JP2003508016A - Nucleic acids encoding CD40 / CD40L chimeric polypeptides, methods for their production and uses thereof - Google Patents

Abstract

(57) Summary i) Mammalian signal peptide and its downstream, ii) CD40L binding and trimerization domain and its downstream, iii) 50-100 amino acid spacer and its downstream, iv) CD40 transmembrane and Nucleic acids encoding chimeric polypeptides comprising nucleic acid fragments encoding a signal transduction domain are useful as gene therapy for the local treatment of solid tumors.

Description

Detailed Description of the Invention

The present invention relates to nucleic acids encoding CD40 / CD40L chimeric polypeptides, methods for their production, pharmaceutical compositions containing said nucleic acids and their use.

[0002] The CD40 receptor (hereinafter also referred to as CD40) is a cell surface receptor from the family of TNF receptors that was first identified on B lymphocytes and is involved in the regulation of proliferation and differentiation. Was functionally characterized. However,
It includes other cell types such as T cells (activation → proliferation stimulation, cytokine secretion), dendritic cells, and monocytes (activation → costimulatory molecule expression, inflammatory cytokines such as TN).
F-α, IL-8, IL-12 secretion) and carcinoma (activation leads to growth inhibition) (Van Kooten et al., Int. Arch. Allergy Immunol. 113 (1997) 393-3).
99) can also be found above. It is a member of the type I membrane proteins, implying an extracellular N-terminus, a transmembrane domain, and an intracellular C-terminus (Laman
et al., Critical Reviews in Immunology 16 (1996) 59-108).

CD40L (= CD154 or gp39) is a ligand for CD40 (WO
93/08207). It tends to be expressed on activated CD4 + T cells, but is also found on activated B cells and dendritic cells. CD40L and CD40
The interaction between and plays a central role in the development of both humoral and cellular immunity. Binding of CD40L to CD40 is a CD40-mediated signal (AP
C (A ntigen P resenting C ell ) to start both the T cell stimulation signals via the (antigen presenting cells) activation) and CD40L itself (T cell activation). CD40L belongs to the type II membrane protein group, which is intracellular N
Mean end, transmembrane region and extracellular C-terminus (Laman et al., Critical R
eviews in Immunology 16 (1996) 59-108). So far, the soluble form of CD40L has also been discussed, meaning that they consist only of the C-terminal extracellular domain and are capable of activating CD40. Thus, the extracellular domain is sufficient for trimer formation, which in turn binds to the extracellular domain of the receptor CD40, and thereby trimerizes and activates this receptor. Qualitative differences are still discussed in the literature regarding mediating signals by soluble and membrane-bound CD40L (Laman et al., Critical Revie.
ws in Immunology 16 (1996) 59-108).

Gires et al. (EMBO J. 16 (1997) 6131-6140) reported that the EBV viral protein LM
We have shown that the P1 and CD40 fusion construct is functionally active in constitutive CD40 signaling.

Hatzivassiliou et al. (J. Immunol. 160 (1998) 1116-1121) reported that LMP-1 and C
Fusion with the cytoplasmic domain of D40 induces epidermal growth factor receptor, NF-κB and LM
It has been described to activate stress activated protein kinases such as P-1.

WO98 / 26061 discloses a cDNA encoding a chimeric protein containing at least part of the mouse CD40L gene together with the same or another CD40L ligand gene from either mouse, human or other species, A method of treating human neoplasia comprising inserting into tumor cells is described. Particularly preferred is a chimeric gene consisting of the transmembrane and cytoplasmic domain of the mouse CD40L gene linked to the extracellular domain of the human CD40L gene.

[0007] It is an object of the present invention to provide novel nucleic acids that are particularly useful in gene therapy and especially for ex vivo treatment of tumor cells and local treatment of solid tumors.

[0008] In accordance Summary of the Invention The present invention, in order to be active, without the need for extraneous components, or strongly requires only extraneous components reduced levels, to generate a constitutively active CD40 It is possible. The present invention is "soluble," that is, CD4 covalently bound to CD40.
A chimera consisting of 0 L of the extracellular domain that is not membrane bound is provided. It comprises the following components: -a signal peptide sequence, preferably that of CD40 or another mammalian signal sequence; -the extracellular domain of CD40L or a part thereof which results in binding to CD40 and trimerization; -a spacer sequence. An extracellular domain of CD40 or a portion thereof that binds to CD40L (optional); a transmembrane domain of CD40; a cytoplasmic domain of CD40 that causes signal transduction.

The invention comprises: i) a mammalian signal peptide and its downstream; ii) a binding and trimerization domain of CD40L and their downstream; iii) a spacer of 50-100 amino acids; iv) transmembrane and signal of CD40. A transduction domain comprising a nucleic acid fragment encoding a chimeric polypeptide, comprising a nucleic acid fragment encoding the transduction domain.

Fragments or modifications of these domains that have substantially the same activity in binding, trimerization and signal transduction are also useful according to the invention. Such modifications are, for example, the exchange of amino acids by other amino acids that do not affect the conformation of the protein. Moreover, such nucleic acids can be used to encode a polypeptide encoded by any of the nucleic acids mentioned above, due to the degeneracy of the genetic code. Particularly preferred are nucleic acids using human codon usage. The signal peptide is preferably the CD40 signal peptide or a fragment thereof.

In a preferred embodiment of the invention (see FIGS. 1 and 2, type A), the encoded polypeptide comprises a signal peptide, a CD40L binding and trimerization domain, no linker or about 1-30. It consists of a short amino acid linker, a ligand binding subdomain of CD40, a spacer of 50-100 amino acids, a transmembrane domain and a signaling domain of CD40. Type A can trimerize without the help of a second cell. The design of the A version of the construct should allow for trimerization without steric hindrance. This means that the CD40L binding region must be well away from the outside of the cell surface. In this case, a spacer, preferably a stalk subdomain of CD40 or CD40L, a chimeric stalk domain consisting of a part of the stalk domain and another amino acid or another polypeptide acting as a spacer, having the lengths mentioned above, is a CD40
It is preferably inserted between the L-binding subdomain and the transmembrane domain of CD40. More preferably, at least 80% of the amino acids are Glu and / or Al.
a spacer composed of a (a linear and movable spacer, Glu / Ala)
Spacer). The CD40 portion of this construct is only required for CD40L binding and signaling. Therefore, other parts of the extracellular domain of CD40 can be deleted. The CD40L portion of this construct is required for binding and trimerization. However, the binding domains of CD40 and CD40L must be guaranteed to be able to bind and trimerize. In this case the linker between both binding domains must be of sufficient length. Therefore, a length of about 8-30 amino acids, preferably 15-30 amino acids is preferred.

In a second preferred embodiment of the invention (see plate B of FIGS. 1 and 3),
After the signal peptide is soluble, ie the CD40L binding and trimerization domain is covalently linked to the transmembrane region of the CD40 receptor, ie the cells of the CD40 receptor required for CD40L binding. The outer region (or at least a portion of the extracellular region that causes binding to CD40L) is deleted. CD4
Between the extracellular domain of 0L and the transmembrane domain of CD40, a spacer approximately 50-100 amino acids in length, such as additional flexibility such as 15-30 amino acids from the stalk domain of CD40 and the hinge region of IgD. It is necessary to introduce the polypeptide. Such spacers are, for example, CD40, as in version A.
Or a stalk domain of CD40L (about 80 amino acids), a part of said stalk domain acting as a spacer and another amino acid or another polypeptide having the above-mentioned length ensuring steric hindrance-free trimer formation. May be a chimeric stalk domain. Here, the Glu / Ala spacer is also preferable.
This aspect offers the advantage that the covalently bound extracellular CD40L domain is trimerized spontaneously and, consequently, causes the covalently bound cytoplasmic CD40 receptor to trimerize and activate it. Another advantage of this embodiment is that the CD40 ligand trimer is still present in unbound form and, as a result, interacts with other natural CD40 receptors on different cells, preserving it. Is that it is capable of undergoing monomer formation and thus activation as well, ie this embodiment not only acts directly on cells expressing the chimeric receptor but also expresses the native CD40 receptor. That is, it may have an activating effect of trans on another cell.

According to the invention, in a modified manner, it induces the presentation of tumor-specific antigens on immunocompetent cells and differentiates and proliferates such immunocompetent cells, such as T cells, macrophages and dendritic cells. Can be induced. The chimeric CD40 / CD40L polypeptide can be labeled with a professional AP, just as CD40L binds to CD40.
It exhibits "trans" activity upon activation of C, ie it is MHC-I and / or
Or MHC-II, costimulatory molecules such as B-7.1, B-7.2, immunostimulatory cytokines such as IL-12, IL-8, and proinflammatory cytokines such as IL-1α, IL-1β. And / or JNK / AP-1, JAK / STAT and I-KB / NF leading to upregulation of the gene encoding IL-6.
Transposes signals through the κB pathway. It also works in "cis",
This means that it acts directly on tumor cells by inducing growth inhibition (Eliopoulos, AG, Oncogene 13 (1996) 2243-2254; van Kooften et al.
, Int. Arch. Allergy Immunol. 113 (1997) 393-399; Young, LS, et al.,
Immunology Today 19 (1998) 502-506).

Multiple proteolytic cleavage sites have been described and indeed are found in human and mouse CD40L (Gauchat, J.-F. et al., FEBS 315.
(1993) 259-266; Armitage, RJ et al., Nature 357 (1992) 80-82; Hsu,
Y.-M. et al., The J. of Biol. Chem. 272 (1997) 911-915; Kato, K. et al.
, J. Clin. Invest. 104 (1999) 947-955).

However, the fusion protein of CD40L and CD40 according to the present invention lacks typical protease cleavage sites. The cleavage site described for CD40L is present in the region of the CD40L molecule that is deleted in the fusion construct. Thus, the CD40 / CD40L chimeric molecule is largely unaffected by proteolytic cleavage, and thereby, less affected by down-regulation of signal transduction abilities by proteolytic cleavage.

In a further preferred embodiment of the invention the nucleic acid is a cytokine such as IL-2,
IL-12, lymphotactin (Dilloo et al., Nature Medicine 2 (1996) 1090
, And Entage, Hum. Gene Therapy 10 (1999) 697-709) and / or a further gene fragment encoding interferon-α.

The invention further comprises a recombinant vector for the expression of said nucleic acid, wherein expression of said nucleic acid is of a mammalian promoter, preferably a CMV promoter or a cytokine-inducible (inflammatory-regulated) promoter. Under control, more preferably under the control of the gene promoter of the acute phase protein, and particularly preferably the human acute phase serum amyloid A gene promoter SAA1 or SAA2 (hereinafter “SAA
Referred to as a "promoter").

The invention further comprises in a preferred embodiment a combination of a vector according to the invention with one or more vectors, which comprises cytokines such as IL-2, I.
It expresses one or more additional genes selected from the group consisting of genes encoding L-12 and interferon-α.

If one or more additional genes are used, said genes are of the same promoter under the control of the same promoter, on the same vector or on different expression vectors, of two identical or different promoters. It can be under control.

The invention further comprises a composition, preferably a pharmaceutical composition, which comprises as an essential component at least one expression vector according to the invention. Said composition comprises a nucleic acid / expression vector according to the invention together with pharmaceutically acceptable excipients and / or preservatives.

Such a composition is produced by the use of the nucleic acid / expression vector according to the invention as an essential constituent of the composition. The composition is useful for activating antigen presenting cells and T cells.

In a preferred embodiment of the invention said composition comprises at least two genes on one or more vectors. Preferred examples of genes encoded by such vectors are listed in Tables 1 and 2 below.

[Table 1]

[0023]   The following combinations are also preferred:

[Table 2]

Particularly preferred is the combination of the chimeric CD40 / CD40L vector with IL-12 (IL-12 as a gene or protein).

The present invention further comprises such an expression vector and a composition comprising such a vector,
Preferably it comprises a method of manufacturing a pharmaceutical composition. The pharmaceutical composition is ex vi
It is used for vo and in vivo treatment, preferably for in vivo treatment of tumor cells of a patient (gene therapy treatment). According to the invention, the chimeric CD40 / CD40L
Vectors containing genes have been found to be preferred under the control of the SAA promoter and to be improved therapeutic agents for the treatment of tumor diseases.

In a further preferred embodiment of the invention, the vector or host cell according to the invention is
Interleukin-2 (IL-2), interleukin-12 (IL-12) and / or interferon-α (preferably interferon α2A) proteins and / or 5-fluorouracil for the treatment of tumor diseases, Preferably combined for use in vivo.

The present invention further includes culturing the mammalian host cells transfected with the expression vector according to the present invention and the host cells of the present invention under conditions that promote the expression of the CD40 / CD40L chimeric gene. And a method for producing a chimeric CD40 / CD40L polypeptide according to the present invention by presenting said polypeptide on a host cell. Transfected host cells can also be used as a pharmaceutical formulation.

The invention further provides the chimeric CD40 / C under conditions where the CD40 / CD40L polypeptide is produced in tumor cells and displayed on the surface of the cells.
A method of producing a modified human tumor cell comprising an expression vector encoding a D40L polypeptide.

A further object of the invention is a chimeric polypeptide encoded by a nucleic acid according to the invention, wherein the extracellular domain of CD40L is from amino acid 47-261 of human CD40L or amino acid 47-260 of mouse CD40L. And / or the intracellular domain of CD40 is amino acids 216-277 of human CD40.
Alternatively, it consists of amino acids 216-289 of mouse CD40.

A further object of the present invention is a process for the preparation of a composition, preferably a pharmaceutical composition, for activating antigen presenting cells and T cells comprising a nucleic acid according to the invention, said composition comprising Is characterized by the use of the nucleic acid according to the invention as a component.

DETAILED DESCRIPTION OF THE INVENTION The promoter according to the invention (also designated as expression control region) causes the expression of DNA and thus the transcription into mRNA and usually has a length of 0.5-5 Kb. It is understood as a nucleic acid region. Such expression control regions can usually include enhancer regions and promoter regions to which transcription factors or repressors can bind. Expression control regions can be regulated through binding of activation or repressors. A regulatory region according to the invention is understood as a region that influences expression by induction by cytokines. On this basis, expression is stimulated.

In a preferred embodiment of the invention, a minimal promoter in combination with an enhancer is used. Minimal promoters and their construction methods are described in, for example, Luckow, B.,
et al., Nucleic Acids Res. 10 (1987) 5490 and Spear, BT, et al., DNA C.
ell Biol. 14 (1995) 635-642. Minimal promoters useful in the expression vectors according to the invention include at least the TATA box, one or more cytokine response factors (CRE) as well as transactivators such as NFκB,
It contains one or more binding sites for CEBP / NF-IL6 and others such as YY1, SAF and AP1. Such binding sites usually consist of 4-12 nucleotides in length. CRE is, for example, Dendorfer, U., Artif. Organs 20 (1
996) 437-444; Birt, DF, et al., J. Nutr, 129 (1999) 25 Suppl., 5715-5.
745; Kang, DC, et al., Int. J. Oncol. 13 (1998) 1117-1126; Weber-Nor
dt, RM, et al., Leuk. Lymphoma 28 (1998) 459-467; Sen, CK, FASEB J.
10 (1996) 709-720.

Useful promoters according to the invention are exogenous viral promoters such as Simian virus 40, Rous sarcoma virus and cytomegalovirus (CMV). Such promoters are constitutive promoters and they require nonspecific inducible signals.

Preferred cytokine-inducible promoters useful in the present invention are highly sensitive to cytokine induction during local or systemic inflammation by the action of proinflammatory proteins such as IL-1β, IL-6 and TNFα. There is. Also, although the expression of these cytokines was revealed in multiple tumors (eg squamous cell carcinoma), the fact that they were absent in normal non-inflammatory tissues resulted in specificity of such promoters in these tumor types. Expression can be found. Multiple tumors expressing high amounts of pro-inflammatory cytokines have been described, for example Knerer et al., Acta Oto-La.
ryngologica 116 (1996) 132-136 shows IL- in squamous cell carcinoma of the head and neck
1 and high levels of TNF-α expression are described. However, other tumors expressing pro-inflammatory cytokines have been described, eg Levy et al., Neurosurger.
y 39 (1996) 823-823 is IL-6 in meningonoma.
And the expression of IL-1β was revealed. Cytokine-induced promoter such preferred SAA2- promoter or sPLA ₂ promoter, tumor tissue,
For example, it may be activated in melanoma, prostate tumor, bladder carcinoma, breast tumor and colon tumor. Therefore, they are particularly suitable for incorporation into constructs designed to facilitate the synthesis of the desired polypeptide. They are especially squamous cell carcinoma,
For example, it is advantageous for treating head and neck cancers.

Of course, gene promoters for acute phase proteins may also be used, which include C-reactive protein, fibrinogen, serum amyloid protein, complement factor 3, orosomucoid, antiprotease α ₁ (antitrypsin) and S.
It contains promoters for other isoforms of AA. The major APRs (acute phase serum amyloid A [A-SAA] and C-reactive protein (CRP) in humans) are extremely responsive to signals that induce them in large numbers during the acute phase response. (Varley, AW, Proc. Natl. Acad. Sci. USA 92 (1995) 5346-5356; US Pat. No. 5,744,304; US Pat. No. 5,851,822; Kushner, I., A.
nn. NY Acad. Sci. USA 389 (1982) 39; Kushner, I., and Mackiewicz, A., D
isease Markers 5 (1987) 1; Fey, GH, and Gauldie, J., In: H. Popper a
nd F. Schaffner (Eds.), Progress in Liver Disease. Vol. 9. (1989) WB Saun
ders, Philadelphia, p89). Thus, the major APR promoter has the potential to be used as an indicator of the ability of natural and synthetic molecules to act as pro-inflammatory and anti-inflammatory agents.

The human acute phase serum amyloid A promoter (SAA2-promoter) is Uh
lar et al., J. Immunol. Methods 203 (1997) 123-130.
The SAA2-promoter has been described to be active in inflamed tissues and can be highly activated by monocyte conditioned medium in vitro and by IL-1β IL-6 and TNF-α IL-6 ( Factor 70). IL
-1β mediated stimulation can be inhibited by IL-1 receptor antagonists (Uhlar et al., J. Immunol. Methods 203 (1997) 123-130 and Steel et al.,
Biochem. Journal 291 (1993) 701-707).

Further preferred is the use in the vector according to the invention of a translational regulator within the SAA2 5'-UTR which plays a crucial role in the regulation of the production of A-SAA. This factor is a cell and / or tissue specific translation enhancer.
Its efficiency can be mediated by intracellular factors that are activated or newly synthesized after cytokine treatment. The sequence of this enhancer element is WO98 / 40506.
Is shown in SEQ ID NO: 4. Preferably, the enhancer is used with an A-SAA promoter (ie, downstream of the promoter and upstream of the gene encoding the product of interest).

A preferred SAA2 promoter according to the invention is a promoter that is highly silenced in healthy non-inflamed tissue but is specifically active in inflamed tissue as well as tumor tissue. The mouse squamous cell carcinoma cell line, eg SCC-VII, is a model system for demonstrating the functionality and tumor specificity of the SAA2 promoter in tumors expressing proinflammatory cytokines, as described for human squamous cell carcinoma. is there.

Therefore, a “SAA promoter” according to the present invention has the function of the A-SAA promoter and is consequently induced by cytokines in a manner virtually identical to the SAA promoter described in WO98 / 40506, and SAA1. as well as/
Or a promoter that is essentially identical to the SAA2 promoter. WO
Also preferred is a promoter encoded by a DNA sequence capable of hybridizing under stringent conditions with SEQ ID NO: 1 shown in 98/40506 and acting as an expression control sequence inducible by cytokines.

The term “hybridizes under stringent conditions” means that two nucleic acid fragments are derived from Sambrook et al., “Expression of cloned genes in E. Col.
i '', Molecular cloning: a laboratory manual (1989), Cold Spring Harbor
It means that they can hybridize to each other under the standard hybridization conditions described in Laboratory Press, New York, USA, 9.47-9.63 and 11.45-11.61. More specifically, “stringent conditions” as used herein refers to hybridization in 6.0 × SSC at about 45 ° C., followed by 2.0 ×.
Means washing at 50 ° C. in SSC. For the selection of stringency,
The salt concentration in the wash step may range, for example, from about 2.0x SSC at 50 ° C for low stringency to about 0.2x at 50 ° C for high stringency.
Up to SSC can be selected. Further, the temperature in the wash step can be increased from room temperature, low stringent conditions at about 22 ° C to high stringent conditions at about 65 ° C.

The term “under control” refers to a position that is related to the position of DNA encoding a desired polypeptide, which causes the promoter to efficiently direct the transcription of the structural gene or genes for the desired polypeptide. Means placed.

The reading direction of DNA and RNA polymerases is always from the 5 ′ end to the 3 ′ end of the DNA chain. The term "downstream" means everything located in the 3'direction on the transcribed DNA strand, and the term "upstream" means everything located in the 5'direction on the DNA strand.

The signal peptide (signal sequence) is required for membrane translocation of the polypeptide,
It is an essential part of membrane-bound or secreted polypeptides that are processed after or during membrane translocation. The signal sequence has a length of about 13-36 amino acids and contains at least one positive residue at the amino terminus. The central part of the signal peptide is a strongly hydrophobic portion of 10 to 15 residues, for example, Nunnari, J., et.
al., Curr. Opin. Cell Biol. 4 (1992) 573-580 and Gilmore, R., et al., Ann.
. NY Acad. Sci. 674 (1992) 27-37. A preferred signal peptide is the CD40, CD40L or TNF-R signal peptide. The signal peptide is cleaved upon integration into the membrane of the target cell.

A signal peptide is useful in the chimeric polypeptide according to the invention, or C
A fragment or modification of the signal peptide of D40 or TNF-R is
Exact cell surface expression of epitope-tagged CD40 / CD40L fusion proteins can be tested by FACS analysis with antibodies to the epitopes to determine whether they have the activity of the CD40 or TNF-R signal peptide, respectively. . Only the signal peptide with the appropriate function can be tagged C as described above.
Allows cell surface expression of the D40 / CD40L fusion protein to be detected. This method is reviewed in Jarvik, JW, et al., Ann. Rev. Genet. 32 (1998) 601-618.

The CD40L domain of the fusion gene is trimerized and results in the activation of receptor-mediated signal transduction in the same manner as the CD40 ligand which is transactivating in the native system. The bound CD40 receptor is trimerized.

To determine whether a fragment or modification of the binding or trimerization domain of human or mouse CD40L has the activity of binding or trimerization domain of human or mouse CD40L, binding of CD154 and Trimerization domain (C
D154 is a ligand for CD40) or modifications thereof can be tested. This indicates that NF-κB, a transcription factor that is one of the major consequences of CD40 signaling.
Can be made by determining the activation of Cells expressing the CD154, CD154 binding and trimerization domain or modifications thereof and a CD40 + NF-κB reporter gene plasmid (eg pNF-κB-CAT, where CAT
The gene has four NF-κB binding sites: 5'GGGAATTTCC (SEQ ID NO: 1)
(Garceau, N., J. Exp. Med. 191 (2000) 381-385; Urban, MB,
et al., Gene Dev. 4 (1990) 1975-1984; and Mihm, S., AIDS 5 (1991) 497-50.
In 3 pNF-κB-CAT is described. Co-incubation with expressing cells and determination of reporter gene expression (eg by CAT-ELISA) allows accurate CD154-mediated transactivation of CD40 signaling to be measured. To

To determine whether a transmembrane and signaling fragment or modification of the human or mouse CD40 polypeptide has human or mouse CD40 transmembrane and signaling activity, CD40, or transmembrane and signaling The functionality of CD40 with modifications in the transduction domain can be tested. This is the NF as described above
It can also be done by determining the activation of κB. Cells expressing CD154, modification of CD40 or transmembrane and / or signal transduction domain, and NF
-Incubation with cells expressing CD40 carrying the κB reporter gene plasmid and determination of expression of said reporter gene (eg C
(By AT-ELISA) allows accurate measurement of CD40 signaling.

To determine whether a fragment or modification of the ligand binding subdomain of CD40 has the activity of the ligand binding subdomain of CD40, the functionality of CD40 or the fragment or modification of the ligand binding subdomain of CD40 is determined. Can be done. This can also be done by determining the activation of NF-κB as described above. Incubation of cells expressing CD154 with CD40 or a fragment or modification of the ligand binding domain, CD40 carrying the NF-κB reporter gene plasmid and determination of expression of said reporter gene (eg by CAT-ELISA) , Enables accurate CD40L binding and thereby accurate CD40 signaling.

CD40 according to the invention is understood as a polypeptide having the activity and biochemical properties of the CD40 receptor. Such a polypeptide is, for example, human or mouse CD40, or a modification thereof such as a mutation, deletion or substitution. A preferred modified form of CD40 polypeptide is human CD40, preferably within the intracellular domain.
Is at least 80% homologous to. Homology according to the invention can be determined with the aid of the computer program Gap or BestFit (University
of Wisconsin; Needleman and Wunsch, J. Mol. Biol. 48 (1970) 443-453, S
Mith and Waterman, Adv. Appl. Math. 2 (1981) 482-489).

The human CD40 molecule is 277 amino acids long (MW = 30,619 Da).
, And a 19 amino acid long hydrophobic signal sequence (positions 1-19),
An extracellular domain of 174 amino acids (positions 20-193), a transmembrane domain of 22 amino acids (positions 194-215), and a cytoplasmic domain of 62 amino acids (positions 216-277) follow. Continued (Swiss-Prot .: P25942 and Stamen
kovic et al., The EMBO J. 8 (1989) 1403-1410). The extracellular domain is important for ligand binding and thereby mediated trimerization of the active complex. It consists of two subdomains (ligand binding subdomain and stalk subdomain). The extracellular domain according to the present invention, as a full-length domain,
Or it should be understood as at least part of which causes ligand (CD40L) binding. The ligand-binding subdomain consists of about 100 amino acids (positions 20-120), and the stalk domain is described by Bajorath et al., Proteins 27 (1997).
) 59-70 consisting of about 73 amino acids (positions 121-193). The Stoke subdomain is a domain required for steric reasons. This subdomain ensures an optimal distance between the binding domain and the cell surface necessary for trimerization.

The cytoplasmic domain mediates signaling of complexes activated by trimerization. Signaling, so-called TRAF protein (T NF- R - A ss
mediated by ociated F actor (TNF receptor-associated factor)),
This binding site is located in the cytoplasmic domain of CD40 and ultimately leads to activation of various transcription factors, the most prominent of these transcription factors being NF-κB. A similar mouse CD40 molecule is 289 amino acids long (MW = 21,111 Da) (Grimaldi et al., The J. of Immunol. 149 (199).
2) 3921-3926 and Swiss-Prot .: P27512), and a 19 amino acid long signal sequence (positions 1 to 19), which has an extracellular domain of 174 amino acids (
20-193), a 22 amino acid long hydrophobic transmembrane domain (194-21)
Position 5), and a 74 amino acid long cytoplasmic domain (positions 216-289).

CD40L (CD154) according to the invention is understood as a polypeptide having the activity and biochemical properties of CD40L. Such a polypeptide is, for example, human or mouse CD40L, or a modification such as a mutation, deletion or substitution. Preferred modified CD40L polypeptides are at least 80% homologous to human CD40L, preferably within the extracellular domain.

The human CD40L protein is 261 amino acids long (Swiss-Prot.: P2
9965, MW = 29,273 Da), and a 22 amino acid long cytoplasmic region (1
~ 22), to which a 24-amino acid signal anchor (type II membrane protein; no typical signal peptide) (23-46) and an 215 amino acid long extracellular signal-mediating domain (47). ~ 261st) continues. CD40L
The extracellular domain of is composed of two subdomains: a stalk domain (positions 47-119) and a trimerization domain (positions 120-261). The mature glycoprotein has a molecular weight of 35 KDa.

Mature CD40L (Swiss-Prot .: P27548) is 260 amino acids long (
MW = 29,396 Da), and a 22 amino acid long cytoplasmic region (1-22).
Position), to which a signal anchor with a length of 24 amino acids (positions 23 to 46) and an extracellular signal-mediating domain with a length of 214 amino acids (positions 47 to 260)
Continues. The mature mouse glycoprotein has a molecular weight of 33 KDa.

The three-dimensional structure of CD40L has been elucidated. The structure of the CD40 / CD40L complex has been derived from the determination of the known homologous structure of TNF-R / TNF-β and the crystal structure of the soluble extracellular domain of the CD40L trimer (Karpusas, M., et al. .,
Structure 3 (1995) 1031-1039). This led to a model in which CD40L trimerizes and, consequently, mediates CD40 trimerization. Human CD40 and mouse CD40 show 76% homology. Human CD40L and mouse CD4
0L indicates 86% homology.

The nucleic acid segment according to the present invention may be introduced ex vivo or in vivo into a mammalian cell or host by any of several methods, including vector transfection. Viral vectors can be used to infect human and animal cells with recombinant DNA; certain adenoviral vectors have proven to be particularly useful. Adenovirus vectors are preferred. Of course DN
The A segment does not necessarily have to be introduced into the cell by a viral vector: direct transfection can be done, for example, by electroporation, gene gun technology, or DNA-liposome complexes. The DNA / liposome complex has been used to introduce DNA encoding a prostaglandin synthase into rabbits, resulting in the production of prostaglandin E2 and prostacyclin (Conary et al., J. Clin. .Invest. 93 (1994) 1834-1840). Suitable vectors include selected proteins, or one or more nucleic acids according to the invention,
It includes genes encoding proteins such as those under the control of promoters active in mammalian, preferably human cells, such as the CMV promoter, SV40 promoter or SAA promoter. The vector is introduced into the host cell by any of the many methods known to those skilled in the art, such as direct introduction of DNA by gene gun technology, transfection of liposomes or direct local injection. Direct injection is preferred in the case of bladder carcinoma and injection with an endoscopic probe is preferred in the case of colon carcinoma.

In a preferred embodiment, the chimeric nucleic acid and the gene encoding IL-2, IL-12 and / or interferon-α are co-expressed on the same or different vectors. This can be achieved by co-transfection or co-introduction of two viral or plasmid vectors carrying both genes or a single vector present in which both genes are encoded. In the latter embodiment, both genes are expressed by separate promoters (promoters can be the same or different from each other), they are linked in expression via an IRES sequence (internal ribosomal translocation site), or It may be present linked through a splice donor sequence before the first gene and a splice acceptor sequence after the first gene, ie before the second gene. In the latter embodiment, in the case of unprocessed mRNA, the first gene will be read, and in the case of processed mRNA, the first gene will be excised and the second gene will be read. .

The gene encoding the chimeric nucleic acid according to the present invention represents an “immunological master gene”, ie after ligand binding via CD40L CD40 induces large amounts of costimulatory, proinflammatory or maintenance factors. , And as a result, induces a strong immune response (Stout et al., Immunology Today 17 (1996) 487-492). CD40 also has a large amount of mediators of inflammation, such as IL-12, IL1-β.
, Induces the NFκB transduction pathway at the end where IL-6 or TNF-α is present. Therefore, a preferred embodiment of the present invention consists of a chimeric nucleic acid under the expression control of a cytokine inducible promoter. The advantage of this embodiment is that it induces an autocrine activation cascade after being inserted into a tumor that expresses only a few proinflammatory cytokines, i.e. the inflammatory cytokine is a promoter on the inserted vector according to the invention. And the chimeric polypeptide will subsequently activate NFκB at the end of activation of the promoter. This effect is due to binding sites for proinflammatory transactivators such as NFκB or NF-IL6 (
For example, the −190 to −78 region of the SAA2 promoter or only the respective binding sites) can be further enhanced by inserting the cytokine-inducible promoter, preferably in multimeric form.

A gene therapeutic agent according to the present invention means a pharmaceutical composition comprising one or more expression vectors according to the present invention as an essential component in an amount required for a tumor patient to ensure effective treatment. To be understood. Such a composition preferably comprises at least one vector together with a non-viral delivery system as an adenovirus vector or a retrovirus vector. In such cases, the transport system or the viral vector itself or the expressed CD40 / CD40L gene will cause a local or systemic inflammatory response in the tumor and / or tissues surrounding the tumor. In such cases, transportation /
Cytokine mobilization resulting from administration of the targeted vehicle will lead to promoter-driven generation of the therapeutic agent at least 100-fold more than its uninduced basal level.

Somatic cell gene therapy can be achieved, for example, by using retroviral vectors, other viral vectors, or by non-viral gene transfer (
Friedman, T., Science 244 (1989) 1275; Morgan 1993, RAC Data Management
See Report, June 1993).

Vector systems suitable for gene therapy include, for example, retroviruses (Mulligan, RC,
(1991), Nobel Symposium 8: Etiology of human disease at the DNA level (
Lindsten, J., and Pattersun, eds.) 143-189, Raven Press), adeno-associated virus (McLughlin, J. Virol. 92 (1988) 1963), adenovirus (Zhang, WW)
et al., Cancer Gene Therapy 6 (1999) 113-118), vaccinia virus (Moss
et al., Ann. Rev. Immunol. 5 (1987) 305), bovine papillomavirus (Rasm
ussen et al., Methods Enzymol. 139 (1986) 642) or viruses from the herpesvirus group, such as the Epstein-Barr virus (Margolskee et al., Mol.
Cell. Biol. 8 (1988) 2937) or herpes simplex virus. However, adenovirus is preferred.

Adenovirus vectors are also described in Fasbender, A. et al., J. Biol. Chem. 272.
(1997) 6479-6489 and Dodds, E., et al., J. Neurochem. 72 (1999) 2105-21.
As described in 12, it can be applied as a formulation of cationic lipids with ADV.

Non-viral delivery systems are also known. For this, usually "nude" nucleic acids, preferably DNA, are used, or auxiliary agents such as transfection reagents (liposomes, dendromers, polylysine transferrin conjugates (Felgner et al., Proc. Natl. Acad. Sci. USA 84 (1987) 7413)).

According to the invention, tumor-specific cells that are cytolytically active for a very long period of time are generated,
And it became clear that it could be activated. CD40 and CD40
The combination of L expression is preferably in combination with the expression of IL-2, IL-12 and / or interferon-α, or in a therapeutically active amount of IL-2, IL-12.
And / or together with the interferon-α polypeptide or 5-fluorouracil, further results in a synergistic enhancement of the level and duration of the activation phase of the tumor-specific T cells.

The composition may be administered parenterally, for example using injectable solutions, preferably for intratumoral injection, and preferably for head and neck cancer (squamous cell carcinoma). For the preparation of such injectable solutions, the vector according to the invention is mixed with an inert, inorganic or organic excipient of pharmaceuticals, buffers and / or preservatives. Such excipients are preferably waters, alcohols, polyols, glycerols, which have a neutral pH value (pH 6-8). Pharmaceutically acceptable buffers are eg phosphates, lactates, phosphate buffers, Tris. The pharmaceutical composition also comprises a preservative,
Toxicity agents, stabilizers, wetting agents, clarifying agents, thickening agents, salts for modifying the osmotic pressure, buffers or antioxidants may also be included. They may also include other therapeutically useful agents.

Preservatives suitable for use in the above formulations include benzalkonium chloride, benzethonium chloride, chlorobutanol, thimerosal and the like. Suitable buffers include boric acid, sodium and potassium bicarbonate, sodium and potassium borate, sodium and potassium carbonate, sodium acetate, sodium bicarbonate, etc. at about pH 6-pH 8.
, Preferably in an amount sufficient to maintain a pH of about pH 7 to pH 7.5. Suitable tonicity agents include dextran 40, dextran 70, dextrose, glycerin,
Potassium chloride, propylene glycol, sodium chloride and the like. Suitable antioxidants and stabilizers include sodium bisulfite, sodium metabisulfite, sodium thiosulfate, thiourea and the like. Suitable wetting and clarifying agents are polysorbate 8
0, polysorbate 20, polyoxamer 282, and tyloxapol.
Suitable thickeners are dextran 40, gelatin, glycerin, hydroxyethyl cellulose, hydroxymethyl propyl cellulose, lanolin, methyl cellulose, petrolatum, polyethylene glycol, polyvinyl alcohol, polyvinyl.
Includes polyvinylpyrrolidone, carboxymethyl cellulose and the like.

[0067]   CD40 on antigen presenting cells (APC) and its ligand CD40L on T cells
Interactions between APCs play a prominent role in the upregulation of APC function
Plays. These include surface expression of B7 and activation of IL-12 synthesis,
The two proteins work together in inducing an effective antitumor response. Basal cells and
And squamous cell carcinoma-derived tumor cells may explain the mechanism of tumor escape
C, which showed down-regulation or dramatic loss of
T cells expressing D154 can no longer kill tumor cells
It was revealed. The ability of the substances according to the invention in inducing an antitumor response is
Non-immunogenic CD40 with the construct^- Tumor cells (eg, squamous cell carcinoma)
Cell line, eg SCC VII)
obtain. Wild type SCC VII, novel control and CD40⁺ Cells are those
It was indistinguishable in growth rate and morphology in vitro. CD40 ⁺  Syngeneic C3H / HeN mice injected subcutaneously with SCC VII had CD40^- Wild type S
Tumor growth was significantly reduced compared to CC VII or the new control. Contrast
In summary, CD40-transfected cells injected into nude mice were
It showed the same tumor growth kinetics as the two controls, which immunocompetent T cells did
It has been shown to be required for tumor activity. CD40⁺ Of tumor-derived tumor explants
CD4 for histological analysis⁺ Predominant infiltration of T cells and CD25 T cell activator
The expression of Kerr was revealed. These changes in the control tumors
Not detected. Chimeras according to the present invention can be used in apoptosis and tumor cells
Induces inhibition of growth rate.

According to the invention, the vector according to the invention can be used for the regulation or treatment of tumor diseases, preferably for the treatment of vascularized tumors. The dosage is a biologically effective amount of the vector and can be varied within wide limits and, of course, will meet the individual requirements in each particular case. Preferred doses of viral vector are 10 ⁸ -10 ¹¹ pFU / injection and 50-1000 μg DNA / injection (Kikuchi
et al., Human Gene Therapy 10 (1999) 1375-1387), preferably 50-200 μg DNA / injection for non-viral vectors.
The preferred volume per injection is 1-10 ml.

Viral vectors are preferably prepared in a pharmaceutical composition comprising a phosphate buffer of pH 7.4 or other buffers of pH 6-8 (Caruso, M., et al., Proc. Natl. Acad.
Sci. USA 93 (1995) 11302-11306). The non-viral vector is preferably 6-
Prepared in a pharmaceutical composition with a pH value of 8 and additionally with liposomes (Yanagihara, I.
, et al., Mol. Cell. Biol. Hum. Dis. Ser. 5 (1995) 64-82; Thierry, AR
, et al., Gene Ther. 4 (1997) 226-237; Gao, X., et al., Gene Ther. 2 (1
995) 710-722; Abdallah, B., et al., Biol. Cell. 85 (1995) 1-7; Treco,
DA, et al., Mol. Med. Today 1 (1995) 314-321).

Patients are preferably treated with 1-2 injections per week administered directly to the tumor over a period of 3-10 weeks, which induces a cytotoxic immune response against tumor cells. . After this period, the extent of tumor change is examined and, if necessary and possible, the tumor removed. This type of treatment is of particular importance for improving the outcome of the treatment, since there is still a significant number of tumor cells after surgery. However, preoperative immunization again elicits a cytotoxic immune response after tumor removal and results in the formation of cytotoxic T cells that can destroy metastatic cells and minimal residual diseased cells.

Further preferred is the individual injection at different sites of the tumor and / or around the tumour, which means that the pharmaceutical agent according to the invention can be used not only for the actual tumor site but also for other tumor tissues. To reach non-tumor cells of, for example, fibroblasts, macrophages, T cells or dendritic cells.

Even more preferred is that the inflammatory response on the tumor is locally enhanced prior to, during or shortly after administration of the pharmaceutical agent according to the invention. This can be achieved, for example, by topical heat treatment (microwave), pressure, or by injection of the pharmaceutical agent at various sites and around the tumor.

The vector according to the invention can be injected directly into the tumor, postoperatively into the hole of the tumor, or systemically, as a formulation with an introduction reagent.

In a preferred embodiment of the invention, the vector-containing pharmaceutical agent according to the invention (preferably no expression vector for IL-2, IL-12 and / or interferon-α) is as an adjuvant, and IL-2, IL-12 and / or
Alternatively, it is administered together with a polypeptide having interferon-α activity. In this case, the vector-containing agent is administered as described above, while the polypeptide is preferably administered systemically. In this connection, it is preferred to administer said polypeptide before (before or after, or up to 12 hours before or after) injection of the drug containing the vector.

Table 3 shows preferred dosing schemes for adjuvant administration of polypeptides.

[Table 3]

5-Fluorouracil is administered at 12 mg / kg body weight per day on the first day of tumor treatment and at 6 mg / kg body weight on days 6, 8, 10 and 12.

According to the invention, it is important to use endotoxin-free DNA,
And to avoid any inflammatory reaction of the tissue. This would induce expression of proinflammatory cytokines, while leading to stimulation of inducible promoters in other cells except squamous cell carcinoma. DNA containing no such endotoxin is W
It can be prepared according to O97 / 29113.

Long-term, high expression of immunostimulatory genes is preferred for this treatment, and plasmids or adenovirus vectors derived from tumors also injected into neighboring tissues or other organs, such as the liver or lungs. Tumor cell specificity that reduces side effects due to efflux is preferred. In addition, tumor-specific treatment can result in systemic injection of a tumor-targeting immunostimulatory plasmid formulation. Tumor specificity can be achieved either by targeting the transfection reagent to the tumor, or by tumor-specific gene expression, or a combination of both, or both. Targeting transfection reagents is not yet possible with sufficient specificity and efficiency, but tumor-specific promoters are available.

According to the description, the invention provides a tumor with a vector encoding an immunostimulatory gene,
It will have a great impact on the treatment of metastases and minimal residual disease. The use of the promoter according to the invention makes it possible to obtain high levels of long-term, strongly inducible / repressible,
And enables tumor-specific expression. Therefore, in a cancer treatment regimen using injection of vector DNA, the possibility of systemic treatment reaching not only the primary tumor but also metastasis as a result of long-term and higher amounts of immunostimulatory proteins in the tumor, and toxic side effects. A wider therapeutic window (higher doses and multiple doses) can be optimized by reducing

A further preferred embodiment of the present invention is the treatment of non-solid tumors of blood origin, such as chronic and acute lymphocytic leukemia, multiple myeloma, chronic and acute myelogenous leukemia. In the case of these non-solid tumors, the treatment will preferably be ex vivo.
This involves the removal of tumor cells from the circulation by separation of leukocytes, the introduction of these tumor cells ex vivo by the vector according to the invention and the reinfusion of the transduced tumor cells into the patient. The method using CD40L gene transfer is described in Kato
et al., J. Clin. Invest. 101 (1998) 1133 and Kikuchi, T., et al., Human
Gene Therapy 20 (1999) 1375-1382. Treatments with the vectors according to the invention can likewise be carried out.

The following examples, references and figures are provided to aid the understanding of the present invention, the scope of which is set forth in the following claims. It is understood that modifications of the method may be made without departing from the spirit of the invention.

Materials and Methods Animals: 6-8 week old female C57BL / 6 mice are provided by Charles River. All animals were housed in individually ventilated cages (IVC) on a daily 12-hour light / 12-hour dark cycle according to international guidelines. C57BL / 6 mouse is B1
6F10 mouse melanoma cells are syngeneic and are used in transplantation experiments with this cell line.

[0083] was purchased a female syngeneic ^{C3H / HeN (H-2K K} ) mouse from Germany of the Charles River. The mice were 8-12 weeks old. Female Balb / c nu / nu
Mice (20 weeks old) were obtained from Bomholtgard Breeding, Denmark.

The B / 6F10 melanoma cell line was grown in syngeneic C57BL / 6 mice (H-2b).

Cell line: B16F10 mouse melanoma cells were obtained from ATCC (CRL6475),
And 10% fetal bovine serum (PAA Laboratories, Austria), 4 mM L-glutamine (Gibco-Life Technologies, MD, USA), DMEM supplemented with 1 mM sodium pyruvate and 4.5 g / l glucose (UK).
Biochrom Ltd.). The culture conditions are 37 ° C. and 5% CO ₂ in a water-saturated atmosphere. The culture was passaged twice a week, using 1x trypsin / EDTA (Roche Diagnostics GmbH, Germany).

HEK293 cells were obtained from ATCC (CRL-1573) and were 10%
Fetal bovine serum (PAA Laboratories, Austria), 2 mM L-glutamine (Gi
It was cultured in DMEM (Biochrom Ltd., UK) supplemented with bco-Life Technologies).

Mouse squamous cell carcinoma cell lines (eg SCC VII / SF; O'Malley, B., et a
l., J. Cancer Res. 56 (1996) 1737-1741 and Snit, HD, et al., Radiation
Research 104 (1985) 47-65) and the mouse colon cancer cell line (eg CT26; Xi
ang, R., et al., Cancer Res. 58 (1998) 3918-3925) are both CD40 ⁻ and CD40L ⁻ , 10% heat inactivated FCS (GIBCO Life Technologies),
DMEM high glutamax ™ medium (GIBCO Life Technol) supplemented with penicillin and streptomycin (Roche Diagnostics GmbH, Germany)
ogies). This cell line can also be used in place of the example B16F10.

DNA Constructs and Transfections: CD40 / CD40L Fusion Proteins: Two types of fusion proteins were expressed: Type A: a) This type was fused to the extracellular portion of mouse CD40L (amino acids 47-260). It comprises the CD40 signal peptide (amino acids 1-21). Hinge region of immunoglobulin D (IgD) (accession number 709448.1, Locus)
MUSIGCD15. EM ROD → ht64-123) -derived linker (Tu
cker, Science 209 (1980) 1353-1360), this part of the remaining CD
40 proteins (amino acids 22 to 277 (human) or 289 (mouse)) are linked.

B) Compared to a), the extracellular part of CD40L leaves amino acids 120 to 260, and is therefore shortened by removing the stalk domain of CD40L.

Type B: This type corresponds to the extracellular part of CD40L (amino acids 120 to 260 (or human C
CD40 signal peptide (amino acid 1) fused to D40L 121-261)
~ 21) directly fused to a shortened form of CD40 (amino acids 122-277 (human) or 289 (mouse)) (no linker or short linker (1-30 amino acids / eg IgD). -A 20 amino acid linker from the hinge)), and consequently A type b) lacking the CD40-CD40L binding domain of CD40)).

The final fusion construct is human / human, mouse / human or mouse / mouse CD40L / CD40. Construction of the mouse / mouse chimeric construct is described in Example 1.

Fusion of different parts of the cDNA construct was performed by standard PCR techniques. The DNA sequence was verified by sequencing the resulting PCR product.

The chimeric cDNA is pcDNA3 (Invitrogen Cor, San Diego, USA).
p.) cloned into a mammalian expression vector. Transfection of HEK293 cells was performed with the transfection reagent FUGENE ™ (Roche Diagnostics GmbH, Germany) consisting essentially of a formulation of lipids (non-liposome formulation).

Flow Cytometry and Antibodies: The cells were transfected with CD40, MHC-I (H) due to expression of surface markers on relevant cells.
^{-2K d ^ BALB / c),} CD40L, ICAM-I (CD154), B7.
1 (CD80), B7.2 (CD86), Fas receptor (Fas, APD-1 /
CD95), Fas ligand (FasL, CD95L) or CD40-CD
Characterized by direct immunofluorescence using a monoclonal antibody conjugated with either PE and / or FITC to a tag (eg, HA tag, Flag tag) (→ CD40-CD40L ^tag ) fused to the N-terminus of 40L fusion protein I attached it. The corresponding isotype control was used for the determination of non-specific binding. Antibody binding was carried out for 20 minutes at room temperature, followed by washing and FACScan ™ (
Analysis was performed using Becton Dickinson (Germany).

In vivo experiments: Tumor cells were expressed in 5 × 10 ⁵ / 200μ on the right rear flank of syngeneic or athymic animals.
Subcutaneous (SC) tumor was applied at a concentration of 1. Tumor growth was quantified three times a week using calipers 5 days after tumor attachment. Tumor volume was calculated by the approximate elliptic formula: 4/3 × π / 8 × (length × width × height).

For experiments on subcutaneous implantation of Ad-CD40-CD40L-introduced B16F10 melanoma cells into C57BL6 mice in vitro, B16F10.
Cells were seeded in 12 well plates (Falcon) at 4 × 10 ⁵ cells / well. When the cells were adherent, appropriately diluted vector in transfer medium (DMEM without additives) was added to the wells. After determination of surface expression by FACS analysis of ^tagged CD40-CD40L ^tag fusion constructs introduced in parallel, 200 μl volume of 5 × 1
0 ⁵ tumor cells were implanted subcutaneously into the abdominal wall side. For this method, mice were anesthetized with ether. Tumor growth was quantified three times a week with calipers 5 days after tumor attachment. Tumor volume was calculated in the approximate elliptical formula: 4/3 x π / 8 x (length x width x height).

Example 1 Construction of Expression Vector Materials and Methods: pcDNA3-CD40-CD40L (type A and B) mouse / mouse fusion constructs: (mouse / human and human / human chimeric constructs were constructed in a similar manner, And tested.) The signal peptide of mouse CD40 comprising amino acids 1-21 is ATG
It was amplified by PCR using two overlapping oligonucleotides with a KpnI restriction site just upstream of the start codon and a BsmBI and HindIII restriction sites at the 3'end of the resulting fragment. Two different types of mCD40 receptor portion of the fusion gene were amplified by PCR using pcDNA3-mCD40 (pcDNA3 with cloned mCD40 cDNA) as template. The first fragment (corresponding to the type B fusion gene) encodes amino acids 122-289 of mCD40 and the second fragment (corresponding to the type A fusion gene) amino acids 21-289 of mCD40. . In the first case, a HindIII site followed by a BsmBI site, followed by 5 nucleotides corresponding to the 3'end of the coding sequence for mCD40L was introduced at the 5'end of the PCR fragment. In both cases a HindIII site followed by a BsmBI site,
Subsequently, 5 nucleotides corresponding to the 3'end of the 20 amino acid IgD hinge region were introduced at the 5'end of the PCR fragment. In both cases, the downstream primer introduced an XhoI site at the 3'end of the PCR fragment with the stop codon immediately upstream.

The IISK + Bluescript vector (Stratagene) was added to Kpn.
It was cleaved with I / XhoI. The PCR fragment of the signal peptide was KpnI /
PC cleaved by HindIII and carrying two different types of mCD40 gene
The R fragment was cleaved with HindIII / XhoI. In ligation of the three fragments, the intermediate constructs pBluescript-signal peptide-CD40 (B type; amino acids 122-289) and pBluescr.
ipt-signal peptide-CD40 (type A; amino acids 21-289) was obtained.

MCD40L binding and trimerization domain (amino acids 120-260) and m
The CD40L stalk + binding and trimerization domain (amino acids 47-260) was also used as template for pcDNA3-mCD40L (cD of cloned mCD40L.
Obtained by PCR amplification using pcDNA3) with NA. In each case, the upstream primer introduced an EcoRI site, followed by a BsmBI restriction site, followed by 5 nucleotides corresponding to the 3'end of the signal sequence at the 5'end of the PCR product. The downstream primer introduced an XbaI restriction site at the 3'end of the PCR fragment with the BsmBI restriction site immediately upstream. PCR generated
The fragment is the IISK + Bluescript vector (Stratage).
The EcoRI / XbaI site was cloned into the EcoRI / XbaI site of ne).

A linker derived from the immunoglobulin D hinge region (IgD-hinge; see above) comprising 20 amino acids was obtained by PCR amplification using two overlapping oligonucleotides. . A flanking EcoRI site at the 5'end was inserted, followed by a BsmBI site, 5 nucleotides corresponding to the 3'end of the coding mCD40L sequence. At the 3'end of the IgD hinge fragment, Xba
The I site was introduced along with the BsmBI site immediately upstream. The resulting fragment was cloned into the EcoRI / XhoI site of the polylinker of IISK + Bluescript.

CD40L (amino acids 47-260) and CD40L (amino acids 120-26)
0) as well as the IgD hinge were excised from pBluescript by BsmBI digestion and further BsmBI in the three fragment ligations.
PBluescript-signal peptide-CD40 (A type; amino acids 21-289) and the BsmBI site-signal peptide-CD40 (B type; amino acids 122-289) of pBluescript that had been cleaved with.

The resulting vector was: -pBluescript-signal peptide-CD40L (amino acids 47-2
60) -hinge-CD40 (A type / length) -pBluescript-signal peptide-CD40L (amino acid 120-
160) -hinge-CD40 (A type / short; hereinafter, the term "A type" is "A type / short"
-PBluescript-signal peptide-CD40L (amino acid 120-
260) -hinge-CD40 (B type).

The CD40-CD40L fusion gene was blue digested with KpnI / XhoI.
Excised from the script vector and subcloned into the polylinker of pcDNA3 (Clontech, CA, USA), where the fusion gene is transcriptionally controlled by the CMV promoter.

PSAA2-CD40-CD40L (types A and B) were constructed by removing Cat from pSAA2-Cat (European Patent Application No. 99107611.8) by NotI digestion and filling the cohesive ends with Klenow enzyme. CD40-CD
The 40L fusion gene was digested with KpnI / XhoI to pcDNA3-CD40-
It was excised from CD40L and the overhanging ends were made blunt by Klenow-polymerase treatment and the blunt ended fragment was ligated to the pSAA2-CAT vector fragment. In the pSAA2-CD40-CD40L construct, the fusion gene is under the control of the SAA2-promoter.

PSAA2-IL2 was constructed by removing the Cat gene in pSAA2-Cat by NotI digestion and inserting the IL2 cDNA amplified in PCR using primers with NotI overhangs.

PcDNA3-IL2 was constructed by cloning the PCR amplified IL2 gene into the mammalian expression vector pcDNA3 (Clontech).

PSAA2-IL12 was isolated from Ca in pSAA2-Cat by NotI digestion.
It was constructed by removing the t gene. The two subunit genes of IL12, p35 and p40, were amplified separately by PCR. Those transcripts are E
Ligation was performed by using the IRES sequence from MCV (from pIRES1neo; Clontech). P35 was amplified with a 5'primer with a NotI site in the overhang and a 3'primer with a SalI site in the overhang. The EMCV IRES was amplified with a 5'primer with a SalI site in the overhang and a 3'primer with an XhoI site in the overhang. P40 is a No.
Amplified by the 3'primer with tI and XhoI sites. The amplified fragment is then pBlue cleaved with NotI and XhoI.
It was cloned into script (Stratagene). Final pBl
uescript-p35-IRES-p40 was subsequently cut with NotI, and the fragment containing p35 and p40 transcriptionally linked by the IRES of EMCV was cloned into pSAA2-Cat instead of the Cat gene.

PcDNA3-IL12 is p35-IRES- derived from pSAA2-IL12.
p40 gene cassette is a mammalian expression vector pcDNA3 (Clontech)
It was constructed by subcloning in.

AdV-CD40-CD40L: Adenovirus vector (AdV) was constructed by cloning the gene to be expressed into an adenovirus transfer vector containing the expression cassette and a region of homology to the adenovirus genome. To be done. The AdV transfer vector and the AdV genome were linearized and 293A cells (ATCC CRL 1573
． 1) is co-transfected. There, they religate to form an AdV vector that packs within the viral particle but can replicate by deletion of their E1 region. This deletion results in a cloning capacity of 7 Kb. The AdV vector is stably transfected with the sheared AdV DNA and thus provides the E1 gene product in trans, 293A.
It can only be propagated in the cell and produced as a result.

The adenovirus vector AdV-CD40-CD40L was constructed using pcDNA3-C.
Adenovirus transfer vector pQBI in which CMV5 promoter / enhancer and globin poly A site of the entire expression cassette derived from D40-CD40L is removed.
-AdCMV5 (QUANTUM Botechnologies Inc.)
Constructed by inserting into. By deleting the appropriate restriction sites, pcDNA3-C
D40-CD40L-derived whole expression cassette (CMV promoter, CD40, C
D40-L, polyA site) was amplified by PCR, and also pQBI-AdCMV5 without the CMV5 promoter / enhancer and globin polyA was amplified by PCR. The two amplified fragments were restricted with PmeI (
The recognition site was provided by the overhang of each PCR primer), and T4-
Ligation was performed with DNA ligase.

The adenovirus transfer vector AdV-CD40-CD40L was linearized by ClaI digestion at the linearization site, and the protocol described (QUANTTU).
M Botechnologies Inc. In accordance with (1), the cells were co-transfected with linear QBI-viral DNA into QBI-293A cells.

At 293A, the transfer vector and the cotransfected adenovirus DNA are religated in their overlapping regions to form the adenovirus vector AdV-CD40-CD40L.

AdV-CD40-CD40L has a deletion of the E1 region of human adenovirus type 5; the CD40-CD40L expression cassette is cloned in place of the deleted E1 region. Due to the deletion of E1, the adenovirus vector cannot replicate alone. Only in 293A cells containing sheared genomic DNA from human adenovirus type 5, the E1A and E1B genes essential for viral replication are provided in trans, so that the virus can be produced in that cell line. The viral vector is purified through multiple plaque purification iterations and is described in the QUANTUM Adeno-Quest kit protocol, or outlined in Graham et al., Mol. Biotechnol. 3 (1995) 207-220. Large-scale isolation from infected 293A cells by the method described above.

The adenovirus vector AdV-IL2 contains pcDNA3-CD40-CD4.
By using the pcDNA3-IL12 expression cassette instead of 0L
Constructed similar to dV-CD40-CD40L.

The adenovirus vector AdV-IL12 contains pcDNA3-CD40-CD
It was constructed similarly to AdV-CD40-CD40L by substituting the expression cassette for pcDNA3-IL-12 in place of 40L.

Co-expression vector: A vector co-expressing two genes and an adenovirus vector can be prepared by using one of the expression plasmids described above, E immediately downstream of the first gene to be expressed.
It was constructed by inserting the IRES sequence from MCV (encephalomyocarditis virus from pIRES1neo (Clontech)) and inserting a second gene to be expressed between the IRES sequence and the poly A sequence. The IRES sequence (internal ribosome entry site) restarts the translation machinery and, consequently, links and expresses both genes.

[0117]   Following this method produces the following vector:     pSAA2-CD40 / CD40L-IL2     pSAA2-CD40 / CD40L-IL12     AdV-CD40 / CD40L-IL2

Example 2 Vector Expression The pcDNA3-CD40 / CD40-chimera was transiently transfected into HEK293 cells. Two days after transfection, expression of the fusion protein on the cell surface resulted in CD40 C-terminal and similar N-terminal Flag.
-Tag or HA-tagged CD40-CD40L chimeric construct (C
Validated by Western blot (see FIG. 4) with anti- <CD40> antibody against D40-CD40L ^tag ) and anti-Flag- or HA.
-Analyzed by FACS analysis using the tag antibody.

The constitutive activity of the fusion protein is one of the major consequences of CD40 signaling.
NF-κB (eg, CAT gene (CAT-ELISA, Roche Diagnostic Gm.
bH, DE) → pNF-κB-CAT) reporter plasmid was tested by cotransfection.

To test the cis activity of the CD40L / CD40 chimera, pcDNA3-C
The D40L / CD40 chimeric plasmid together with the reporter plasmid
Co-transfection into EK293 cells and 48 hours later CAT
The activity was tested.

[0121]   FIG. 5 shows the results of an assay for CD40-CD40L cis activity.

To test the transactivity of the CD40L / CD40 chimera, the reporter plasmid pNF-κB-CAT was cotransfected with pcDNA3-CD40 (expression of the CD40 gene alone) into HEK293 cells and pcDNA3-. The CD40-CD40L chimeric plasmid was separately transfected into HEK293 cells. After 24 hours and after verification of cell surface expression of the CD40 / CD40L chimera, both cell clones were incubated together for a further 24 hours and subsequently tested for CAT expression.

FIG. 6a / b shows the assay design / results for CD40-CD40L transactivity.

Example 3 In Vitro Proliferation and Surface Expression of CD40-CD40L Chimera SCCVII, CT26 and B16-F10 cells express CD40-CD40L chimera or cell surface molecules such as the low affinity nerve growth factor receptor (LNGFR). Expressed reporter gene or indicator gene, eg lac as control
Infected with adenovirus expressing Z. Related cell surface markers (CD40
-CD40L, MHCI, ICAM-1 etc.) in response to viral infection and expression of fusion proteins. In addition, the growth kinetics and apoptotic behavior of infected cells were examined.

Non-Viral Transfections: Cells were seeded in 6-well plates at a density of 1-2 × 10 ⁵ cells / well 16 hours before transfection. Transfection was performed using FuGENE ™ 6 Transfection Reagent (Roche Diagnostics Gm, Germany).
bH). As a result, 100 μl of 8 μl FuGENE ™ 6 was added.
1 of serum-free DMEM medium was added, incubated for 5 minutes at room temperature and then mixed with 2 μg of DNA. Incubate this mixture for 15 minutes at room temperature,
And 2 ml of cell culture medium (DMEM, 10% FCS, 2 mM glutamine, 4
． 5 g / l glucose) was added drop-wise to the cells. For transient expression assays, gene expression after 2 days; addition of G418 (0.5 to testing cells for stable transfection and selecting for neo ^R marker gene expression).
Cells were selected according to a concentration of 1 mg / ml).

Example 4 In B16F10-Derived Tumor in Isogenic Mice (C57BL / 6)
Growth in vitro To test the tumor growth kinetics of the mouse B16F10 cell line, which does not express CD40 or CD40L or CD40-CD40L chimera, 5 × 10 ⁵ cells / 200 μl were injected per mouse. Tumor formation occurred within 10-15 days. Tumor growth was quantified with calipers.

Example 5 Ad-CD40L / CD40 transduced B16F10 melanoma cells subcutaneously into C57BL6 mice Ad-CD40-CD40L transduced B16F10 in vitro.
For experiments in subcutaneous implantation of melanoma cells into C57BL6 mice, B16
F10 cells were seeded in 12 well plates (Falcon) at 4 × 10 ⁵ cells / well. Vector preparation of Ad-CD40-CD40L chimera, Ad-empty (control) and Ad-lacZ (control), appropriately diluted with transduction medium (DMEM without additives) when cells are adherent. In addition, a vector-free one (mock control) was added to the wells. Infection is 37 ° C and 5%
Performed in a CO ₂ incubator. Two hours after removing the transduction medium, the culture medium with its additives was added and the incubation was continued for another 2 days.

Cell surface expression is expressed in parallel under the same conditions, tagged Ad.
It was indirectly verified by FACS analysis of the CD40-CD40L ^tag .

On day 0, a 200 μl volume of 5 × 10 ⁵ tumor cells was implanted subcutaneously in the lateral abdominal wall.
For this method, mice were anesthetized with ether. Tumor growth was quantified three times a week using calipers 5 days after tumor attachment. Body weight was monitored three times a week. Common disorders were examined daily. Tumor volume is approximately elliptical formula: 4/3 x π / 8 x
It was calculated by (length x width x height).

Example 6 Effect of Ad-CD40-CD40L Administration on Primary Tumor Growth Mouse B16F10 cells were injected in an amount of 5 × 10 ⁵ cells / 200 μl per mouse. Ad-CD40-CD40L (14- 16 days after tumor cell tumor tumor adhesion
Adtypes A and B) or AdCMV-LacZ as a control were injected intratumorally and tumor size was measured every 3 days for approximately 21 days. Different doses by the density of the viral vector ^{(1 × 10 7 ~1 × 10} 9 / 50μl) dependent curves and 1
A single versus multiple injection comparison was made. At the end of the experiment, histopathology was performed on the explanted tumor and examined for activated T cells (C4, CD25).

[0131]   From these studies, optimal measures can be established.

Example 7 T Cell Memory Experiments Example 6 was performed according to Example 5, but in the case of tumor regression, treated mice are exposed contralaterally with intraperitoneal tumor cells. Secondary tumor growth is measured to monitor memory T cell responses.

Example 8 Vaccine Carcinoma Experiments In a further approach, Ad-CD40-CD40L-, ex vivo.
Alternatively, the tumor cells transduced with AdCMV-LacZ were irradiated (5000 rads),
They were then transplanted at a remote site in syngeneic mice bearing the existing B16F10 tumor (confirmed according to Example 4). Tumor growth of the primary tumor was monitored, demonstrating antigenic specificity of the antitumor effect. Different (eg CT-26) tumor cells transduced with Ad-CD40-CD40L- or AdCMV-LacZ as controls, which did not give anti-B16F10 response, were transplanted.

Example 9 In Vivo Tumor Growth of Athymic Mice Demonstrate Whether Tumor-Specific CTL (Cytotoxic T Cell) Activity Plays a Central Role in Reducing Tumor Growth In Vivo For this purpose, the Balb / c nu / nu athymic mouse strain lacking mature T cells was used. B16F10
Cells were engrafted in the same pattern of time as described for syngeneic C57BL / 6 mice. Kinetics of tumor growth were measured under the conditions described in Example 5.

Example 10 Pharmaceutical composition a) Non-viral vector For the preparation of a pharmaceutical composition 100 μg of the non-viral vector according to the invention is added to 1 ml of phosphate buffer (PBS: 8 g sodium chloride per 1 L H ₂ O). 1.
44 g disodium hydrogen phosphate, 0.24 g potassium dihydrogen phosphate, pH 7.
Dissolve in 4).

B) Viral vector For the manufacture of a pharmaceutical composition according to the invention 10 ¹⁰ pFU in a 1 ml volume of PBS.
1 ml of liposomal transfection reagent (
Roche Diagnostics GmbH of Germany; DOSPER: 1,
3-dioleoyloxy-2- (6-carboxy-spermyl) propylamide /
PBS), incubated at room temperature for 15 minutes, and stored at 4 ° C.

[Table 4]

[Table 5]

[Table 6]

[Brief description of drawings]

[Figure 1]   FIG. 1 shows a schematic comparison of type A and type B.

[Fig. 2]   FIG. 2 represents a CD40-CD40L fusion construct (type A).

[Figure 3]   FIG. 3 represents the CD40-CD40L fusion construct (type B).

FIG. 4. pcDNA3-transfected into HEK293 cells and stained with a monoclonal anti- <CD40> antibody against the intracellular region of CD40.
3 shows Western blots of CD40-CD40L chimeric constructs A and B. Control was pcDNA3 transfected into HEK293 cells
-Indicates CD40. A-type forms a band at approximately 40 KD; B-type forms a band at approximately 47 KD. 1 = CD40 (0.1 μg); 2 = CD40 (0.4 μg); 3 = CD40 (1
． 6 = g); 4 = control; 5 = CD40 (0.1 μg); 6 = type A (0.
1 μg); 7 = B type (0.1 μg); 8 = CD40 (0.4 μg); 9 = A type (
0.4 μg); 10 = B type (0.4 μg); 11 = CD40 (1.6 μg); 1
2 = A type (1.6 μg); 13 = B type (1.6 μg).

FIG. 5 shows in vitro functional analysis of CD40L / CD40 fusion constructs. Cis activity test; pcDNA3-CD40-CD40L and pNF
The -κB-CAT reporter plasmid was co-transfected into human 293 cells and tested 48 hours later for CAT expression (CAT-ELISA).

FIG. 6a: Type B in vitro of CD40L / CD40 fusion construct.
Functional analysis at o is shown. Trans Signal Transduction Test: pcDNA3-CD40-
293 cells were transfected with CD40L chimera alone and pcDNA3-CD40 + pNF-κB-CAT, respectively. 24 from transfection
After hours, both pools of cells were incubated together for 24 hours and then C
Tested for AT expression.

FIG. 6b is an in vitro study of Form B of the CD40L / CD40 fusion construct.
The functional analysis in ro is shown. Trans activity test: pcDNA3-CD40-CD4
0L alone and pcDNA3-CD40 + pNF-κB-CAT 293 respectively
The cells were transfected. Twenty-four hours post transfection, pools of both cells were incubated together for 24 hours and then tested for CAT expression.

─────────────────────────────────────────────────── ─── Continuation of front page (51) Int.Cl. ⁷ Identification code FI theme code (reference) A61P 35/00 C12P 21/02 C 4H045 C07K 14/725 C12N 15/00 ZNAA 19/00 5/00 B C12N 5/10 A61K 37/02 C12P 21/02 37/66 G (81) Designated country EP (AT, BE, CH, CY, DE, DK, ES, FI, FR, GB, GR, IE, IT, LU , MC, NL, PT, SE), OA (BF, BJ, CF, CG, CI, CM, GA, GN, GW, ML, MR, NE, SN, TD, TG), AP (GH, GM, K) E, LS, MW, SD, SL, SZ, TZ, UG, ZW), EA (AM, AZ, BY, KG, KZ, MD, RU, TJ, TM), AE, AG, A , AM, AT, AU, AZ, BA, BB, BG, BR, BY, CA, CH, CN, CR, CU, CZ, DE, DK, DM, DZ, EE, ES, FI, GB, GD, GE, GH, GM, HR, HU, ID, IL, IN, IS, JP, KE, KG, KP, KR, KZ, LC, LK, LR, LS, LT, LU, LV, MA, MD, MG , MK, MN, MW, MX, NO, NZ, PL, PT, RO, RU, SD, SE, SG, SI, SK, SL, TJ, TM, TR, TT, TZ, UA, UG, US, UZ, VN, YU, ZA, ZW (72) Inventor Fries, Thomas Germany, Day 82152 Planek, Riesle-Karlstad-Strasse 21 (72) Inventor Mars, Gerd Germany, Day-82377 Penz Berg, Bechler Strasse 3 Ar (72) Inventor Pessara, Ulrich Germany, De-82362 Beilheim, Behrenmuhle 1 (72) Inventor Schoer, Berner Germany, De-82393 Ifeldorf, Gut Steinbach (72) Inventor Zebel , Stefan Germany, Day-82377 Penzberg, Saaranger 34 F-term (reference) 4B024 AA01 AA11 BA63 CA04 CA07 DA02 DA03 EA02 EA04 FA18 GA11 HA17 4B064 AG20 CA10 CA19 CC01 CC24 DA05 DA13 4B065 AA91X AA91 CA14 CA46 CA01 CAACA2 CAA02 4C084 AA02 BA44 DA12 DA14 DA22 MA02 NA05 ZB26 4C086 AA01 AA03 BC43 EA16 MA02 MA03 MA04 NA05 ZB26 4H045 AA10 AA20 AA30 BA10 BA42 CA40 DA50 DA86 EA20 EA50 FA74

Claims (14)

[Claims] 1. A mammalian signal peptide and its downstream, ii) a binding and trimerization domain of CD40L and its downstream, iii) a spacer of 50-100 amino acids and its downstream, iv) transmembrane and signal of CD40. A nucleic acid encoding a chimeric polypeptide comprising a nucleic acid fragment encoding a transduction domain. 2. The nucleic acid according to claim 1, wherein the spacer is a stalk domain of CD40 or CD40L. 3. The method of claim 1 or 2 which encodes between the binding and trimerization domain of CD40L and the spacer, the extracellular region of CD40 or at least a portion of said region that results in binding to CD40L. The described nucleic acid. 4. An additional spacer encoding 1 to 30 amino acids is inserted between the binding and trimerization domain of CD40L and the extracellular region of CD40 or said portion thereof. The nucleic acid according to claim 3. 5. A recombinant expression vector comprising the nucleic acid according to claims 1-4. 6. A mammalian host cell transformed or transfected with the expression vector of claim 5. 7. A host cell transformed or transfected with the expression vector comprising the nucleic acid of any one of claims 1 to 4 is used to express the nucleic acid encoding the chimeric polypeptide. And a method for preparing a chimeric CD40 / CD40L polypeptide, which comprises culturing under conditions that promote the expression and display the polypeptide on the surface of the host cell. 8. A human tumor cell having an expression vector comprising a nucleic acid according to any one of claims 1 to 4, wherein a CD40 / CD40L polypeptide is produced in said tumor cell and the surface of said cell. A method for producing modified human tumor cells, characterized in that they are cultured under the conditions presented above. 9. Chimeric polypeptide encoded by a nucleic acid according to any one of claims 1 to 4, characterized in that the extracellular domain of CD40L is followed by the signal peptide of CD40 or a fragment thereof. 10. The intracellular domain of CD40 consists of amino acids 216-277 of human CD40L or amino acids 216-289 of mouse CD40L, and / or the extracellular domain of CD40L comprises amino acids 47-47 of human CD40L.
Chimeric polypeptide according to claim 9, characterized in that it consists of amino acids 47 to 260 of 261 or mouse CD40L. 11. Chimeric polypeptide according to claim 8 or 9, characterized in that at least 80% of the polypeptide spacer consists of the amino acids Glu and / or Ala. 12. A composition featuring a nucleic acid according to any one of claims 1 to 4 in combination with a pharmaceutically acceptable buffer, excipient and / or preservative. 13. The method of claim 1 for activating antigen presenting cells and T cells.
Use of the nucleic acid according to any one of claims 1 to 4 as a method for producing a composition comprising the nucleic acid according to any one of claims 4 to 4, as an essential constituent of the composition. A method characterized by. 14. A tumor treatment comprising, as an essential component, the vector according to claim 5 and a substance selected from the group consisting of interleukin-2, interleukin-12, interferon-α and 5-fluorouracil. A composition for the treatment of a patient.