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 (VH, NL. 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 VH and VL 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' VH region. The second contains a degenerate sequence for the 3' part of the VH region followed by a sequence encoding a ((Gly)4Ser)3 linker
and the 5' part of the VL regions. The third is a degenerated primer having homology
with the 5' part of the VL 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 VH
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 VH-aTC/15AA-linker/VL-aCD40 and
VH-aCD40/15AA-linker/VL-aTC is replaced by the 5 residue linker (Gly4Ser) by using
sites located in the 3 '-part of VH and the 5 '-part of VL. 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.