with "C and NAD- which bridges the substrates together to yield ADP on EF-2. As a positive control in this assay, we used PE [List Laboratories, California];
rsT4.3 served as a negative control. We also used a rabbit reticulocyte lysate as the source for EF-2.
AB illustrated in Figure 14, we observed no significant difference in the ADP ribosylation activity of the PE40 and PE43 containing proteins (PEx45 and PEx46). Moreover, the activity did not change when each o~ the PE43 containing fusion proteins were reduced prior to the assay (see for example PEx46 in Pigure 14). This indicates that the status of the ~usion protein's disulfide bridges, located in the translocation domain and in the gpl20 binding domain, may not exert any influence on the ADP ribosylation actlvity.
Exam~l~ 10 ADDITIONAL ASSAYS OF THE ACTIVITIES
OF THE IMMUNOTOXINS OF THIS INVENTION
According to the design of the rsT4-PE fusion protein, the fusion protein should bind to cells which -express gpl20/160 on the surface of HIV infected cells.
The bound fusion protein should be taken up by endocytosis as with PE. Once in the endosome or lysosome, the fusion~protein is then exposed to low pH
which should induce a conformational change in the translocation domain. Thus, presumably hydrophobic stretches are exposed to the surface thereby interacting with the membrane of the transport vesicle.
This should result in a translocation of the ADP
ribosylation domain of PE into the cytoplasm.
The enzymatically active domain of the fusion PE portion of the immunotoxins of this invention ;~0~09~g carries out the ADP ribosylation of EF-2, facilitating the blocking of protein translation. Eventually, cell death occurs. Cells which do not bear gpl20 on the surface should not be affected by the hybrid toxin.
We tested in cell assays the potency of immunotoxins, PEx45 and PEx46, to kill gpl20/160 bearing cells. In the first assay, we used gpl60 expressing Chinese Hamster Ovary (CHO) cells as target cells; as control cells, we used MIS expressing CHO
cells. In the second assay, we used HIV-infected H9 cells and uninfected H9 cells as controls. H9 specifies a CD4 positive human cell line. We used 3H-thymidine incorporation as a measure of viabllity.
We refer to this type of assay as a proliferation assay.
In a third assay, we used CHO9P160 cells as a target cell line and CHO~'' cells as a control cell line.
We determined the ef~ect of the toxin by plating out and counting the number of surviving cells after they had been treated with either PEx45 or PEx46. We refer to this type of cell assay as a plating efficiency assay. The design of the rsT4-PE immunotoxin should permit binding to cells which express gpl20/160 on the surface of the cell lines in the above-described cell assays.
Proliferation Assav We used a gpl60 expressing CHO cell line ;~
(CHo9~'~0) as target cells and MIS (Mullerian inhibiting ~ubstance) expressing CHO cell line (CHO~'s) served as control cells in a cell proliferation assay. We treated a defined number of cells with a sample of immunotoxin for a period of about 24 - 48 hours in ~-MEM, supplemented with GLN and 10% FBS. We then added . .
" ' "" :' ' ~ .~ , ,'.

zoioosss 3H-labelled thymidine to the medium and the cells were allowed to incubate for a period of about another 24 hours. We harvested the cells and then washed them on a standard glass-fiber filter paper. We then counted the cell-associated radioactivity and used the obtained value as a direct measure in determining the viability of the cells to incorporate labelled thymidine. As illustrated in Figure 15, the effects of Pseudomonas Exotoxin A (PE), PEx45, and PEx46 on CH0~'~ and CHO~'s cells after treatment for a period of about 48 hours is presented by plotting "% thymidine incorporation"
versus the concentration of the protein.
It is evident from Figure 15, that PE itself does not discrlminate between gpl20/160 expressing cells and MIS expre~sing cells. Both cell lines show a strongly reduced ability to incorporate 'H-thymidine.
PEx45 has similar cell killing potential to PE when it le used on expressing CH0 cells. This demonstrate6 that PEx45 actually binds to the cells, and is then ;-translocated into the cytoplasm thereby inactivating protein translation. Cells which are missing the T4 speci~ic receptor gpl60 (CHo~'5) provide the "therapeutic window" of this drug (illustrated by the light shadowed bar in Figure 10). As depicted in Figure 15, PEx46 shows less toxicity on CHOr'~'~ cells than PEx45. The nonspecific toxicity of this fusion protein is also reduced which results in approximately the same therapeutic window as for PEx45 (illustrated by the dark shadowed bar in Figure 10).

Z0{)0989 ~ ~:

These results are listed below in Table 1.
~able 1 TOXIN CONCENTRATIONS AT WHICH 50%
'H THYMIDINE INCORPORATION_IS OBSERVED*
~fter 48 h Treatment ~fter 24 h Tro~tment T4 PEÇHoDl CH0~ 5 c~O~D~ao CH~Is PEJ~45 181 43 1 x 10-~ 7 x 10'g 2 x 10'~
P~46 181 402 x 10~~ 5 x 10'~ 4 x 10'~ 1 x 10~~
2 x 10'~ 1 x 10 0 PEX56 181 40 4 X 10-l7 1 x 10-~
PE 1 x 10~" 2 x 10~"

3 x 10 ~ 4 x 10 ~ 2 x 10 ~ 3H-thymidine incorporation of untreated cells i9 100 The concentrations at which a 50% 3H-thymidine lncorporation i~ observed are compared in Table 1. As shown by Table 1, PEx45 shows a 10 fold higher potency than either PEx46 or PEx56 and thus resembles the potency profile of native PE. Both PEx45 and PEx46 show a difference between specific and nonspecific killing of about 5 x 10~ M. As illustrated in Table 1, when cells are treated for shorter time period~, the "therapeutic window" for PEx46 increased to at least about 2.5 x 103 M. This increase may be due to the inactivation of the toxin during the longer treatment, ~
25 re~ulting in a partial recovery of cell viability. ;
We demonstrated the necessity of the presence , of a freely accessible receptor (gpl20/160) on the surface of cells for the activity of the fusion proteins in a rsT4.3 blocking experiment. We treated ~;
CHOW'~ cells with PE, PEx45, or PEx46 in the absence or presence of rsT4.3 at a concentration of about 5.4 x 10-7 M. In the absence of rsT4.3, PE and each of the immunotoxins, PEx45 and PEx46, demonstrated similar killing abilitles (see Figure 15). As shown in ,. .: ' '~' :~

, ::

Z~009B~

Figure 10, rsT4.3 reduced the killing activity of PEx46 by blocking the receptor. The activity of PE was not influenced by the presence of rsT4. These results demonstrate that PEx45 and PEx46 enter the cells via binding to gpl20/160 on the cell surface.
HIV-infected H9 cells were similarly treated with PEx45 and PEx46. We used HIV strain IIIB.
Unin~ected H9 cells served as a control in this cell assay. H9 cells are human CD4 positive T-lymphocyte cell~. We have shown that these HIV-infected H9 cells express about 32,000 molecules of gpl20 on the cell surf2ce. We demonstrated this by using l25I-labelled anti-gpl20 antibody (DURDA, Dupont, Delaware).
Comparable results to those described above with the CHo90''0 cell line were obtained in this assay.
El~lng ~fficiencv Assay As depicted in Figure 24, a small number of di~persed cells, 1000 CHOgD'~ cells or 300 CHo~'5 cells/21 cm2 plate, were incubated for a period of about 12-24 hours and were treated with the protein sample ~or a period of about 24 hours. Cells were then dispersed and incubated in fresh medium for seven days. After ~ixing the cells with paraformaldehyde and staining with hematoxylin, the number of surviving cell colonies were counted. This assay may determine the "killing"
, ability of the toxin more directly than the proliferation assay. Preliminary data from this assay indicated that treatment of CHo90'60 cells with the rsT4-PE fusion protein results not only in a temporary suppression of 3H-thymidine incorporation, but also in cell death.

2000sas ~xample 11 FUSION PROTEINS (~A~Ç11 AND TANG12~
We cloned and characterized the DNA sequence encoding angiogenin. We then genetically fused angiogenin to a soluble T4-PE polypeptide to prepare additional immunotoxin fusion proteins according to this invention. To test for angiogenin 1 8 ability to ~unction as a toxin, we cut the ADP ribo3ylation domain o~ the PE portion ~ound in the soluble T4-PE fusion plasmid, pEX46, corresponding to AA494 of PE. We replaced the remaining portion of PE (AA495-AA613) with angiogenin, with and without its signal sequence.
As illustrated in Figures 17A and 17B, we screenQd a ~ EMBL3 library constructed from DNA of human mutant fibroblast line 49, XXXXX GM5009 (a gift of Dr. Mark Pa~ek, Biogen, Inc., Cambridge, Mao~achusetts) with an oligonucleotide probe, ANG-86, having the ~ollowing nucleotide sequence: pATA GTG CTG
GGT CAG GAA GTG TGT GTA CCT GGA GTT ATC. This sequence is based on the amino acid sequence of angiogenin illustrated in Figure 16. We chose one of several clones identified -- EMBL Angl -~ for further study.
Based upon the published sequence of angiog~nin lKurachi, et al, ~iQchemistrY, 24, pp. 5494-499 (1985)],~ we knew that that the structural gene for the protein is found on a 736 bp ~glII to EcoRV fragment. We cut EMBL Angl with ~g1II and EcoRV~ -to obtain the 736 bp fragment containing the angiogenin gene. We then ligated the ~g1II-EcoRV fragment into pHC79 [Boehringer Mannheim, Indianapolis, Indiana] ;
which, had been previously cut with ~lII and EÇ~RV and -;
screened with ANG-86. The resulting plasmid, pANG1, contained angiogenin in the BglII to EcoRV sites of pHC79.

200~989 In order to express the mature form of angiogenin in E.coli, we introduced a ClaI site immediately upstream of the coding sequence of mature angiogenin. To accomplish this, we mutagenized pANGl with the oligonucleotide ANGI0-1 to yield pANG3.
ANGI0-1 has the following nucleotide sequence: CTG GGT
CTG GGT GGA TCC AAA TCG ATT TGG ATG CAG CAG GAT AAC
TCC. We then cut the plasmid pANG3, having angiogenin flanked by ClaI sites, with ClaI. We then cloned the resulting Çl~I fragment into the ÇlgI site of the lambda pL expression vector pl97.12, which had been previously cut with ClaI and transformed E.coli A89.
We selected for tetracycline resistant colonies and screened with ANG-86. As a result, we isolated plasmid pANG4 which contained mature angiogenin under the lambda pL promoter. We then transformed pANG4 into the E.Coli strain A89. The resulting strain was induced and the angiogenin purified by conventional methods as described by P. Denefle et al., "Chemical Synthesis of a Gene Coding for Human Angiogenin, Its Expression in E~coli and Conversion of Its Product into Its Active Form", Gene, 56, pp. 61-70 (1987).
We next engineered the angiogenin gene such that it could be genetically fused to other proteins, such as soluble T4 protein and pseudomonas Exotoxin A.
We designed two different versions -- one with and one without the angiogenin signal sequence.
The plasmid pTANGll contains parts of three different proteins. It carries a soluble T4 polypeptide of the formula AA3-A~183 of Figure 2 linked to a portion of PE comprising the translocation domain and a portion of the ADP ribosylation domain (AA253-AA494) of Pseudomonas Exotoxin A, followed by the gene for mature angiogenin. The fusion protein TANGll comprises rsT4 (AAl-AA182), the translocation domain of "', '~'~"

200(~989 PE (AA184-AA300), the binding domain (Ib) of PE
(AA301-AA340), a linker from the ADP ribosylation domain of PE (AA341-AA429), and angiogenin (AA433-AA559) (as depicted in Figure 19~.
We obtained pTANGll as illustrated in Figure 17B. We mutagenized pANG1 with the oligonucleotide ANGI0-2 to yield pANG7 which has a ~glII site immediately upstream of the mature angiogenin sequence. ANGI0-2 has the following nucleotide sequence: GGT CTG GAT CTG ACC AAG ATC TTG
GGG GGG CCG GGG CAG GAT AAC TCC ACG. We then introduced a ~m~I site downstream of the angiogenin gene by cutting pANG7 with EcoRV and subsequently ligating in a ~m~I linker to yield pANG9. The plasmid pANG9 has mature angiogenin flanked by a ~g1II site upstream and a ~gmHI site downstream.
We also digested plasmid pEX46 (as prepared in Example 6) with ~m~I which cuts at AA494 of PE.
This site i5 within the ADP ribosylation domain of the PE gene and thus should remove the ADP ribosylation function of pEX46. We then digested pANG9 with Eg~
and ~mHI to yield a 600 bp fragment containing ~-angi~genin. We purified the fragment and ligated it into the ~mHI-digested pEX46, to produce pTANG11.
We obtained pTANG12, which contains angiogenin with its signal sequence, by introducing a BalII site immediately upstream of the intiating Met of the angiogenin signal peptide (see Figure 17B).
Specifically, we mutagenized pANG1 with oligonucleotide ANGI0-3 in order to produce pANG8. ANGI0-3 has a nucleotide sequence: CCG CAG GAG CCT GTG TAG ATC TTT
ATG GTG ATG GGC CTG. We then introduced a BamHI site ;
downstream of the angiogenin gene by cutting pANG8 with ~ ;
EcoRV and ligating in a BamHI linker to produce plasmid pANG10. We then digested pANG10 with BalII and BamHI
, .. :.

20~0989 to yield a 600 bp fragment containing angiogenin. This fragment was purified and then ligated into the ~m~I
digested pEX46, to produce pTANG12.
The plasmid pTANG12 contains angiogenin, complete with signal peptide fragment, substituted for the ADP ribosylation cell inactivating domain of PE.
We transformed the plasmids pTANGll and pTANG12 into E.coli strain A89. We can then renature and purify the protein after temperature shift induction of the promoter. The fusion protein TANG12 comprises rsT4 (AA1-AA182), the translocation domain of PE
-AA300)~ the binding domain ~Ib) of PE
~AA301-AA340), a linker from the cell inactivation ~ ~AA341 AA429), the angiogenin signal q (AA431 AA454), and angiogenin (AA455-AA ) (as depicted in Figure 18).
Ex~mple 12 E~QIEI~S (TANG13 AND TANG14~
In order to test ~or the ability of soluble T4-angiogenin fusion proteins to function as toxins without a portion of PE, we linked angiogenin directly to the C terminal end of a soluble T4 protein having he formula AA3-AA183 f Figure 2- As illustrated in Figure 20, we obtained plasmid pTANG13 by cutting pEX46 (as prepared in Example 6) with EcoRI and electroeluted the resulting expression vector. We also cut pEX46 with EcoRI and ~alII, and electroeluted the 615 bp fragment containing the rsT4 gene.
We next cut plasmid pANG9 (as prepared in ~ -Example 11) with EcoRI and BalII, and electroeluted the 740 bp fragment containing the angiogenin gene. We then linked that fragment with the expression vector fragment and the 615 bp T4 fragment previously isolated ~ ~

', , from pEX46. Following transformation into E.coli A89, we identified the pTANG13 plasmid so produced.
The plasmid pTANG13 comprises DNA encoding a soluble T4 polypeptids of the formula AA3-AA183 of Figure 2 linked to mature angiogenin. The protein may be purified and renatured after temperature shift induction of the promoter. It may then be tested for T4 binding, selective killing of gpl60 producing cells tas described in Examples 9 and 10), and RNase activity. ~he fusion protein TANG13 comprises rsT4 (AA1-AA182) and angiogenin (AA19l-AA3l4) (as depicted in Figure 21).
We also produced a soluble T4-angiogenin 4usion protein in which angiogenin carries its signal sequence. We digested the plasmid pANG10 (as described in Example 11) with EcoRI and ~glII, and electroeluted -~
the 800 bp angiogenin-containing fragment. We then ligated that fragment with an expression vector fragment and the 615 bp fragment previously isolated from pEX46 to produce plasmid pTANG14.
The plasmid pTANG14 comprises DNA encoding a soluble T4-polypeptide of the formula AA~-AA183 of Figure 2 linked to angiogenin and the angiogenin signal sequence. We transformed E~coli A89 with pTANG14. The resulting protein may be renatured and purified after temperature shi~t induction of the promoter. Fusion protein TANG14 compri'ses rsT4 (AA1-AA182), the angiogenin signal sequence (AA184-AA210), and g (AA211 AA334) (as depicted in Figure 22) TANG14 can then be tested for its ability to selectively kill gpl60 cells.
Microorganisms and recombinant DNA molecules prepared by the processes of this invention are exem-plified by cultures deposited in the In Vitro ~

,. "
: .
: ' zooo9~9 ~:

International, Inc. culture collection, in Linthicum, Maryland, on September 29, 1988, and identified as:
DH5 : E.coli DH5 - IVI 10186 GM2929 : E.coli GM2929 - IVI 10187 5 HBlOlIQ : E.coli HBlOlIQ - IVI 10188 211.11 : E.coli DH5/p211.11 - IVI 10190 199.7 : ~coli DH5/pl99.7 - IVI 10191 197.12 : E.coli DH5/pl97.12 - IVI 10192 BG391 : E.coli DH5/pBG391 - IVI 10193 SA307 : E.coli DH5/pSA307 - IVI 10194 SA313 : E.coli DH5/pSA313 - IVI 10195 SA400 : E.coli DH5/pSA400 - IVI 10196 EX27 : E.coli DH5/pEX27 - IVI 10197 EX39 : ~coli DH5/pEX39 - IVI 10198.
Such microorganisms and recombinant DNA
molecule~ are exemplified by culture~ deposited in the ~ ;
In Vitro International, Inc. culture collection on October 16, 1989 and identified as:
EX45 : E.coli DH5/pEX45 - IVI 10208 EX46 : E.coli DH5/pEX46 - IVI 10209 EX56 : E.coli DH5/pEX56 - IVI 10210 ANG4 : E~coli DH5/pANG4 - IVI 10211 ANG9 : E.coli DH5/pANG9 - IVI 10212 ANG10 : E.coli DH5/pANG10 - IVI 10213 TANG11 : E~coli DH5/pTANG11 - IVI 10214 A89 : E.coli A89 - IVI 10215.

While we have hereinbefore described a number of embodiments of this invention, it is apparent that our basic constructions can be altered to provide other ~;;
embodiments which utilize the processes and compositions of this invention. Therefore, it will be appreciated that the scope of this invention includes ~ -all alternative embodiments and variations which are defined in the foregoing specification and by the ~,:
:' 20009~39 claims appended hereto; and the invention is not to be limited by the specific embodiments which have been presented herein by way of example.

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Claims (52)

1. An immunotoxin comprising a soluble T4 protein conjugated with a toxin.

2. The immunotoxin according to claim 1, wherein the toxin is selected from the group consisting of ricin, abrin, angiogenin, Pseudomonas Exotoxin A, pokeweed antiviral protein, saporin, gelonin and diptheria toxin, and toxic portions thereof.

3. The immunotoxin according to claim 1, wherein the toxin is Pseudomonas Exotoxin A or a toxic portion thereof.

4. The immunotoxin according to claim 1, wherein the toxin comprises the translocation domain and the ADP ribosylation cell inactivating domain of Pseudomonas Exotoxin A.

5. The immunotoxin according to claim 4, wherein the toxin further comprises the boundary region of Pseudomonas Exotoxin A.

6. The immunotoxin according to claim 1, wherein the toxin is angiogenin or a portion thereof.

7. The immunotoxin according to claim 6, further comprising the translocation domain and the ADP
ribosylation cell inactivating domain of Pseudomonas Exotoxin A.

8. The immunotoxin according to claim 1, wherein the soluble T4 protein is selected from the group consisting of a polypeptide of the formula AA-23-AA362 of Figure 2, a polypeptide of the formula AA1-AA362 of Figure 2, a polypeptide of the formula Met-AA1-AA362 of Figure 2, a polypeptide of the formula AA
23-AA374 of Figure 2, a polypeptide of the formula AA1-AA374 of Figure 2, a polypeptide of the formula Met-AA1-374 of Figure 2, a polypeptide of the formula AA1-AA377 of Figure 2, a polypeptide of the formula Met-AA1-377 of Figure 2, a polypeptide of the formula AA-23-AA377 of Figure 2, or portions thereof.

9. The immunotoxin according to claim 1, wherein the soluble T4 protein is selected from the group consisting of a polypeptide of the formula AA-23-AA182 of Figure 2, a polypeptide of the formula Met-AA1-AA182 of Figure 2, a polypeptide of the formula AA1- AA182 of Flgure 2, a polypeptide of the formula AA3-AA183 of Figure 2, a polypeptide of the formula AA-23-AA182 of Figure 2, followed by the amino acids asparagine-leucine-glutamine-histidine-serine-leucine, a polypeptide of the formula AA1- AA182 of Figure 2, followed by the amino acids asparagine-leucine-glutamine-histidine-serine-leucine, a polypeptide of the formula Met-AA1-182 of Figure 2, followed by the amino acids asparagine-leucine-glutamine-histidine-serine-leucine, a polypeptide of the formula AA-23-AA111 of Figure 2, a polypeptide of the formula AA1-AA111 of Figure 2, a polypeptide of the formula Met-AA1-AA111 of Figure 2, a polypeptide of the formula M
23-AA113 of Figure 2, a polypeptide of the formula AA1-AA113 of Figure 2, a polypeptide of the formula Met-AA1-113 of Figure 2, a polypeptide of the formula AA-23-AA131 of Figure 2, a polypeptide of the formula AA1-AA131 of Figure 2, a polypeptide of the formula Met-AA1-l31 of Figure 2, a polypeptide of formula AA-23-AA145 of Figure 2, a polypeptide of the formula AA1-AA145 of Figure 2, a polypeptide of the formula Met-AA1-AA145 of Figure 2, a polypeptide of the formula AA-23-AA166 of Figure 2, a polypeptide of the formula AA1-AA166 of Figure 2, a polypeptide of the formula Met-AA1-AA166 of Figure 2, or portions thereof.

10. The immunotoxin according to claim 1, wherein the soluble T4 protein is selected from the group consisting of a polypeptide of the formula AA1-AA362 of mature T4 protein, a polypeptide of the formula Met-AA1-AA362 of mature T4 protein, a polypep tide of the formula AA1-AA374 of mature T4 protein, a polypeptide of the formula Met-AA1-AA374 of mature T4 protein, a polypeptide of the formula AA1-AA377 of mature T4 protein, a polypeptide of the formula Met-AA1-AA377 of mature T4 protein, a polypeptide of the formula AA-23-AA374 of mature T4 protein, a polypeptide of the formula AA-23-AA377 of mature T4 protein, or portions thereof.

11. The immunotoxin according to claim 1, wherein the soluble T4 protein is selected from the group consieting of a polypeptide of the formula AA-23-AA182 of mature T4 protein, a polypeptide of the formula AA3-AA1830f mature T4 protein, a polypeptide of the formula AA1-AA182 of mature T4 protein, a polypeptide of the formula Met-AA1-AA182 protein, a polypeptide of the formula AA 23-AA182 of mature T4 protein, followed by the amino acids asparagine-leucine-glutamine-histidine-serine-leucine, a polypeptide of the formula AA1-AA182 of mature T4 protein, followed by the amino acids asparagine-leucine-glutamine-histidine-serine-leucine, a polypeptide of the formula Met-AA1-AA182 of mature T4 protein, followed by the amino acids asparagine-leucine-glutamine-histidine-serine-leucine, a polypeptide of the formula AA-23-AA113 of mature T4 protein, a polypeptide of the formula AA1-AA113 of mature T4 protein, a polypeptide of the formula Met-AA1-AA113 of mature T4 protein, a polypeptide of the formula AA-23-AA111 of mature T4 protein, a polypeptide of the formula AA1-AA111 of mature T4 protein, a polypeptide of the formula Met-AA1-AA111 of mature T4 protein, a polypeptide of the formula AA23-AA131 of mature T4 protein, a poly-peptide of the formula AA1-AA131 of mature T4 protein, a polypeptide of the formula Met-AA1-AA131 of mature T4 protein, a polypeptide of the formula AA-23-AA145 of mature T4 protein, a polypeptide of the formula AA1-AA145 of mature T4 protein, a polypeptide of the formula Met-AA1-AA145 of mature T4 protein, a polypep-tide of the formula AA-23-AA166 of mature T4 protein, a polypeptide of the formula AA1-AA166 of mature T4 protein, a polypeptide of the formula Met-AA1-AA166 of mature T4 protein, or portions thereof.

12. A hybrid DNA sequence coding for an immunotoxin, said DNA sequence comprising a first DNA
sequence coding on expression for a soluble T4 protein and a second DNA sequence coding on expression for a toxin.

13. The hybrid DNA sequence according to claim 12, wherein said second DNA sequence codes on expression for a toxin selected from the group con-sisting of ricin, abrin, angiogenin, Pseudomonas Exotoxin A, pokeweed antiviral protein, saporin, gelonin and diptheria toxin, and toxic portions thereof.

14. The hybrid DNA sequence according to claim 12, wherein the first DNA sequence is selected from the group consisting of:
(a) the DNA inserts of p199.7, pBG391 and p211.11;
(b) DNA sequences which hybridize to one or more of the foregoing DNA inserts and which code on expression for a soluble T4-like polypeptide; and (c) DNA sequences which code on expression for a soluble T4-like polypeptide coded for on expression by any of the foregoing DNA inserts and sequences.

15. The hybrid DNA sequence according to claim 13, wherein said second DNA sequence codes on expression for Pseudomonas Exotoxin A or a toxic portion thereof.

16. The hybrid DNA sequence according to claim 15, wherein said second DNA sequence codes on expression for the translocation domain of Pseudomonas Exotoxin A.

17. The hybrid DNA sequence according to claim 15, wherein said second DNA sequence codes on expression for the translocation domain and the ADP
ribosylation cell inactivating domain of Pseudomonas Exotoxin A.

18. The hybrid DNA sequence according to claim 16, wherein said second DNA sequence codes on expression for the translocation domain of Pseudomonas Exotoxin A, the ADP ribosylation cell inactivating domain of Pseudomonas Exotoxin A and the boundary region of Pseudomonas Exotoxin A.

19. The hybrid DNA sequence according to claim 12, wherein said first DNA sequence codes on expression for a soluble T4 polypeptide of the formula AA3-AA183 of Figure 2 and said second DNA sequence codes on expression for the translocation domain of Pseudomonas Exotoxin A.

20. The hybrid DNA sequence according to claim 12, wherein said first DNA sequence codes on expression for a soluble T4 polypeptide of the formula AA3-AA183 of Figure 2 and said second DNA sequence codes on expression for the translocation domain of Pseudomonas Exotoxin A, and the ADP ribosylation cell inactivating domain of Pseudomonas Exotoxin A.

21. The hybrid DNA sequence according to claim 12, wherein said first DNA sequence codes on expression for a soluble T4 polypeptide of the formula AA3-AA183 of Figure 2 and said second DNA sequence codes on expression for the translocation domain of Pseudomonas Exotoxin A, the ADP ribosylation cell inactivating domain of Pseudomonas Exotoxin A and the boundary region of Pseudomonas Exotoxin A.

22. The hybrid DNA sequence according to claim 12, wherein said first DNA sequence codes on expression for a soluble T4 polypeptide of the formula AA3-AA183 of Figure 2 and said second DNA sequence codes on expression for angiogenin.

23. The hybrid DNA sequence according to claim 21, said second DNA sequence further comprising a third DNA sequence which codes on expression for the translocation domain of Pseudomonas Exotoxin A.

24. A recombinant DNA molecule comprising a hybrid DNA sequence selected from the group consisting of the hybrid DNA sequences of any one of claims 12-23, wherein said DNA sequence is operatively linked to an expression control sequence in the recombinant DNA
molecule.

25. The recombinant DNA molecule according to claim 24, said molecule being selected from the group consisting of p218, pEX11, pEX27, pEX44, pEX45, pEX46, pEX56, pTANG11, pTANG12, pTANG13 and pTANG14.

26. An immunotoxin coded for on expression by a recombinant DNA molecule according to claim 24.

27. A method for producing an immunotoxin comprising the step of culturing a host transformed with a recombinant DNA molecule according to claim 24.

28. A host transformed with a recombinant DNA molecule according to claim 24.

29. The host according to claim 28, said host being selected from the group consisting of strains of E.coli, Pseudomonas, Bacillus, Streptomyces, fungi, such as yeasts, and animal cells, such as CHO
and mouse cells, African green monkey cells, such as COS1, COS7, BSC1, BSC40, and BMT10, insect cells, and human cells and plant cells in tissue culture.

30. The immunotoxin according to claim 1, wherein the soluble T4 protein is chemically fused to the toxin.

31. The immunotoxin according to claim 30, wherein the soluble T4 protein is a polypeptide of the formula AA1-AA124 of Figure 3 and the toxin is ricin A.

32. The immunotoxin according to claim 1, wherein the soluble T4 protein is genetically fused to the toxin.

33. The immunotoxin according to claim 26, wherein the soluble T4 protein is a full length soluble T4 protein, and the toxin is Pseudomonas Exotoxin A, or a toxic portion thereof.

34. The immunotoxin according to claim 26, wherein the soluble T4 protein is a soluble T4 polypeptide of the formula AA3-AA183 of Figure 2 and the toxin is Pseudomonas Exotoxin A, or a toxic portion thereof.

35. A hybrid DNA sequence coding on expression for an immunotoxin, said DNA sequence comprising a first DNA sequence coding for a streptavidin-like polypeptide, a second DNA sequence coding on expression for a soluble T4 protein, and a third DNA sequence coding on expression for a toxin.

36. The hybrid DNA sequence according to claim 35, wherein the first DNA sequence is selected from the group consisting of:
(a) pSA307;

(b) DNA sequences encoding polypep-tides which hybridize to the foregoing DNA sequence, and which code on expression for the polypeptide; and (c) DNA sequences which code on expression for a polypeptide coded for on expression of any of the foregoing DNA sequences.

37. The hybrid DNA sequence according to claim 35, said DNA sequence comprising a sufficient portion of a signal DNA sequence to cause, upon expression of the DNA sequence, secretion of the immunotoxin coded for by the hybrid DNA sequence across the cell membrane of a host transformed with said hybrid DNA sequence.

38. The hybrid DNA sequence according to claim 35, wherein said third DNA sequence codes on expression for a toxin selected from the group con-sisting of ricin, abrin, angiogenin, Pseudomonas Exotoxin A, pokeweed antiviral protein, saporin, gelonin and diptheria toxin, and toxic portions thereof.

39. The hybrid DNA sequence according to claim 35, wherein said second DNA sequence is selected from the group consisting of:
(a) the DNA inserts of p199.7, pBG391 and p211.11;
(b) DNA sequences which hybridize to one or more of the foregoing DNA inserts and which code on expression for a soluble T4-like polypeptide; and (e) DNA sequences which code on expression for a soluble T4-like polypeptide coded for on expression by any of the foregoing DNA inserts and sequences.

40. The hybrid DNA sequence according to claim 38, wherein said third DNA sequence codes on expression for Pseudomonas Exotoxin A or a toxic portion thereof.

41. The hybrid DNA sequence according to claim 40, wherein said third DNA sequence codes on expression for the translocation domain and the ADP
ribosylation cell inactivating domain of Pseudomonas Exotoxin A.

42. The hybrid DNA sequence according to claim 41, wherein said third DNA sequence codes on expression for the translocation domain, the ADP
ribosylation cell inactivating domain, and the boundary region of Pseudomonas Exotoxin A.

43. The hybrid DNA sequence according to claim 38, wherein said third DNA sequence codes on expression for angiogenin, or a toxic portion thereof.

44. The hybrid DNA sequence according to claim 43, wherein said third DNA sequence further comprises a fourth DNA sequence which codes on expression for the translocation domain of Pseudomonas Exotoxin A.

45. A recombinant DNA molecule comprising a hybrid DNA sequence selected from the group consisting of the hybrid DNA sequences of any one of claims 36-44, wherein the hybrid DNA sequence is operatively linked to an expression control sequence in the recombinant DNA molecule.

46. The recombinant DNA molecule according to claim 45, said molecule being pEX39.

47. An immunotoxin coded for on expression by a recombinant DNA molecule according to claim 45.

48. A host transformed with a recombinant DNA molecule according to claim 45.

49. The host according to claim 46, said host being selected from the group consisting of strains of E.coli, Pseudomonas, Bacillus, Streptomyces, fungi, such as yeasts, and animal cells, such as CHO
and mouse cells, African green monkey cells, such as COS1, COS7, BSC1, BSC40, and BMT10, insect cells, and human cells and plant cells in tissue culture.

50. A method for producing an immunotoxin comprising the step of culturing a host transformed with a recombinant DNA molecule according to claim 24 or 45.

51. A pharmaceutically acceptable compo-sition comprising an immunotherapeutic or immuno-suppressive amount of an immunotoxin according to any one of claims 1, 30 or 47, and a pharmaceutically acceptable carrier.

52. A method for treating or preventing AIDS, ARC or HIV infection comprising the steps of administering to a patient, in a pharmaceutically acceptable manner, a pharmaceutical composition according to claim 51.