WO1999061057A2

WO1999061057A2 - Molecules targeting cd40 and tumor cells

Info

Publication number: WO1999061057A2
Application number: PCT/US1999/011421
Authority: WO
Inventors: David W. Thomas
Original assignee: Tanox, Inc.
Priority date: 1998-05-23
Filing date: 1999-05-21
Publication date: 1999-12-02
Also published as: AU4009899A; WO1999061057A3

Abstract

Disclosed are molecules having one specificity for CD40 and the other for a tumor cell antigen. The molecules may be bispecific, or multispecific, antibodies. The molecules are useful for treating cancer and tumors. Where the tumor cell specificity is directed against a tumor cell surface antigen of sufficient density, upon in vivo administration, a number of bispecific molecules will bind to the tumor cell surface and present, in sum, multivalency for the CD40-expressing cells. The multivalent anti-CD40 can now ligate CD40 on adjacent CD40-expressing cells (e.g., macrophages, dendritic cells, B-lymphocytes, and others), resulting in activation of these CD40 cells, which aid in eliminating the tumor.

Description

Molecules Targeting CD40 and Tumor Cells

Field of the Invention

This invention relates to molecules and bispecific molecules, having one specificity for CD40 and another for a tumor cell surface antigen.

Background of the Invention

The CD40 pathway is a major immunoregulatory pathway for both humoral and

cellular immune responses. CD40 is expressed by B lymphocytes, macrophages and

dendritic cells of the immune system, as well as by several other non-immune cell types.

CD40-mediated cellular activation requires ligation of the cell surface CD40 through

binding to the homotrimer CD40 ligand, which is transiently expressed primarily by

activated CD4 T lymphocytes. CD40-expressing cells can also be artificially activated

by certain agonist anti-CD40 monoclonal antibodies, provided that bivalency is

preserved to ligate the CD40.

CD40 activation of macrophages has been shown to stimulate tumoricidal

activity. In addition, CD40 ligation on macrophages and dendritic cells is a major

stimulus for interleukin-12 generation, which is a major cytokine involved in cellular

immune responses protective against cancerous cells. Further, CD40 has a role in the

generation of immunological memory, needed for generating long-term tumor

immunity. Thus, if macrophages and other leukocytes can be activated via CD40 at the

tumor site, it is predicted that both immediate and tumoricidal activity will be generated,

as well as stimulating the immune response to generate long-term immunity against the

tumor cell antigens. Summary of the Invention

The invention includes bispecific molecules, including bispecific antibodies or diabodies, having one specificity for CD40 which has agonist activity, and the other for

a tumor cell surface antigen. A number of different bispecific molecules or antibodies

can be used, one example being two single chain Fv antibodies linked together. Such

bispecific antibodies (diabodies) are described in U.S. Patent No. 5,534,254 (Creative

Biomolecules, Inc.).

The bispecific molecules of the invention will not activate isolated CD40-

expressing cells because, although the one specificity for CD40 has agonist activity,

monovalent CD40 binding does not provide the ligation required for CD40-mediated

cellular activation. However, if the second specificity is directed against a tumor cell surface antigen of sufficient density, upon in vivo administration, a number of the

bispecific molecules will bind to the tumor cell surface and present, in sum,

multivalency for the CD40-expressing cells. The multivalent anti-CD40 can now ligate

CD40 on adjacent CD40-expressing cells (e.g., macrophages, dendritic cells, B-

lymphocytes, and others), resulting in activation of these CD40 cells.

If the binding affinity of the tumor-specific binding specificity is relatively high

and the anti-CD40 binding affinity is lower, then the bispecific molecule should be

localized at the tumor site, thereby enhancing the efficacy of the treatment and reducing

the product requirements. Therefore, the bispecific approach provides a potentially

superior immunotherapy to a monospecific approach because:

1. The immune responses are highly specific and localized only to the vicinity of the cancer cells;

2. there is immediate generation of tumoricidal activity; and 3. tumor specific cellular and humoral memory immune responses are generated for long lasting tumor immunity.

Making and Using the Invention One embodiment of the invention is a bispecific molecule formed by fusion of

two single chain antibodies (one derived from an agonist antibody specific for CD40

and the other for a tumor cell antigen). Another embodiment is a fusion protein

including a monoclonal antibody to CD40, or fragments thereof, and a monoclonal

antibody to a tumor cell antigen, or a fragment thereof. The monoclonal antibodies

used to form the bispecific molecule include chimeric antibodies, humanized

antibodies, human antibodies, single-chain antibodies and fragments, including Fab,

F(ab') , Fv and other fragments which retain the antigen binding function of the

parent antibody. Single chain antibodies ("ScFv") and the method of their

determination and construction are described in U.S. Patent No. 4,946,778. Single chain antibodies from camels have also been made, and are another alternative.

Chimeric antibodies are produced by recombinant processes well known in the art, and have an animal variable region and a human constant region. Humanized antibodies correspond more closely to the sequence of human antibodies than do chimeric antibodies. In a humanized antibody, only the complementarity determining regions (CDRs) which are responsible for antigen binding and specificity are animal derived and have an amino acid sequence corresponding to the animal antibody, and substantially all of the remaining portions of the molecule (except, in some cases, small portions of the framework regions within the variable region) are human derived and have a corresponding amino acid sequence to a human antibody. See L. Riechmann et al., Nature, 1988; 332: 323-327; United States Patent No. 5,225,539 (Medical Research Council); U.S. Patent Nos. 5,585,089; 5,693,761; 5,693,762 (Protein Design Labs, Inc.). 530,101. Delmmunised™ antibodies are antibodies in which the potential T and B cell

epitopes have been eliminated, as described in International Patent Application

PCT/GB98/01473. Therefore, immunogenicity in humans is expected to be

eliminated or substantially reduced when they are applied in vivo. Human antibodies can be made by several different methods, including by use

of human immunoglobulin expression libraries (Stratagene Corp., La Jolla, California;

Cambridge Antibody Technology Ltd., London, England) to produce fragments of

human antibodies (V_H, N_L. FV, Fd, Fab, or (Fab') ₂), and using these fragments to

construct whole human antibodies by fusion of the appropriate portion thereto, using techniques similar to those for producing chimeric antibodies. Human antibodies can

also be produced in transgenic mice with a human immunoglobulin genome. Such

mice are available from Abgenix, Inc., Fremont, California, and Medarex, Inc.,

Annandale, New Jersey. In addition to connecting the heavy and light chain Fv

regions to form a single chain peptide, Fab can be constructed and expressed by similar means (M.J. Evans et al., J. Immunol. Meth., 1995; 184: 123-138). mice with a

human immunoglobulin genome. Such mice are available from Abgenix, Inc.,

Fremont, California, and Medarex, Inc., Annandale, New Jersey.

All of the wholly and partially human antibodies are less immunogenic than

wholly murine or animal-derived antibodies, as are the fragments and single chain

antibodies. All these molecules are therefore less likely to evoke an immune or

allergic response. Consequently, they are better suited for in vivo administration in

humans than wholly animal antibodies, especially when repeated or long-term administration is necessary, as may be needed for treatment with the bispecific

antibodies of the invention.

Bispecific antibodies and the method of making them are described in U.S.

Patent No. 5,534,254 (Creative Bimolecules, Inc.). Different embodiments of

bispecific antibodies described in the patent include linking single chain Fv with

peptide couplers, including Ser-Cys, (Gly) -Cys, (His)₆-(Gly) -Cys, chelating agents,

and chemical or disulfide couplings including bismaleimidohexane and

bismaleimidocaproyl. All such linkers and couplings can be used with the diabodies

of the invention.

The bispecific molecules of the invention are administered as a pharmaceutical

composition at a concentration that is therapeutically effective to treat, shrink or prevent

growth of tumors. The dosage and mode of administration will depend on the

formulation, the cancer type being treated, and other factors which can be readily

determined during routine human clinical trials or through extrapolation from animal

studies.

Typically, the pharmaceutical composition is administered by injection, either

intravenously or intraperitoneally. It may also be possible to obtain compositions which

may be topically or orally administered, or which may be capable of transmission across

mucous membranes. Before administration to patients, formulants are preferably added to the

pharmaceutical composition. For example, these formulants may include oils, polymers,

vitamins, carbohydrates, amino acids, salts, buffers, albumin, surfactants, or bulking

agents. Carbohydrates may include sugar or sugar alcohols such as mono, di, or polysaccharides, or water soluble glucans. The saccharides or glucans can include

fructose, dextrose, lactose, glucose, mannose, sorbose, xylose, maltose, sucrose, dextran, pullulan, dextrin, alpha and beta cyclodextrin, soluble starch, hydroxethyl starch and carboxymethylcellulose, or mixtures thereof. Sugar alcohols include C to C₈

hydrocarbon having an -OH group, for example, galactitol, inositol, mannitol, xylitol,

sorbitol, glycerol, and arabitol. The sugars or sugar alcohols mentioned above may be

used individually or in combination at any concentration which allows them to be soluble in the aqueous preparation, which concentration may be between 1.0 w/v% and

7.0 w/v%, and, perhaps preferably, between 2.0 and 6.0 w/v%.

Amino acids may also be present, including levorotary (L) forms of carnitine,

arginine, and betaine; however, other amino acids may also be added. Polymers may

include polyvinylpyrrolidone (PVP) with an average molecular weight between 2,000

and 3,000, or polyethylene glycol (PEG) with an average molecular weight between

3,000 and 5,000. A buffer in the composition may also be used to minimize pH changes

in the solution before lyophihzation or after reconstitution. Any physiological buffer

may be used, but citrate, phosphate, succinate, and glutamate buffers or mixtures thereof

are often preferred, at a concentration from 0.01 to 0.3 molar. Surfactants that can be added to the formulation are described in EP Patent Nos. 270,799 and 268,110.

Additionally, pharmaceutical compositions can be chemically modified by

covalent conjugation to a polymer to increase their circulating half-life. Polymers, and

methods to attach them to peptides, are shown in U.S. Patent Nos. 4,766,106; 4,179,337; 4,495,285; and 4,609,546, and include polyoxyethylated polyols and PEG. PEG is

soluble in water at room temperature and has the general formula: R(O-CH -CH₂)_nO-R where R can be hydrogen, or a protective group such as an alkyl or alkanol group. The

protective group may have between 1 and 8 carbons, and, perhaps more preferably, it is

methyl.

Water soluble polyoxyethylated polyols can also be used in the present

invention. They include polyoxyethylated sorbitol, polyoxyethylated glucose, or,

preferably, polyoxyethylated glycerol (POG), because the glycerol backbone of POG is

the same backbone occurring naturally in, for example, animals and humans in mono-,

di-, and triglycerides. Therefore, this branching would not necessarily be seen as a

foreign agent in the body. The POG may preferably have a molecular weight in the

same range as PEG. The vehicles for POG formulations are saline, Ringer's solution,

dextrose solution, and Hanks' solution. Nonaqueous (J. Bio. Chem. 263:15064, 1988)),

and a discussion of POG/IL-2 conjugates is found in U.S. Patent No. 4,766.

Administration can be parentally, in a unit dosage injectable form such as a

solution, suspension or emulsion, in association with a pharmaceutically acceptable

parental vehicle. Such vehicles are inherently nontoxic and nontherapeutic, and include

fixed oils, ethyl oleate, or, preferably, 5% dextrose in saline. The vehicle may contain

minor amounts of additives, including substances that enhance isotonicity and chemical

stability, e.g., buffers and preservatives.

The invention will now be described with reference to the following examples.

Example I

Generation of single chain antibody fragments from monoclonal antibodies to human CD40 and a Tumor Cell Antigen.

The techniques described in this Example I were used to generate a single chain antibody fragment (ScFv) of the anti-CD40 monoclonal antibody 5D12 (deposited at the ATCC, Rockville, Maryland under Accession number HB 11339, as

described in US Patent No. 5,677,165). The 5D12 antibody is an antagonist antibody,

and therefore not suitable for use in the invention, where agonist anti-CD40 activity is needed. However, similar methods to those used for generation of the 5D12-derived

single chain antibody fragment could be used to produce a single chain antibody

fragment with agonist activity for anti-CD40 when organised on a solid support or

other assay allowing multivalent binding. These methods are described below, along

with methods for generating a single chain antibody fragment for a tumour cell

antigen.

Both the V_H and V_L region are amplified by PCR, followed by a second

assembly PCR to connect both regions. Four primers are designed. The first contains

a Hindlll and Sfil restriction site for cloning purposes followed by a degenerated

sequence annealing to the 5' V_H region. The second contains a degenerate sequence for the 3' part of the V_H region followed by a sequence encoding a ((Gly)₄Ser)₃ linker

and the 5' part of the V_L regions. The third is a degenerated primer having homology

with the 5' part of the V_L region, while the last primer contains a Notl restriction site

and anneals to the 3' part of the V region.

As a template for this PCR reaction, one can use a plasmid containing the V_H

or V regions of the antibody of interest. The cDNA obtained in this PCR step is gel

purified and used in an assembly PCR resulting in the linkage of the V region through

the ((Gly) Ser)₃ linker. Subsequently the obtained single chain construct is digested

with the restriction enzymes Hindlll and Notl, followed by ligation in pGEM-13Zf (Promega, Madison, USA). The ligation is transformed in DH5 and plated on LB

plates. By sequencing of several clones, a correct ScFv clone is found.

For the generation of ScFvs reactive with a human tumor cell antigen,

following isolation of the cell line of interest, one would use an analogous primer set

and repeat the steps above.

Example II

Construction of bi-specific diabody molecules capable of binding to human CD40 and a human tumor cell antigen:

Bispecific bivalent molecules can be generated by shortening the flexible

linker sequence in the anti-CD40 ScFv and in the anti-tumor cell ScFv, from fifteen

residues to five (Gly₄Ser) and by cross-pairing the variable heavy and light chain

domains from the two single chain Fv fragments with the different antigen

recognition. The construction is preferably performed in three steps. The light chain

variable fragments are exchanged in the ScFv constructs from anti-tumor cell antigen

("aTC") and anti-CD40 ("aCD40") by using restriction enzyme sites located in the 5'-

end and just outside the 3 '-part of the light chain variable gene. In the following step

the 15 -residue linker of the chimeric constructs V_H-aTC/15AA-linker/V_L-aCD40 and

V_H-aCD40/15AA-linker/VL-aTC is replaced by the 5 residue linker (Gly₄Ser) by using

sites located in the 3 '-part of V_H and the 5 '-part of V_L. Finally, both chimeric cassettes

are combined in the vector pUC119-fabsol (a pUC119 derivative similar to

pUC119His6mycXba (Low et al, J. Mol. Biol. 260:359 (1996), but with all Apall-

sites in the vector backbone deleted by in vitro mutagenesis) containing a bi-cistronic

expression cassette. A diabody producing clone containing both ScFv-cassettes is

identified and used for expression of the recombinant diabody molecule. Example III

Expression, isolation and characterization of diabodies capable of binding to human CD40 and a human tumor cell antigen:

The plasmid containing the aTC/aCD40-bicistronic expression cassette

described in Example II is used and expressed in a suitable host cell. The functionality

of the produced diabody is tested in BIAcore. Purified aTC-Ig is immobilized on the

surface of a CM-chip, and tested to determine if it yields the required Response Units

for a coupled protein. Injection of the periplasmic fraction of the diabody for 120

seconds with a flow rate of 10 μl/min results in the capture of diabody. Subsequently,

CD40-Ig is injected under the same conditions as the diabody resulting in the binding

of additional antigen. This experiment demonstrates the capability of the produced

diabody molecule to bind CD40 and the tumor cell antigen simultaneously. Further in

vitro and in vivo characterization can also be performed, including determining the

relative affinity for CD40 and tumor cell antigen.

Claims

WHAT IS CLAIMED IS:

1. A molecule having one specificity for CD40 and the other for a tumor cell antigen.

2. The molecule of claim 1 which is bispecific.

3. The molecule of claim 1 which comprises antigen binding regions from two

different antibodies linked together, wherein the antibody specific for CD40 is an

agonist.

4. The molecule of claim 2 wherein the antibodies are linked by a peptide, or a

chemical or disulphide coupling.

5. The molecule of any of claims 1 to 4 included in a pharmaceutical composition with

a pharmaceutical excipient.

6. Recombinant vectors comprising the nucleic acid sequences encoding the

recombinant antibody fragments according to claims 3 or 4, and the necessary

control elements to enable the expression of said recombinant antibody fragments in

a suitable host cell.

7. A method for producing the bispecific molecule, comprising the steps of culturing a

host cell which is transformed with a vector according to claim 6, under conditions

enabling the expression of said the bispecific molecule in said host cell and

subsequently purifying said the bispecific molecule.

8. A method for treating tumors or cancers, the method comprising administering to a

patient in need of such treatment a therapeutically effective amount of a

pharmaceutical composition according to any of claims 1 to 5.

9. A method for treating tumors or cancers, comprising administering a pharmaceutical

composition according to any of claims 1 to 5 into a tumor or cancerous site.

10. A method for treating tumors or cancers, comprising administering gene constructs

encoding a molecule having one specificity for CD40 and the other for a tumor cell

antigen 16.

11. The method of claim 10 wherein the molecule is an antibody or fragment thereof, or

a peptide.

12. Cells transfected or infected with the gene construct of claims 10 or 11.

13. The method of claims 10 or 11 wherein transfection or infection of the gene constructs is done ex vivo or in vivo.

14. The method of claim 13 wherein the transfection is done ex vivo by

electroporation, calcium phosphate transfection, micro-injection or by incorporating

the gene constructs into suitable liposomes.

15. The method of claim 14 wherein the infection is done in vivo or ex vivo by

incorporating the gene constructs into a retrovirus, adenovirus or a parvovirus vector,

or by incorporating the gene constructs, or the gene constructs with a viral or plasmid

vector, into a suitable liposome.

SEQUENCE LISTING

<110> Mark de Boer Cornells J.M. Melief

Rienk Offinga Rene Toes Stephen P. Schoenberger <120> CD40 Binding Molecules and CTL Peptides for Treating Tumors

<130> 98-4 <150> 06/086,625

<151> 1998-05-23

<160> 2 <170> FastSEQ for Windows Version 3.0

<210> 1 <211> 9 <212> PRT <213> mouse

<400> 1 Val Asn lie Arg Asn Cys Cys Tyr lie

<210> 2 <211> 10 <212> PRT <213> mouse

<400> 2 Ser Gly Pro Ser Asn Thr Pro Pro Glu lie 1 5 10