Intracellular Immunity
The present invention relates to compounds which are conjugates of a ligand specific for
a pathogen, and a RING domain. In one embodiment, the RING domain is derived from
a TRIM polypeptide, such as TRIM21.
Viruses and their hosts have been co-evolving for millions of years and this has given rise
to a complex system of immunity traditionally divided into innate and adaptive
responses 1. Innate immunity comprises germ-line encoded receptors and effector
mechanisms that recognise pathogen -associated molecular patterns, or PAMPs2. The
advantage of innate immunity is that it is fast and generic; however viruses are adept at
avoiding recognition by inhibiting innate immunity or by changing their molecular patterns.
In contrast, adaptive immunity can 'cure' a host of infection and provide protection against
future infection. Unlike the PAMP receptors of innate immunity, adaptive immunity uses
proteins such as antibodies to target pathogens. Antibodies are unique in the human
body in that they evolve during the lifetime of an individual and can continue to target
evolving pathogens 3. The weakness of adaptive immunity is that it can take 1-2 weeks to
reach full effectiveness. Furthermore, the dogma of antibody immunity for the last 100
years has been that antibodies only provide extracellular protection 1. It is thought that
once a virus has entered the cytosol of a cell, antibodies are unable to prevent its
infection.
Intracellular antibodies have been developed; for example, see Moutel S, Perez F., Med
Sci (Paris). 2009 Dec; 25(12):1 173-6; Stocks M., Curr Opin Chem Biol. 2005
Aug;9 (4):359-65. However, results using intracellular antibodies, or intrabodies, have
been mixed. In general, attmpts to develop intracellular antibodies have focussed on
single chain antibody fragments, such as scFvs and single domain antibodies, such as
antibodies and dAbs.
Antibodies and immune sera have long been used for the treatment of pathogenic
infections. Fore example, horse antiserum was used in the 1890s to treat tetanus and
diphtheria. However, antisera are seen as foreign by the human immune system, which
reacts by producing antibodies against them, especially on repeat doses. During most of
the 20th C, the adverse effect of animal antibodies prompted the use of human antiserum
from donors who had recovered from disease, typically for prophylaxis of respiratory and
hepatitis B infections following a reduction in the popularity of antibody therapy due to
problems with toxicity, humanised and human antibodies have eliminated such concerns,
and led to a return of such therapeutic approaches. See Casadevall et al., Nature
Reviews Microbiology 2 , 695-703 (September 2004), for a review. Diseases which have
been targeted using antibody therapy include anthrax, whooping cough, tetanus,
botulism, cryptococcosis, cryptosporidiosis, enterovirus gastrointestinal-tract infections,
group a streptococcal infections, necrotizing fasciitis, hepatitis B, measles, tuberculosis,
meningitis, aplastic anaemia, rabies, RSV infection, pneumonia, shingles, chickenpox
and pneumonia due to VZV, and smallpox. Despite these developments, however,
antibody therapy is considered only when no other suitable therapies are available,
requiring high doses of antibody and producing unpredictable results.
The effectiveness of antibodies against pathogens is understood to be at least partly
dependent on the Fc portion of the antibody, which is responsible for mediating the
effects of complement. Therefore, antibody fragments have not been generally proposed
for antiviral therapy, despite their advantages of small size and lower cost of production.
The primary therapy for viral diseases remains vaccination, which is a prophylactic
approach. It is believed that viral antigens, processed by antigen-presenting cells such
as dendritic cells, are presented to the immune system and induce naive T-cells to
differentiate into memory and effector T-cells. Memory T-cells are responsible for the
more aggressive and immediate immune response to a secondary infection, mediating
the benefits of vaccination. For a review, see Kaech et al., Nature Reviews Immunology,
volume 2, April 2002, 25 .
Another immunologically-based approach to the therapy of infections disease is the use
of cytokines, including inteferons. Interferon was first proposed for the treatment of
cancer and multiple sclerosis, as well as viral infections. It has been licensed for the
treatment of hepatitis C since 1998. Moreover, low dose oral or intranasal interferon is
administered for the treatment of colds and flu, especially in eastern Europe. However,
the mechanism of its action is not known, since the doses used are believed to be lower
than the doses at which an antiviral effect could be observed. O'Brien et al., J Gen Virol.
2009 Apr;90(Pt 4):874-82, used interferon as an adjuvant to an adenovirus-delivered
vaccine against VEEV; they observed a decrease in protection against the virus, but an
increase in the immune response to the viral vector.
Recently, we described an intracellular cytosolic protein called TRIM21 that is capable of
binding to an invariant region of antibody molecules via its PRYSPRY domain 4.We found
this activity to be structurally, thermodynamically and kinetically conserved across
mammals5. Hypotheses for the function of TRIM21 have been suggested, including its
involvement in apoptosis and a role in directing of unfolded IgG made in B-cells to the
proteasome.
Summary of the Invention
Antibodies are extracellular proteins, as are all known mammalian IgG receptors (with the
exception of FcRn, which is intracellular but not cytosolic). It therefore seemed
incongruous to us that TRIM21 should be a universally conserved intracellular protein,
and yet be a high affinity, highly specific IgG receptor. We hypothesized that perhaps
current understanding of antibody immunity is incomplete and that there is a 'missing'
system of immunity taking place inside cells, mediated by TRIM21. Data presented herein
demonstrates the existence of this missing immune system and its operation in
preventing infection by two unrelated viruses - dsDNA Adenovirus and ssRNA Coxsackie
virus.
In a first aspect of the invention, therefore, there is provided a compound comprising:
(a) a ligand which binds, directly or indirectly, specifically to an antigen of a pathogen,
provided that said ligand is not the PRYSPRY domain of TRIM21 ; and
(b) a RING domain and/or an inducer of TRIM21 expression.
We have shown that TRIM21 is a high-affinity ligand for immunoglobulins the RING
domain of TRIM 2 1 is an E3 ligase, which is ubiquitinated and directs the
immunoglobulins, together with bound antigens, to the proteasome.
In accordance with the present invention, at least a RING domain, such as the RING
domain of a TRIM polypeptide, can be bound to a ligand for an antigen. Such a ligand
preferably binds directly to the antigen, and may comprise at least part of an
immunoglobulin molecule; however, other ligands may be used, including peptides,
peptide and nucleic acid-based aptamers, naturally-occurring ligands, receptors, and
binding fragments thereof.
In another embodiment, the ligand binds indirectly to the antigen. For example, the
ligand may bind to immunoglobulins non-specifically, such as to the Fc portion of an
immunoglobulin. In such an embodiment, the ligand is not the TRIM21 PRYSPRY
domain. Exemplary ligands include Protein A, Protein G, Protein L, peptides, for instance
peptides which recognise immunoglobulin Fc regions, anti-Fc antibodies and fragments
thereof, and the like. The target specificity is provided, in this case, by an antibody or
antibody fragment which is specific for the antigen of the pathogen. This antibody may
be coadministered with the compound of the invention, or may be naturally occurring.
In the context of the present invention, the term "ligand" is used to refer to either half of a
binding pair.
Where the ligand is an immunoglobulin, it can be any immunoglobulin molecule, for
example an immunoglobulin molecule selected from the group consisting of an IgG, IgA,
IgM, IgE, IgD, F(ab')2, Fab, Fv, scFv, dAb, VH . IgNAR, a TCR, and multivalent
combinations thereof. Multivalent antibodies include, for instance, bivalent antibodies
and antibody fragments, bispecific antibodies and antibody fragments, trivalent versions
thereof, and proprietary formats such as diabodies. Single domain antibodies, such as
dAbs and V H antibodies, are particularly suitable for combining to form multivalent and/or
multispecific molecules.
Where the ligand is an antibody, the antibody molecule comprises at least one of a VH
domain and a VL domain, or the equivalent thereof.
In one embodiment, the TRIM polypeptide is selected from the group consisting of
TRIM5ct, TRIM19, TRIM21 and TRIM28. Although TRIM21 is preferred due to its
antibody-binding properties, if the polypeptide, or a domain thereof, is bound to the
antigen itself, or a ligand specific for the antigen, the antibody-binding ability is no longer
required. In such an instance, the RING domain from a TRIM polypeptide other than
TRIM21 can be used, to the same effect. Advantageously, another domain, such as the
B-box domain or the coiled coil domain can also be added. The coiled coil domain is
responsible fo TRIM21 dimerisation.
Preferably, the RING domain is present in two or more copies on the compound
according to the invention. Dimerisation of TRIM21 occurs through its coiled coil domain,
and assists in the targeting of the protein to the proteasome through E3-mediated ligation
of ubiquitin.
In one embodiment, the compound of the invention comprises a substantially intact TRIM
polypeptide, wherein the PRYSPRY (B30.2) domain has been replaced with an antigen
or an antigen -specific ligand. For example, it can be replaced with an antibody,
comprising at least one of a VH domain and a VL domain.
In a further embodiment, the compound of the invention comprises an inducer of TRIM
expression instead of, or as well as, a TRIM domain. TRI M21 expression is upregulated
by interferon, so the inducer of TRIM expression is advantageously interferon or an
interferon inducer. A variety of interferon inducers, including bacterial polysaccharides
and nucleoside analogues such as poly l:C, are known in the art.
Interferon inducers can act intracelluarly, or at the cell surface. Where the interferon
inducer acts at the cell surface, at least a proportion of the compound which is
administered to a subject will be retained on the cell surface, bound to the interferon
inducer receptor. In an advantageous embodiment, the interferon inducer may be bound
to the compound by a labile linkage, for example a linkage with a limited half-life under
physiological conditions. For example, the half life would be sufficient for the ligand to bid
to the pathogen and to the interferon receptor, but not significantly longer.
In a second aspect, there is provided method for treating a pathogenic infection,
comprising administering to the subject a compound according to the first aspect of the
invention.
Similarly, there is provided the use of a compound according to the first aspect of the
invention, for inducing an immune response in a subject.
In a third aspect, the invention provides a method for treating an infection in a subject,
comprising co-administering to the subject an antibody specific for an antigen of a
pathogen causing said infection, and a polypeptide comprising a ligand which binds to
said antibody and a RING domain.
Similarly, there is provided the use of an antibody specific for an antigen of a pathogen
causing an infection in a subject, and a polypeptide comprising a ligand which binds to
said antibody and a RING domain, for the treatment of said infection.
We have demonstrated that treatment of cells with a virus-specific antibody and wild-type
or modified TRIM21 leads to inhibition of viral infectivity, even in cells in which
endogenous TRIM21 has been knocked down. Accordingly, the coadministration of
TRIM21 can be used to enhance antiviral therapy used for the treatment of an infectious
disease.
In a fourth aspect, there is provided a method for treating an infection in a subject
suffering from such an infection, comprising administering to the subject a therapeutically
effective amount of a polypeptide comprising a ligand which binds, indirectly, to an
antigen of a pathogen and a RING domain.
Similarly, there is provided the use of a polypeptide comprising a ligand which binds,
indirectly, to an antigen of a pathogen and a RING domain for the treatment of an
infectious disease in a subject.
Preferably, the polypeptide comprising the PRYSPRY domain of TRI 21 and a RING
domain comprises further domains of TRIM polypeptides, such as from TRIM21. In one
embodiment, the polypeptide comprises a coiled coil domain and/or a B-box domain. In
one embodiment, the polypeptide is TRIM21, preferably human TRIM21.
TRIM21 has not previously been proposed to possess anti-infective properties. However,
as shown herein, it binds with very high affinity to the Fc receptor of IgG and IgM, and
directs the antibody plus any bound antigen to the proteasome. Exogenous TRIM21,
therefore, potentiates an endogenous antibody response to a pathogen.
Our results reveal that there is a missing system of intracellular immunity through which
antibodies mediate the neutralisation of virus inside the cytosol of infected cells. This
intracellular system combines features traditionally associated exclusively with either
adaptive or innate immunity. Pathogen targeting is provided by adaptive immunity in the
form of antibodies whereas neutralisation is provided by an intracellular receptor
(TRIM21) and innate degradation pathway. TRIM21 is distinct from other antibody
effector mechanisms, which are systemic and based on immune surveillance. TRIM21 is
expressed in most cells and not just professional immune cells, which means that every
infection event is an opportunity for neutralisation. Encapsulating immunity within host
cells may be crucial to inhibiting viral spread. Finally, TRIM21 utilises both IgM and IgG
suggesting that it operates alongside both innate immunity during the early stages of
infection and adaptive immunity to provide long-term protection.
TRIM21 may have been contributing to many antibody neutralisation experiments over
the last 100 years. Indeed, as we see that TRIM21 mediates a potent antibody
neutralisation of adenovirus it will be important to reassess whether the antibody
neutralisation of other viruses is caused by a block to entry or is TRIM21 -dependent. This
may be an important consideration in vaccine design, as effective vaccines may need to
stimulate TRIM21 immunity. e suggest that a good predictor of TRIM21 involvement in
the antibody neutralisation of other viruses will be a synergistic relationship between
interferon and antibody. Indeed unexplained synergy between interferon and antibody
has been reported for herpes simplex virus8, enterovirus 708 and sindbis virus 9. TRIM21
may also contribute to viral neutralisation in experiments where no antibody is added
since the calf serum used in routine tissue culture contains a repertoire of antibodies of
potentially cross-reactive specificity.
The existence of a TRIM21/antibody intracellular immune response may help to resolve
several unexplained observations in viral infection. It has been reported that antibody
neutralisation of both poliovirus 10 and respiratory syncytial virus 1 occurs even when
viruses are allowed to pre-adhere to target cells. It has also been observed that a single
IgG is sufficient to mediate neutralisation of poliovirus 12 and adenovirus 13 and only 5-6
IgG molecules are required for rhinovirus 14 . Finally, there are numerous reports of intact
antibodies being far more effective than their proteolysed fragments, even than Fab2 that
maintain bivalent antigen binding. For instance, Fab2 fragments have been shown to be
less effective than intact IgG in the neutralisation of Yellow fever virus 15 , HSV16 and
Influenza 17 , suggesting an Fc domain effector function for efficient neutralisation.
TRI 21-mediated degradation may explain all these phenomena.
Brief Description of the Figures
Figure 1: TRIM21 mediates intracellular antibody neutralisation. (A) Confocal
microscopy images of adenovirus infected HeLa cells. Adenovirus pre-coated in antibody
and detected after infection with an Alexa-fluor546 secondary (red) can be seen inside
cells. Endogenous TRIM21 localisation is shown in green and DAPI stained nucleus in
blue. A merge of these channels shows localisation of TRIM21 to antibody-coated
virions. The images are a Z-projection and the scale bars are 10 m and 2 m in the
zoomed box. (B) Cells treated with IFNa, TRI 21 siRNA (KD), siRNA control (HeLa) or
IFNa & TRIM21 siRNA (IFNa KD) were infected with GFP adenovirus at different
polyclonal antibody concentrations. The level of infection was determined by measuring
the percentage of GFP positive cells and for each condition normalised to the levels in the
absence of antibody. Adenovirus infection is reduced by 2-logs in cells expressing the
highest levels of TRIM21. (C) Western blot of TRIM21 protein levels in each condition. (D)
Infection of treated cells with coxsackievirus in the presence of increasing concentrations
of human serum IgG. IFNa and antibody operate synergistically to neutralise virus. This
effect is reversed by specifically knocking-down TRIM21 .
Figure 2: TRIM21 neutralises infection independent of cell-type and antibody. (A)
Neutralisation of TRIM21 is reversed by knockdown, independent of siRNA sequence or
siRNA vs shRNA, in the presence of antibody. (B) TRIM21 neutralises adenovirus
infection in three different cell lines. Neutralisation is enhanced by IFNa and reversed by
knockdown (KD). (C) TRIM21 neutralises adenovirus infection when using different
polyclonals or an anti-hexon monoclonal IgG. (D) Entry neutralisation is minimal in
comparison with TRI 2 1-mediated neutralisation. Antibody-dependent TRIM21
neutralisation of virus requires the presence of the Fc domain. Fab2 fragments are
bivalent and have the same potential for entry-neutralisation as intact IgG yet they show a
limited affect on infection. This is confirmed by knockdown of TRIM21, which reverses
IgG neutralisation. (E) TRIM21 binds to serum Ig . Binding of Ig to TRIM21 was
measured as a change in fluorescence anisotropy and fit to a standard quadratic
expression (Materials & Methods) to give an affinity of 16.8mM ± 1.5mM. (F) Serum IgM
antibodies can also be used by TRIM21 to neutralise virus. Knockdown of TRIM21 (KD)
reverses this effect and IFNa increases it. Error bars in all panels were calculated from
triplicate experiments. (G) IgA antibodies can moreover be used by TRIM21 to neutralise
virus. Knockdown of TRIM21 using siRNA (siTRIM21) reverses this effect and IFNa
increases it. Control siRNA (siControl) has no effect.
Figure 3: Mechanism of TRIM21 neutralisation. (A) SEC MALS chromatograms of
TRIM21 (black), IgG (light gray) and TRIM21 in complex with IgG (dark gray). The
continuous traces represent the Rl signal (left-hand axis) and are an indication of protein
concentration. The short horizontal lines represent the calculated mass at each sampling
interval (1s) within each peak (right-hand axis). Analysis indicates that TRIM21 is dimeric
with a mass of 107kDa, that IgG has a mass of 154kDa, and that TRIM21:lgG complex
yields a peak corresponding to free IgG and a 1: 1 complex with mass -280kDa. (B&C)
Steady-state fluorescence titration of IgG with full-length TRIM21 (left-hand) and ARING-
DBOC TRIM21 (right-hand). Titrations were fit to a standard quadratic expression
(Materials & Methods) to give an affinity of full-length TRIM21 to antibody of 0.6 ± 0.1 nM
(B) and 0.9 ± 0.2 nM for ARING-ABOX TRIM21 (C). (D) TRIM21 neutralisation is reversed
by the proteasome inhibitor MG132 but not the autophagy inhibitor 3-MA. Error bars were
calculated from triplicate experiments. (E) Direct correlation between MG132
concentration and reversal of TRIM21 neutralisation. MG132 only reverses neutralisation
in the presence of antibody. (F) Proteasomal degradation, TRIM21 and antibody are
necessary factors in the same pathway of viral neutralisation. For example, knockdown
of TRIM21 obviates the MG132 affect.
Figure 4 : TRIM21 E3 ubiquitin ligase function is essential for viral neutralisation (A)
Recombinant full-length TRIM21 neutralises virus but TRIM21 lacking the RING and B
Box domains does not. (B) TRIM21 is an active E3 ligase but deletion of RING and B Box
domains prevents autoubiquitination. (C) TRIM21 does not directly ubiquitinate hexon or
its associated antibody during infection. (D) Confocal microscopy Z-projection showing
HeLa cells infected with antibody-coated adenovirus. TRIM21 co-localised virions are
positive for ubiquitin. (E) Western blots of hexon, antibody and TRIM21 protein levels 1-6
hours post-infection. Adenovirus hexon protein and antibody are rapidly degraded in a
TRIM 1-dependent manner. Addition of MG132 partially rescues degradation.
Degradation does not significantly affect the cellular pool of TRIM21 .
Figure 5: Intracellular antibody-coated beads recruit TRIM21 and are ubiquitinated.
Streptavidin-conjugated latex beads are coated with anti-streptavidin antibody and
transfected into cells. Intracellular beads are recognised by TRIM21 and colocalise with
ubiquitin. The scale bars represent 10 m and 5 pm in the zoomed box.
Detailed Description of the Invention
Unless otherwise stated, all technical and scientific terms used herein have the same
meanings as commonly understood by one of ordinary skill in the art to which this
invention belongs. Any methods and materials similar or equivalent to those described
herein can be used in the practice or testing of the present invention. Methods, devices,
and materials suitable for such uses are now described. All publications cited herein are
incorporated herein by reference in their entirety for the purpose of describing and
disclosing the methodologies, reagents, and tools reported in the publications that might
be used in connection with the invention.
The practice of the present invention employs, unless otherwise indicated, conventional
methods of chemistry, biochemistry, molecular biology, cell biology, genetics,
immunology and pharmacology, known to those of skill of the art. Such techniques are
explained fully in the literature. See, e. g. , Gennaro, A. R., ed. (1990) Remington's
Pharmaceutical Sciences, 18th ed., Mack Publishing Co.; Hardman, J. G., Limbird, L. E.,
and Gilman, A. G., eds. (2001) The Pharmacological Basis of Therapeutics, 10th ed.,
McGraw-Hill Co.; Colowick, S. et al., eds., Methods In Enzymology, Academic Press, Inc.;
Weir, D. M. , and Blackwell, C. C , eds. (1986) Handbook of Experimental Immunology,
Vols. I-IV, Blackwell Scientific Publications; Maniatis, T. et al., eds. (1989) Molecular
Cloning: A Laboratory Manual, 2nd edition, Vols. I-III, Cold Spring Harbor Laboratory
Press; Ausubel, F. M. et al., eds. (1999) Short Protocols in Molecular Biology, 4th edition,
John Wiley & Sons; Ream et al., eds. (1998) Molecular Biology Techniques: An Intensive
Laboratory Course, Academic Press; Newton, C. R., and Graham, A., eds. (1997) PCR
(Introduction to Biotechniques Series), 2nd ed., Springer Verlag.
In the context of the present invention, administration is performed by standard
techniques of cell culture, depending on the reagent, compound or gene construct to be
administered. For instance, administration may take place by addition to a cell culture
medium, introduction into cells by precipitation with calcium phosphate, by
electroporation, by viral transduction or by other means. If the method of the invention
employs a non-human mammal as the test system, the mammal may be transgenic and
express the necessary reagents in its endogenous cells.
An antigen, in the context of the present invention, is a molecule which can be recognised
by a ligand and which possesses an epitope specific for a pathogen. Typically, an
antigen is an antigenic determinant of a pathogen, such as a virus or a bacterium, and is
exposed to binding by ligands such as antibodies under physiological conditions.
Preferred antigens comprise epitopes targeted by known neutralising antibodies or
vaccines specific for a pathogen.
A pathogen may be any foreign body, such as an organism, for example a bacterium or a
protozoan, or a virus, which can infect a subject. Advantageously, the pathogen is a
virus. Viruses may be enveloped or non-enveloped. In one embodiment, the pathogen is
a non-enveloped virus.
A ligand which binds directly to an antigen is a ligand which is capable of binding
specifically to an antigen under physiological conditions. As used herein, the term
"ligand" can refer to either part of a specific binding pair; for instance, it can refer to the
antibody or the antigen in an antibody-antigen pair. Antibodies are preferred ligands, and
may be complete antibodies or antibody fragments as are known in the art, comprising for
example IgG, IgA, Ig , IgE, IgD, F(ab')2, Fab, Fv, scFv, dAb, VHH, IgNAR, a modified
TCR, and multivalent combinations thereof. Ligands may also be binding molecules
based on alternative non-immunoglobulin scaffolds, peptide aptamers, nucleic acid
aptamers, structured polypeptides comprising polypeptide loops subtended on a nonpeptide
backbone, natural receptors or domains thereof.
A ligand which binds indirectly to an antigen is a ligand which binds to the antigen via a
second ligand. For instance, it is a ligand which binds to an antibody. The ligand binds
the antibody in a manner independent of the binding specificity of the antibody; for
instance, it can bind the Fc region. In one embodiment, the ligand is selected from the
group comprising Protein G, protein A, Protein L, the PRYSPRY domain of TRI 2 1, an
anti-immunoglobulin antibody, and peptides which specifically recognise antibodies, for
example in the Fc region .
The PRYSPRY domain of TRIM21 is comprised of the PRY and SPRY regions,
respectively at positions 286-337 and 339-465 of the human TRI 2 1 amino acid
sequence, as set forth in SEQ ID No. 1.
The RING domain is of human TRIM21 between amino acids 15 and 58 of the human
TRIM21 amino acid sequence, as set forth in SEQ ID No. 1.
The BBOX domain is of human TRIM21 between amino acids 9 1 and 128 of the human
TRIM21 amino acid sequence, as set forth in SEQ ID No. 1.
The Coiled Coil domain is of human TRI 2 1 between amino acids 128 and 238 of the
human TRI 2 1 amino acid sequence, as set forth in SEQ ID No. 1.
The term "immunoglobulin" refers to a family of polypeptides which retain the
immunoglobulin fold characteristic of antibody molecules, which contains two beta sheets
and, usually, a conserved disulphide bond. Members of the immunoglobulin superfamily
are involved in many aspects of cellular and non-cellular interactions in vivo, including
widespread roles in the immune system (for example, antibodies, T-cell receptor
molecules and the like), involvement in cell adhesion (for example the ICAM molecules)
and intracellular signalling (for example, receptor molecules, such as the PDGF receptor).
The present invention is applicable to all immunoglobulin superfamily molecules which
possess binding domains. Preferably, the present invention relates to antibodies.
An antigen is specific to a pathogen if targeting the antigen results in substantially
exclusive targeting of the pathogen under physiological conditions.
The variable domains of the heavy and light chains of immunoglobu lins, and the
equivalents in other proteins such as the alpha and beta chains of T-cell receptors, are
responsible for determining antigen binding s pecificity. VH and V L domains are capable of
binding antigen independently, as in V and V L dAbs. References to V H and L domains
include modified versions of V H and V L domains, whether synthetic or naturally occurring.
For example, naturally occurring V H variants include camelid V HH domains, and the heavy
chain immunoglobulins IgNAR of cartilaginous fish.
A TRIM polypeptide is a member of the tripartite motif (TRIM) family of proteins, which
comprises 70 members in the human genome, including TRIM21 (Ro52). TRIM proteins
are involved in diverse cellular processes, including cell proliferation, differentiation,
development, oncogenesis, and apoptosis. TRIM proteins are multidomain, so-called
because of their conceived N-terminal RBCC domains: a RING finger encoding E3
ubiquitin ligase activity, a B-box, and a coiled-coil domain mediating oligomerization. The
C-terminal PRYSPRY or B30.2 domain commonly determines function of different TRIM
polypeptides, by acting as a targeting module. See Nisole et al., Nature Reviews
Microbiology 3 , 799-808 (October 2005). The RING domain is defined by a regular
arrangement of cysteine and histidine residues that coordinate two atoms of zinc, and is
found in a large variety of proteins. It is characterised by the structure C-X2-C-X(9-39)-CX(
1-3)- H-X(2-3)-(N/C/H)-X2-C-X(4-48)C-X2-C, and associated with a B-box domain in
TRIM polypeptides. See Freemont, Curr Biol. 2000 Jan 27;10(2):R84-7.
A domain is a folded protein structure which retains its tertiary structure independently of
the rest of the protein. Generally, domains are responsible for discrete functional
properties of proteins, and in many cases may be added, removed or transferred to other
proteins without loss of function of the remainder of the protein and/or of the domain. The
RING, B-box, Coiled Coil and PRYSPRY domains of TRIM polypeptides are examples
thereof. By antibody variable domain is meant a folded polypeptide domain comprising
sequences characteristic of antibody variable domains. It therefore includes complete
antibody variable domains and modified variable domains, for example in which one or
more loops have been replaced by sequences which are not characteristic of antibody
variable domains, or antibody variable domains which have been truncated or comprise
N- or C-terminal extensions, as well as folded fragments of variable domains which retain
at least in part the binding activity and specificity of the full-length domain.
An inducer of TRIM expression is an agent which increases intracellular levels of a
desired TRIM polypeptide. Preferably, the polypeptide is TRIM21 . Type I interferon in an
inducer of TRIM21 expression.
As referred to herein, coadministration is the simultaneous, simultaneous separate or
sequential administration of two agents, such that they are effective at the same time at
the site of interest. In the context of the coadministration of an antibody and a TRIM21
polypeptide, therefore, the two agents should be administered such that the antibody is
bound by the TRIM21 polypeptide prior to internalisation by the cell. Thus, the antibody
and the TRIM21 polypeptide can be admixed prior to administration, or separately
administered such that they are present in the circulation at the same time.
Antibodies target pathogens before they infect cells. We show herein that upon infection
these antibodies remain bound to pathogens and direct an intracellular immune response
that is present inside every cell. We demonstrate that each cell posses a cytoplasmic IgG
receptor, TRI 21, which binds to antibodies with a higher affinity than any other IgG
receptor in the human body. This enables TRIM21 to rapidly recruit to intracellular
antibody-bound virus and target it for degradation in the proteasome via its E3 ubiquitin
ligase activity. At physiological antibody concentrations, TRIM21 completely neutralises
viral infection. These findings represent an unprecedented system of broad-spectrum
immunity, revealing that the protection mediated by antibodies does not end at the cell
membrane but continues inside the cell to provide a last line of defence against infection.
The PRYSPRY domain of TRIM21 is responsible for antibody binding, and in this sense
TRIM21 appears to be unique in the TRIM polypeptide family. However, the TRIM
domains which is responsible for proteasome targeting, the RING domain, is not specific
to TRIM21 ; rather, it is common to proteins including the TRIM family.
Induction of TRIM21 expression in cells is dependent on interferon, which is subject to
delay and to interference by viral mechanisms. Therefore, the present invention provides
antigen-specific ligands which are fused to a RING domain, such that when the pathogen
is internalised by the cell, ligands bound to the pathogen immediately direct it to the
proteasome for degradation. This effectively allows the cell to cure itself of the pathogen
infection.
1. Ligands
Any ligand which can bind to a pathogen -associated antigen under physiological
conditions, and be internalized by a cell, is suitable for use in the present invention. The
natural immune system uses antibodies as ligands for pathogens, and antibodies or
antibody fragments are ideal for use in the present invention. Other possibilities include
binding domains from other receptors, as well as engineered peptides and nucleic acids.
1a. Antibodies
References herein to antigen- or pathogen -specific antibodies, antigen- or pathogenbinding
antibodies and antibodies specific for an antigen or pathogen are coterminous
and refer to antibodies, or binding fragments derived from antibodies, which bind to
antigens which are present on a pathogen in a specific manner and substantially do not
cross-react with other molecules present in the circulation or the tissues.
An "antibody" as used herein includes but is not limited to, polyclonal, monoclonal,
recombinant, chimeric, complementarity determining region (CDR)-grafted, single chain,
bi-specific, Fab fragments and fragments produced by a Fab expression library. Such
fragments include fragments of whole antibodies which retain their binding activity for the
desired antigen, Fv, F(ab'), F(ab')2 fragments, and F(v) or VH antibody fragments as well
as fusion proteins and other synthetic proteins which comprise the antigen-binding site of
the antibody. Furthermore, the antibodies and fragments thereof may be human or
humanized antibodies, as described in further detail below.
Antibodies and fragments also encompass antibody variants and fragments thereof.
Variants include peptides and polypeptides comprising one or more amino acid sequence
substitutions, deletions, and/or additions that have the same or substantially the same
affinity and specificity of epitope binding as the antigen-specific antibody or fragments
thereof.
The deletions, insertions or substitutions of amino acid residues may produce a silent
change and result in a functionally equivalent substance. Deliberate amino acid
substitutions may be made on the basis of similarity in polarity, charge, solubility,
hydrophobicity, hydrophilicity, and/or the amphipathic nature of the residues. For
example, negatively charged amino acids include aspartic acid and glutamic acid;
positively charged amino acids include lysine and arginine; and amino acids with
uncharged polar head groups having similar hydrophilicity values include leucine,
isoleucine, valine, glycine, alanine, asparagine, glutamine, serine, threonine,
phenylalanine, and tyrosine.
Conservative substitutions may be made, for example according to the Table below.
Amino acids in the same block in the second column and preferably in the same line in
the third column may be substituted for each other:
ALIPHATIC Non-polar G A P
1L V
Polar - uncharged C S T M
N O.
Polar - charged D E
K R
AROMATIC H F W Y
Homologous substitution (substitution and replacement are both used herein to mean the
interchange of an existing amino acid residue, with an alternative residue) may occur i.e.
like-for-like substitution such as basic for basic, acidic for acidic, polar for polar etc. Nonhomologous
substitution may also occur i.e. from one class of residue to another or
alternatively involving the inclusion of unnatural amino acids - such as ornithine
(hereinafter referred to as Z), diaminobutyric acid ornithine (hereinafter referred to as B),
norleucine ornithine (hereinafter referred to as O), pyriylalanine , thienylalanine,
naphthylalanine and phen ylglycine.
Thus, variants may include peptides and polypeptides comprising one or more amino
acid sequence substitutions, deletions, and/or additions to the antigen specific antibodies
and fragments thereof wherein such substitutions, deletions and/or additions do not
cause substantial changes in affinity and specificity of epitope binding. Variants of the
antibodies or fragments thereof may have changes in light and/or heavy chain amino acid
sequences that are naturally occurring or are introduced by in vitro engineering of native
sequences using recombinant DNA techniques. Naturally occurring variants include
"somatic" variants which are generated in vivo in the corresponding germ line nucleotide
sequences during the generation of an antibody response to a foreign antigen.
Variants of antibodies and binding fragments may also be prepared by mutagenesis
techniques. For example, amino acid changes may be introduced at random throughout
an antibody coding region and the resulting variants may be screened for binding affinity
for the target antigen , or for another property. Alternatively, amino acid changes may be
introduced into selected regions of the antibody, such as in the light and/or heavy chain
CDRs, and/or in the framework regions, and the resulting antibodies may be screened for
binding to the target antigen or some other activity. Amino acid changes encompass one
or more amino acid substitutions in a CDR, ranging from a single amino acid difference to
the introduction of multiple permutations of amino acids within a given CDR. Also
encompassed are variants generated by insertion of amino acids to increase the size of a
CDR.
The antigen-binding antibodies and fragments thereof may be humanized or human
engineered antibodies. As used herein, "a humanized antibody", or antigen binding
fragment thereof, is a recombinant polypeptide that comprises a portion of an antigen
binding site from a non-human antibody and a portion of the framework and/or constant
regions of a human antibody. A human engineered antibody or antibody fragment is a
non-human (e.g. , mouse) antibody that has been engineered by modifying (e.g. , deleting,
inserting, or substituting) amino acids at specific positions so as to reduce or eliminate
any detectable immunogenicity of the modified antibody in a human.
Humanized antibodies include chimeric antibodies and CDR-grafted antibodies. Chimeric
antibodies are antibodies that include a non-human antibody variable region linked to a
human constant region. Thus, in chimeric antibodies, the variable region is mostly nonhuman,
and the constant region is human . Chimeric antibodies and methods for making
them are described in, for example, Proc. Natl. Acad. Sci. USA, 8 1: 6841 -6855 ( 1984).
Although, they can be less immunogenic than a mouse monoclonal antibody,
administrations of chimeric antibodies have been associated with human immune
responses (HAMA) to the non-human portion of the antibodies.
CDR-grafted antibodies are antibodies that include the CDRs from a non-human "donor"
antibody linked to the framework region from a human "recipient" antibody. Methods that
can be used to produce humanized antibodies also are described in, for example, US
5,721 ,367 and 6 ,180,377.
"Veneered antibodies" are non-human or humanized (e.g., chimeric or CDR-grafted
antibodies) antibodies that have been engineered to replace certain solvent-exposed
amino acid residues so as to reduce their immunogenicity or enhance their function.
Veneering of a chimeric antibody may comprise identifying solvent-exposed residues in
the non-human framework region of a chimeric antibody and replacing at least one of
them with the corresponding surface residues from a human framework region.
Veneering can be accomplished by any suitable engineeri ng technique.
Further details on antibodies, humanized antibodies, human engineered antibodies, and
methods for their preparation can be found in Antibody Engineering, Springer, New York,
NY, 2001.
Examples of humanized or human engineered antibodies are IgG, IgM, IgE, IgA, and IgD
antibodies. The antibodies may be of any class (IgG, IgA, IgM, IgE, IgD, e c.) or isotype
and can comprise a kappa or lambda light chain. For example, a human antibody may
comprise an IgG heavy chain or defined fragment, such as at least one of isotypes, lgG1,
lgG2, lgG3 or lgG4. As a further example, the antibodies or fragments thereof can
comprise an lgG1 heavy chain and a kappa or lambda light chain.
The antigen specific antibodies and fragments thereof may be human antibodies - such
as antibodies which bind the antigen and are encoded by nucleic acid sequences which
may be naturally occurring somatic variants of human germline immunoglobulin nucleic
acid sequence, and fragments, synthetic variants, derivatives and fusions thereof. Such
antibodies may be produced by any method known in the art, such as through the use of
transgenic mammals (such as transgenic mice) in which the native immunoglobulins have
been replaced with human V-genes in the mammal chromosome.
Human antibodies to target a desired antigen can also be produced using transgenic
animals that have no endogenous immunoglobulin production and are engineered to
contain human immunoglobulin loci, as described in WO 98/24893 and WO 91/00906.
Human antibodies may also be generated through the in vitro screening of antibody
display libraries (J. Mol. Biol. (1991) 227: 381). Various antibody-containing phage
display libraries have been described and may be readily prepared. Libraries may contain
a diversity of human antibody sequences, such as human Fab, Fv, and scFv fragments,
that may be screened against an appropriate target. Phage display libraries may
comprise peptides or proteins other than antibodies which may be screened to identify
agents capable of selective binding to the desired antigen.
Phage-display processes mimic immune selection through the display of antibody
repertoires on the surface of filamentous bacteriophage, and subsequent selection of
phage by their binding to an antigen of choice. One such method is described in WO
99/10494. Antigen-specific antibodies can be isolated by screening of a recombinant
combinatorial antibody library, preferably a scFv phage display library, prepared using
human VL and V cDNAs prepared from mRNA derived from human lymphocytes.
Methodologies for preparing and screening such libraries are known in the art. There are
commercially available kits for generating phage display libraries.
As used herein, the term "antibody fragments" refers to portions of an intact full length
antibody - such as an antigen binding or variable region of the intact antibody. Examples
of antibody fragments include Fab, Fab', F(ab') , and Fv fragments; diabodies; linear
antibodies; single-chain antibody molecules (e.g ., scFv); multispecific antibody fragments
such as bispecific, trispecific, and multispecific antibodies (e.g. , diabodies, triabodies,
tetrabodies); binding-domain immunoglobu lin fusion proteins; camelized antibodies;
minibodies; chelating recombinant antibodies; tribodies or bibodies; intrabodies;
nanobodies; small modular immunopharmaceuticals (SMIP), V H containing antibodies;
and any other polypeptides formed from antibody fragments.
The antigen binding antibodies and fragments encompass single-chain antibody
fragments (scFv) that bind to the desired antigen. An scFv comprises an antibody heavy
chain variable region (VH) operably linked to an antibody light chain variable region (VL)
wherein the heavy chain variable region and the light chain variable region, together or
individually, form a binding site that binds to the antigen . An scFv may comprise a V
region at the amino-terminal end and a VL region at the carboxy-terminal end.
Alternatively, scFv may comprise a VL region at the amino-terminal end and a VH region
at the carboxy-terminal end. Furthermore, although the two domains of the Fv fragment,
VL and V , are coded for by separate genes, they can be joined, using recombinant
methods, by a synthetic linker that enables them to be made as a single protein chain in
which the V and VH regions pair to form monovalent molecules (known as single chain
Fv (scFv). An scFv may optionally further comprise a polypeptide linker between the
heavy chain variable region and the light chain variable region.
The antigen binding antibodies and fragments thereof also encompass immunoadhesins.
One or more CDRs may be incorporated into a molecule either covalently or
noncovalentl y to make it an immunoadhesin. An immunoadhesin may incorporate the
CDR(s) as part of a larger polypeptide chain, may covalently link the CDR(s) to another
polypeptide chain, or may incorporate the CDR(s) noncovalently. The CDRs permit the
immunoadhesin to specifically bind to the desired antigen .
The antigen binding antibodies and fragments thereof also encompass antibody mimics
comprising one or more antigen binding portions built on an organic or molecular scaffold
(such as a protein or carbohydrate scaffold). Proteins having relatively defined threedimensional
structures , commonly referred to as protein scaffolds, may be used as
reagents for the design of antibody mimics. These scaffolds typically contain one or more
regions which are amenable to specific or random sequence variation, and such
sequence randomization is often carried out to produce libraries of proteins from which
desired products may be selected. For example, an antibody mimic can comprise a
chimeric non-immunoglobulin binding polypeptide having an immunoglobulin-like domain
containing scaffold having two or more solvent exposed loops containing a different CDR
from a parent antibody inserted into each of the loops and exhibiting selective binding
activity toward a ligand bound by the parent antibody. Non-immunoglobulin protein
scaffolds have been proposed for obtaining proteins with novel binding properties.
Antigen specific antibodies or antibody fragments thereof typically bind to the desired
antigen with high affinity (e.g. , as determined with BIAcore), such as for example with an
equilibrium binding dissociation constant (KD) for the antigen of about 15nM or less, 10
nM or less, about 5 nM or less, about 1 nM or less, about 500 p or less, about 250 pM
or less, about 100 pM or less, about 50 pM or less, or about 25 pM or less, about 10 pM
or less, about 5 pM or less, about 3 pM or less about 1 pM or less, about 0.75 pM or less,
or about 0.5 pM or less.
1b Peptide Ligands
Peptides, such as peptide aptamers, can be selected from peptide libraries by screening
procedures . In practice, any vector system suitable for expressing short nucleic acid
sequences in a eukaryotic cell can be used to express libraries of peptides. In a
preferred embodiment, high-titer retroviral packaging systems can be used to produce
peptide aptamer libraries. Various vectors, as well as amphotropic and ecotropic
packaging cell lines, exist that can be used for production of high titers of retroviruses
that infect mouse or human cells. These delivery and expression systems can be readily
adapted for efficient infection of any mammalian cell type, and can be used to infect tens
of millions of cells in one experiment. Aptamer libraries comprising nucleic acid
sequences encoding random combinations of a small number of amino acid residues,
e.g., 5 , 6 , 7, 8, 9, 0 or more, but preferably less than 100, more preferably less than 50,
and most preferably less than 20, can be expressed in retrovirally infected cells as free
entities, or depending on the target of a given screen, as fusions to a heterologous
protein, such as a protein that can act as a specific protein scaffold (for promoting, e.g. ,
expressibility, intracellular or intracellular localization, stability, secretability, isolatablitiy,
or detectability of the peptide aptamer. Libraries of random peptide aptamers when
composed of, for example 7 amino acids, encode molecules large enough to represent
significant and specific structural information, and with 107 or more possible
combinations is within a range of cell numbers that can be tested .
Preferably, the aptamers are generated using sequence information from the target
antigen.
In identifying an aptamer, for example, a population of cells is infected with a gene
construct expressing members of an aptamer library, and the ability of aptamers to bind
to an antigen is assessed, for instance on a BIAcore platform. Coding sequences of
aptamers selected in the first round of screening can be amplified by PCR, re-cloned,
and re-introduced into naive cells. Selection using the same or a different system can
then be repeated in order to validate individual aptamers within the original pool.
Aptamer coding sequences within cells identified in subsequent rounds of selection can
be iteratively amplified and subcloned an the sequences of active aptamers can then be
determined by DNA sequencing using standard techniques.
1c Structured polypeptides
Polypeptides tethered to a synthetic molecular structure are known in the art (Kemp, D.
S. and McNamara, P. E., J. Org. Chem, 1985; Timmerman , P. et al. , ChemBioChem,
2005). Meloen and co-workers had used tris(bromomethyl)benzene and related
molecules for rapid and quantitative cyclisation of multiple peptide loops onto synthetic
scaffolds for structural mimicry of protein surfaces (Timmerman, P. et al.,
ChemBioChem, 2005). Methods for the generation of candidate drug compound s
wherein said compound s are generated by linking cysteine containing polypeptides to a
molecular scaffold as for example tris(bromomethyl)benzene are disclosed in WO
2004/077062 and WO 2006/0781 6 1.
WO2004/077062 discloses a method of selecting a candidate drug compound. In
particular, this document discloses various scaffold molecules comprising first and
second reactive groups, and contacting said scaffold with a further molecule to form at
least two linkages between the scaffold and the further molecule in a coupling reaction.
WO2006/0781 6 1 discloses binding compounds, immunogenic compounds and
peptidomimetics. This document discloses the artificial synthesis of various collections
of peptides taken from existing proteins. These peptides are then combined with a
constant synthetic peptide having some amino acid changes introduced in order to
produce combinatorial libraries. By introducing this diversity via the chemical linkage to
separate peptides featuring various amino acid changes, an increased opportunity to find
the desired binding activity is provided. Figure 7 of this document shows a schematic
representation of the synthesis of various loop peptide constructs.
International patent application WO2009098450 describes the use of biological selection
technology, such as phage display, to select peptides tethered to synthetic molecular
structures. In this approach, peptides are expressed on phage, and then reacted under
suitable conditions with molecular scaffolds, such that a structurally constrained peptide
is displayed on the surface of the phage.
Such structured peptides can be designed to bind to any desired antigen, and can be
coupled to a RING domain in order to direct the antigen-ligand complex to the
proteasome inside a cell.
1d Indirect ligands
Indirect ligands bind to the antigen via a second ligand, which recognises the antigen
specifically. For example, the second ligand is an antibody which is specific to the
antigen. Ligands described in sections 1a-1c above may be prepared which are specific
for immunoglobulins, but which bind thereto in a manner which is not dependent on the
binding specificity of the target immunoglobulin. For instance, anti-Fc antibodies,
peptides and structured peptides may be prepared. Antibody-binding peptides such as
Protein A, Protein G and Protein L can be used.
2. RING domains
Tripartite motif (TRIM) proteins constitute a protein family based on a conserved domain
architecture (known as RBCC) that is characterized by a RING finger domain, one or two
B-box domains, a Coiled-coil domain and a variable C-terminus.
TRIM proteins are implicated in a variety of cellular functions, including differentiation,
apoptosis and immunity. A number of TRIM proteins have been found to display antiviral
activities or are known to be involved in processes associated with innate immunity. As
noted by Carthagena, et al., PLoS One (2009) 4, 3:e4894, TRIM5a is responsible for a
species-specific post-entry restriction of diverse retroviruses, including N-MLV and HIV-1,
in primate cells, whereas TRIM1/MID2 also displays an anti-retroviral activity which
affects specifically N-MLV infection. TRIM22, also known as Staf50, has been shown to
inhibit HIV-1 replication, although it is still unclear at what step the block occurs. TRIM28
restricts MLV LTR-driven transcription in murine embryonic cells. Furthermore, the
inhibition of a wide range of RNA and DNA viruses by TRI 19/P has been reported.
The most extensive screen performed to date showed that several TRIM proteins,
including TRIM1 1, TRIM31 and TRIM62, can interfere with various stages of MLV or HIV-
1 replication . Finally, TRIM25 has been shown to control RIGI- mediated antiviral activity
through its E3 ubiquitin ligase activity.
The RING finger of TRIM21 , as set forth herein, is responsible for directing bound
antibody/antigen complexes to the proteasome. This is due to the E3 ubiquitin ligase
activity of the RING domain. Advantageously , therefore, the RING domain used in the
present invention has an E3 ligase activity.
The replacement of RING domains with heterologous TRIM domains, exchanging them
between IM proteins, is known in the art. See Li et al., J. Virol. (2006) 6 98-6206.
RING domains were described by Freemont et al Cell. 1991 Feb 8;64(3):483-4. The
domains are believed to function as E3 ligases; see Meroni & Roux, BioEssays 27, 11:
1147- 57 (2005). They are members of the RING-finger (Really Interesting New Gene)
domain superfamily, a specialized type of Zn-finger of 40 to 60 residues that binds two
atoms of zinc; defined by the 'cross-brace' motif C-X2-C-X(9-39)-C-X(1 -3)- H-X(2-3)-
(N/C/H)-X2-C-X(4-48)C-X2-C. There are two variants within the family, the C3HC4-type
and a C3H2C3-type (RI NG-H2 finger) , which have a different cysteine/histidine pattern .
Preferred RING domains are derived from TRIM proteins, and may be part of TRIM
proteins. In one embodiment, the present invention provides a TRIM polypeptide in
which the B30.2 domain, which imparts its specificity, is replaced with an antigen-specific
binding domain. At least the PRYSPRY (B30.2) domain is replaced; other domains may
be replaced or omitted, as long as the RING domain E3 ligase function is conserved.
3. Induction of TRIM expression
Instead of, or in addition to, coupling the RING domain of a TRIM polypeptide to the
desired antigen, it is possible to stimulate the expression of endogenous TRI M21 within a
cell. TRIM21 binds to antibodies with high affinity, and directs the antibody and any
bound antigen to the proteasome.
Since TRIM21 binds to the Fc portion of the antibody, if endogenous TRI M21 expression
is stimulated by conjugating the ligand to an inducer of TRIM expression, the ligand
comprises a binding site for the PRYSPRY domain of TRIM21 . Preferably, it comprises
an antibody Fc region, and in one embodiment it is an antibody. For example, the
antibody can be an IgG or Ig antibody.
TRIM21 expression is induced by interferon. In one embodiment, therefore, the inducer
of TRIM expression is interferon, or an interferon inducer.
Interferon is preferably type I interferon , for example alpha interferon or beta interferon.
Interferons are known in the art in a number of therapeutic applications, but especially
intherapy for HBV and HCV. Interferon derivatives, such as peginterferon (pegylated
interferon) and albuferon (interferon conjugated to HSA) are coadministered with antiviral
agents, such as nucleoside analogues.
Interferon inducers are known in the art. In general, many vaccine adjuvants act as
interferon inducers. These include substances that have been known to act as vaccine
adjuvants for many years, including viral antigens, bacterial antigens such as LPS,
synthetic polymers usch as poly l:C (e.g. Ampligen®) . More recently, it has been shown
that agonists of Toll-like receptors (TLRs) are effective inducers of interferon . For
example, a number of interferon inducers are known from US201 0 1207 99;
US201 0048520; US201 001 8 134; US201 001 8 1 32; US201 001 8 13 1; US201 001 8 130;
US201 0003280. Moreover, small molecule interferon inducers are being developed, for
instance as set forth in Musmuca et al., J. Chem. Inf. Model. , 2009, 49 (7), pp 1777-
1786.
4. Antibody conjugates
Methods for attaching a drug or other small molecule pharmaceutical to an antibody
fragment are well-known , various peptide conjugation chemistries are established in the
art and include bifunctional chemical linkers such as N- succinimidyl (4-iodoacetyl)-
aminobenzoate ; sulfosuccinimidyl (4-iodoacetyl)-aminobenzoate; 4-succinimidyloxycarbonyl-
[alpha]- (2-pyridyldithio) toluene; sulfosuccinimidyl-6-[[alpha]- methyl-
[alpha]- (pyridyldithiol)- toluamidojhexanoate; N-succinimidyl-3- (-2-pyridyldithio)-
proprionate ; succinimidyl- 6 -[3(-(-2-pyridyldithio)-proprionamido] hexanoate;
sulfosuccinimidyl-6- [3(-(-2-pyridyldithio)- propionamido] hexanoate; 3-(2-pyridyldithio)-
propionyl hydrazide, Ellman's reagent, dichlorotriazinic acid, S-(2-thiopyridyl)-L-cysteine,
and the like. Further bifunctional linking molecules are disclosed in U.S. Pat. Nos.
5,349, 066; 5,61 8,528; 4,569,789; 4,952,394; and 5,1 37, 877, as well as Corson et al.,
ACS Cemical Biology 3, 11, pp677-692, 2008.
The RING domains and polypeptide ligands, including antibodies, may be conjugated via
functional or reactive groups on one (or both) polypeptide (s). These are typically formed
from the side chains of particular amino acids found in the polypeptide polymer. Such
reactive groups may be a cysteine side chain, a lysine side chain, or an N-terminal amine
group or any other suitable reactive group.
Reactive groups are capable of forming covalent bonds to the ligand to be attached.
Functional groups are specific groups of atoms within either natural or non-natural amino
acids which form the functional groups .
Suitable functional groups of natural amino acids are the thiol group of cysteine, the
amino group of lysine, the carboxyl group of aspartate or glutamate, the guanidinium
group of arginine, the phenolic group of tyrosine or the hydroxyl group of serine. Nonnatural
amino acids can provide a wide range of functional groups including an azide, a
keto-carbonyl, an alkyne, a vinyl, or an aryl halide group. The amino and carboxyl group
of the termini of the polypeptide can also serve as functional groups to form covalent
bonds to a desired ligand.
Alternatives to thiol-mediated conjugations can be used to attach a ligand to a
polypeptide via covalent interactions. These methods may be used instead of (or in
combination with) the thiol mediated methods by producing polypeptides bearing
unnatural amino acids with the requisite chemical functional groups, in combination small
molecules that bear the complementary functional group, or by incorporating the
unnatural amino acids into a chemically or recombinantly synthesised polypeptide when
the molecule is being made after the selection/isolation phase.
The unnatural amino acids incorporated into peptides and proteins on phage may include
1) a ketone functional group (as found in para or meta acetyl-phenylalanine) that can be
specifically reacted with hydrazines, hydroxylamines and their derivatives (Addition of the
keto functional group to the genetic code of Escherichia coli. Wang L, Zhang Z, Brock A,
Schultz PG. Proc Natl Acad Sci U S A. 2003 Jan 7;100(1 ):56-61 ; Bioorg Med Chem Lett.
2006 Oct 5;16(20):5356-9. Genetic introduction of a diketone-containing amino acid into
proteins. Zeng H, Xie J, Schultz PG), 2) azides (as found in p-azido-phenylalanine) that
can be reacted with alkynes via copper catalysed "click chemistry" or strain promoted
(3+2) cyloadditions to form the correspond ing triazoles (Addition of p-azido-Lphenylalanine
to the genetic code of Escherichia coli. Chin JW, Santoro SW, Martin AB,
King DS, Wang L, Schultz PG. J Am Chem Soc. 2002 Aug 7;124(31 ):9026-7; Adding
amino acids with novel reactivity to the genetic code of Saccharomyces cerevisiae.
Deiters A, Cropp TA, Mukherji M, Chin JW, Anderson JC, Schultz PG. J Am Chem Soc.
2003 Oct 1;125(39):1 1782-3), or azides that can be reacted with aryl phosphines, via a
Staudinger ligation (Selective Staudinger modification of proteins containing pazidophenylalanine.
Tsao ML, Tian F, Schultz PG. Chembiochem. 2005 Dec;6(12):2147-
9), to form the corresponding amides, 4) Alkynes that can be reacted with azides to form
the corresponding triazole (In vivo incorporation of an alkyne into proteins in Escherichia
coli. Deiters A, Schultz PG. Bioorg Med Chem Lett. 2005 Mar 1;15(5):1521-4), 5) Boronic
acids (boronates) than can be specifically reacted with compounds containing more than
one appropriately spaced hydroxyl group or undergo palladium mediated coupling with
halogenated compounds (Angew Chem Int Ed Engl. 2008;47(43):8220-3. A genetically
encoded boronate-containing amino acid., Brustad E, Bushey ML, Lee JW, Groff D, Liu
W, Schultz PG), 6) Metal chelating amino acids, including those bearing bipyridyls, that
can specifically co-ordinate a metal ion (Angew Chem Int Ed Engl. 2007;46(48):9239-42.
A genetically encoded bidentate, metal-binding amino acid. Xie J, Liu W, Schultz PG).
Unnatural amino acids may be incorporated into proteins and peptides by transforming E.
coli with plasmids or combinations of plasmids bearing: 1) the orthogonal aminoacyltRNA
synthetase and tRNA that direct the incorporation of the unnatural amino acid in
response to a codon, 2) The phage D A or phagemid plasmid altered to contain the
selected codon at the site of unnatural amino acid incorporation (Proc Natl Acad Sci U S
A. 2008 Nov 18; 105(46): 17688-93. Protein evolution with an expanded genetic code. Liu
CC, Mack AV, Tsao ML, Mills JH, Lee HS, Choe H, Farzan M, Schultz PG, Smider W ; A
phage display system with unnatural amino acids. Tian F, Tsao ML, Schultz PG. J Am
Chem Soc. 2004 Dec 15; 126(49): 15962-3). The orthogonal aminoacyl-tRNA synthetase
and tRNA may be derived from the Methancoccus janaschii tyrosyl pair or a synthetase
(Addition of a photocrosslinking amino acid to the genetic code of Escherichiacoli. Chin
JW, Martin AB, King DS, Wang L, Schultz PG. Proc Natl Acad Sci U S A. 2002 Aug
20;99(17):1 1020-4) and tRNA pair that naturally incorporates pyrrolysine (Multistep
engineering of pyrrolysyl-tRNA synthetase to genetically encode N(epsilon)-(oazidobenzyloxycarbonyl)
lysine for site-specific protein modification. Yanagisawa T, Ishii
R, Fukunaga R, Kobayashi T, Sakamoto K, Yokoyama S. Chem Biol. 2008 Nov
24;1 5(1 1):1 87-97; Genetically encoding N(epsilon)-acetyllysine in recombinant proteins.
Neumann H, Peak-Chew SY, Chin JW. Nat Chem Biol. 2008 Apr;4(4):232-4. Epub 2008
Feb 17). The codon for incorporation may be the amber codon (UAG) another stop
codon (UGA, or UAA), alternatively it may be a four base codon. The aminoacyl-tRNA
synthetase and tRNA may be produced from existing vectors, including the pBK series of
vectors, pSUP (Efficient incorporation of unnatural amino acids into proteins in
Escherichia coli. Ryu Y, Schultz PG.Nat Methods. 2006 Apr;3(4):263-5) vectors and
pDULE vectors (Nat Methods. 2005 May;2(5):377-84. Photo-cross-linking interacting
proteins with a genetically encoded benzophenone. Farrell IS, Toroney R, Hazen JL,
Mehl RA, Chin JW). The E.coli strain used will express the F' pilus (generally via a tra
operon) . When amber suppression is used the E. coli strain will not itself contain an
active amber suppressor tRNA gene. The amino acid will be added to the growth media,
preferably at a final concentration of 1mM or greater. Efficiency of amino acid
incorporat ion may be enhanced by using an expression construct with an orthogonal
ribosome binding site and translating the gene with ribo-X(Evolved orthogonal ribosomes
enhance the efficiency of synthetic genetic code expansion. Wang K, Neumann , Peak-
Chew SY, Chin JW. Nat Biotechnol. 2007 Jul;25(7):770-7). This may allow efficient multisite
incorporation of the unnatural amino acid providing multiple sites of attachment to the
ligand.
Such methods are useful to attach RING domains to antibodies and other ligands ,
including non-peptide ligands. They are also useful for attaching small molecule
interferon inducers, and other inducers of TRIM21 expression.
Techniques for conjugating antibodies to drugs and other compounds are also described
in Carter & Senter, Cancer Journal: May/June 2008 - Volume 14 - Issue 3 - pp 154-1 69;
Ducry and Stump, Bioconjugate Chem. , 201 0 , 2 1 ( 1) , pp 5-1 3.
Alternatively, bispecific antibodies may be used. For example, bispecific domain
antibodies are known in the art, and are useful for targeting both a desired antigen and a
RING domain, or a polypeptide comprising a RING domain.
The half-life of antibody conjugates in the serum is dependent no a number of factors, but
smaller antibody fragments tend to be eliminated quickly from the circulation.
Accordingly , smaller constructs, for example comprising a domain antibody and a RING
domain, are advantageously coupled to a polypeptide which increases serum half-life.
For example, they can be coupled to HSA. Preferably, the bond to HSA is labile, for
example having a defined half life, such that the construct is released from the HSA when
bound to a cell, and is internalised without the HSA. A useful approach is to use a
multispecific ligand construct, such that the ligand also binds HSA, maintaining it in
circulation. The affinity of the ligand for HSA can be tailored such that the ligand can be
internalised by the cell as appropriate.
5. Coadministration of TRIM21 and Antibodies
Therapeutic antibodies are well known in the art. TRIM21 binds to the Fc portion of IgG
and Ig antibodies, and coadministration thereof to a subject is effective in promoting the
destruction of pathogens by cells.
Table 1 sets forth existing antibody drugs which are available for the treatment of
pathogenic infections. Coadministration of TRIM21 is indicated for treatment with such
drugs.
The polypeptide coadministered with the antibody drug preferably comprises a TRIM21
PRYSPRY domain and a RING domain, capable of acting as an E3 ligase. However,
other immunoglobulin-specific ligands can be used, such as protein A, protein G or
protein L, or anti-Fc peptides, which bind to immunoglobulins in a manner independent of
the antibody's target specificity.
Preferably, the polypeptide also comprises a coiled coil domain and/or a B-box domain.
In a preferred embodiment, it is a substantially complete TRIM21 polypeptide.
TRI 21 is preferably human TRIM21, as set forth in SEQ ID No. 1; See Tanaka.M., et al.,
Histochem. Cell Biol. 133 (3), 273-284 (2010).
The invention encompasses modified derivatives of TIM21, which conserve at least the
antibody-binding and E3 ligase functions. For example, the invention encompasses
substitutions, additions or deletions within the amino acid sequence of TRIM21 , as long
as the required functions are sufficiently maintained. Polypeptides may share at least
50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%
sequence identity (homology) with SEQ ID NO. 1.
Mutation on the polypeptides of the inventin can be targeted to certain domains thereof.
Higher levels of conservation of sequence identity are required, for instance, in the
PRYSPRY domain. This domain is responsible for antibody binding by the polypeptide.
Lower levels of identity are generally required, for example, in the RING domain. RING
domains are widespread in the genome, and have a conserved E3 ligase fun ction.
Advantageously, the consensus sequence, C-X2-C-X(9-39)-C-X(1 -3)- H-X(2-3)-(N/C/H)-
X2-C-X(4-48)C-X2-C, is maintained.
Table 1: antibody anti-infective drugs

EPI-1 9 EPIcyte Respiratory syncytial virus
Pharmaceutical Inc infection
SYM-003 Symphogen A/S Respiratory syncytial virus
infection
CR-301 Crucell NV SARS coronavirus infection
SARS coronavirus spike glycoprotein National Institutes SARS coronavirus infection
antibodies (SARS), National Institutes of of Health
Health
urtoxazumab Teijin Ltd Escherichia coli infection;
Hemolytic uremic syndrome;
Hemorrhagic enteritis
tefibazumab Inhibitex Inc Staphylococcus aureus
infection
anti-S aureus humanized monoclonal Intercell AG Staphylococcus aureus
antibodies, Merck & Co infection
anti-Staphylococcus enterotoxin B Integrated Staphylococcus infection
hyperimmune globulin, Integrated BioTherapeutics
BioTherapeutics Inc
ETI-21 Elusys Staphylococcus aureus
Therapeutics Inc infection
superantigen toxin therapy (antibodies), Synergy Sepsis; Shock;
Callisto Pharmaceuticals Staphylococceae infection;
Inc Streptococcaceae infection;
Toxicity
Tetagam CSL Behring Clostridium tetani infection
KBAB-401 Kenta Biotech Ltd Acinetobacter infection
anti-anthrax antibodies, Verenium Verenium Corp Bacillus anthracis infection
polyclonal anti-RcPA IgG , NEXTherapeutics Bacillus anthracis infection
NEXTherapeutics Inc
ACE-71 0 ACE Biosciences Aspergillus fumigatus
A/S infection
SYM-006 Symphogen A/S Bacterial infection
immune globulin (Burkholderia, Cangene Corp Burkholderia infection
monoclonal), Cangene
CAL-401 Calmune Corp Candida albicans infection
XTL-Cand-MAb XTL Candida infection
Biopharmaceuticals
Ltd
Candistat-G Let's Talk Candida albicans infection
Recovery Inc
monoclonal antibodies (C albicans), Inhibitex Inc Candida albicans infection
Inhibitex
OPHD-001 Ophidian Clostridium difficile infection
Pharmaceuticals
Inc
immunotoxin (INAx, C V infection), Inagen Aps Cytomegalovirus infection
Inagen
sevirumab Novartis AG Bone marrow
transplantation; CMV
retinitis; Cytomegalovirus
infection
human monoclonal antibody (CMV Humabs LLC Cytomegalovirus infection
infection), Humabs
human monoclonal antibodies (CMV Theraclone Cytomegalovirus infection
infection in organ transplantation), Sciences Inc
Theraclone Sciences
CytoGam Medlmmune LLC Cytomegalovirus infection
truly human monoclonal antibodies (CMV RiboVax Cytomegalovirus infection
infection), RiboVax Biotechnologies Biotechnologies SA
human monoclonal antibodies Evec Inc Cytomegalovirus infection
(cytomegalovirus), Evec
regavirumab Teijin Ltd Cytomegalovirus infection;
Immune disorder
monoclonal antibody (CMV), Scotgen Scotgen Cytomegalovirus infection
Biopharmaceuticals
Inc
SDZ-89-1 04 Novartis AG Bone marrow
transplantation;
Cytomegalovirus infection
immunoglobulin (CMV), Cangene Cangene Corp Cytomegalovirus infection
CryptoGAM ImmuCell Corp Cryptosporidium infection
bovine immunoglobulin concentrate-C Let's Talk Diarrhea; Parasitic infection
parvum, GalaGen Recovery Inc
Sporidin-G Let's Talk Cryptosporidium infection;
Recovery Inc Diarrhea
therapeutic antibody program (dengue Sentinext Dengue virus infection
virus infection), Sentinext Therapeutics Sdn
Bhd
monoclonal antibody (dengue virus), MacroGenics Inc Dengue virus infection
MacroGenics
Enterostat-G Alcon Inc Diarrhea; Escherichia coli
infection
BWPT-302 Biomune Systems Escherichia coli infection
Inc
TravelGAM ImmuCell Corp Escherichia coli infection
E coli antibody, Mutabilis Mutabilis SA Escherichia coli infection
anti-Ebola virus monoclonal antibodies Mapp Ebola virus infection
(Ebola virus infection), MAPP Biopharmaceutical
Inc
immune globulin (Ebola/Marburg, Cangene Corp Filovirus infection
polyclonal), Cangene
immune globulin (Ebola/Marburg, Cangene Corp Filovirus infection
monoclonal), Cangene
Enterococcus MAb, Inhibitex Inhibitex Inc Enterobacteriaceae infection
F 10 (neutralizing antibody, group 1 Harvard Medical Influenza virus infection
influenza A infection), Harvard Medical School
School/Dana-Farber Cancer
Institute/XOMA/SR I International
anti-hantavirus monoclonal antibody, Huazhong Hantavirus infection
Huazhong University University of
Science and
Technology
HAV human antibody, Aprogen Aprogen Inc Hepatitis A virus infection
Beriglobin Aventis Behring Hepatitis A virus infection
LLC
HepaGam B Cangene Corp Hepatitis B virus infection
tuvirumab Novartis AG Hepatitis B virus infection;
Transplant rejection
Omri-Hep-B OMRIX Hepatitis B virus infection
Biopharmaceuticals
SA
HBV humanized antibodies, Aprogen Aprogen Inc Hepatitis B virus infection
anti-HBV antibody, AltaRex AltaRex Medical Hepatitis B virus infection
Corp
Hepatitis B Hyperimmune Kedrion SpA Hepatitis B virus infection
C-lmmune Virionics Corp Hepatitis C virus infection
fully-human monoclonal antibodies Stanford University Hepatitis C virus infection
(hepatitis C virus), Crucell
human monoclonal antibodies (HCV Theraclone Hepatitis C virus infection
infection), Theraclone Sciences Sciences Inc
Civacir Nabi Hepatitis C virus infection
Biopharmaceuticals
truly human monoclonal antibodies (HCV RiboVax Hepatitis C virus infection
infection), RiboVax Biotechnologies Biotechnologies SA
anti-HCV hyperimmune, Cangene Cangene Corp Hepatitis C virus infection
Pylorimune-G Chiron Vaccines Gastrointestinal ulcer;
Co Helicobacter pylori infection
bovine antibody-based immunotherapy Erasmus Helicobacter pylori infection
(Helicobacter pylori infection), Erasmus Universiteit
Universiteit Rotterdam Rotterdam
KD-247 Chemo-Sero- HIV infection
Therapeutic
Research Inst
MEDI-488 Medlmmune LLC HIV infection
F-1 05 Centocor Ortho HIV infection
Biotech Inc
TG-1 02 TargetGen HIV infection
Biotechnology
sCD4-1 7b National Institute of HIV infection
Allergy and
Infectious Diseases
PassHIV, Verigen Verigen Inc HIV infection
HIV-IG Nabi HIV infection
Biopharmaceuticals
G3.51 9-PAP-S Tanox Inc HIV infection
hN 01 Nissin Foods HIV infection
Holdings Co Ltd
R7V therapeutic antibody program, URRMA HIV infection
URRMA Biopharma Inc
monoclonal antibodies (HIV), SRD Scotgen HIV infection
Pharmaceuticals Biopharmaceuticals
Inc
HIV-NeutraGAM Nabi HIV infection
Biopharmaceuticals
human monoclonal antibody (HIV Humabs LLC HIV infection
infection), Humabs
HIV monoclonals, Roche Roche Holding AG HIV-1 infection
HIV neutralizing antibodies (HIV infection), Biotherapix SLU HIV infection
Biotherapix
anti-HIV therapy, EPIcyte EPIcyte HIV infection
Pharmaceutical Inc
A-221 -Immune cHIV Stratus Research HIV infection
Labs Inc
HIV antibodies, Unviersity of Southern University of HIV-1 infection
California Southern California
HIV immune globulin, Chiron. Chiron Corp HIV infection
STAR fusion agents (viral infections), Altor Massachusetts Cytomegalovirus infection;
General Hospital HIV infection; Hepatitis C
virus infection; Infection
rhinovirus therapy, EPIcyte EPIcyte Rhinovirus infection
Pharmaceutical Inc
monoclonals, Cambridge Biotech Cambridge Biotech Cytomegalovirus infection;
Corp Herpes simplex virus
infection; Viral infection
AC-8 Calmune Corp Herpetic keratitis
CAL-1 02-R Calmune Corp Herpes simplex virus
infection; Ocular disease
CAL-1 03-R Calmune Corp Herpes simplex virus
infection; Ocular disease
SMART anti-HSV MAb, PDL Novartis AG Herpes simplex virus
infection
monoclonal antibody therapy (H N1/H5N1 Celltrion Inc Influenza virus infection
influenza), Celltrion
influenza therapy, NanoViricides NanoViricides Inc Influenza virus infection
H5N1 influenza mAb therapy, MacroGenics Inc Influenza virus infection
MacroGenics
human monoclonal antibody (H5N1 Humabs LLC Influenza virus infection
infection), Humabs
camel-derived anti-influenza virus Canopus Influenza virus infection
antibodies, Canopus BioPharma Inc
ABC-1 20 AERES Biomedical Plasmodium infection
Ltd
CAL-201 Calmune Corp Metapneumovirus infection
metapneumovirus antibody, Medlmmune ViroNovative BV Viral respiratory tract
infection
monoclonal antibodies (meningitis), Affitech A S Neisseria meningitidis
Affitech infection
nebacumab Centocor Ortho Neisseria meningitidis
Biotech Inc infection; Sepsis
henipavirus-neutralizing antibody, NCI National Cancer Henipavirus infection
Institute
immunotoxins (INAx, parasitic infections), Inagen Aps Parasitic infection
Inagen
CAL-202 Calmune Corp Parainfluenza virus infection
PCP-Scan Immunomedics Inc Fungal pneumonia; Fungal
respiratory tract infection;
Pneumocystis carinii
infection
antibacterial antibodies, InterMune InterMune Inc Pseudomonas aeruginosa
infection; Staphylococcus
aureus infection
polyclonal IgY antibody (oral, Immunsystem IMS Pseudomonas aeruginosa
Pseudomonas aeruginosa), Immunsystem AB infection
XTL-Pseudomonas-MAb XTL Pseudomonas aeruginosa
Biopharmaceuticals infection
Ltd
monoclonal antibody (P aeruginosa), Scotgen Pseudomonas aeruginosa
Scotgen Biopharmaceuticals infection
Inc
Pseudomonas aeruginosa MAb, Millenium Cytovax Pseudomonas aeruginosa
Biotechnologies Inc infection
HyperGAM, Nabi Nabi Pseudomonas aeruginosa
Biopharmaceuticals infection
MS-705 Mitsui Pseudomonas aeruginosa
Pharmaceuticals infection
Inc
monoclonal antibody vaccine (rabies), Massachusetts Rabies virus infection
MBL/Serum Institute of India Biologic
Laboratories
foravirumab + rafivirumab Crucell NV Rabies virus infection
human anti-rabies mAb, Molecular Molecular Rabies virus infection
Targeting Technology/North China Targeting
Pharmaceutical Group Corp Technologies Inc
Berirab CSL Behring Rabies virus infection
monoclonal antibody (rabies), Scotgen Scotgen Rabies virus infection
Biopharmaceuticals
Inc
Rotastat-G Alcon Inc Diarrhea; Viral infection
RespiGam Medlmmune LLC Respiratory syncytial virus
infection
ALX-01 7 1 Ablynx NV Respiratory syncytial virus
infection
anti-RSV mAb, Trellis/Medlmmune Trellis Bioscience Respiratory syncytial virus
Inc infection
HumaRESP Intracel Corp Respiratory syncytial virus
infection
RSV human antibodies, Aprogen Aprogen Inc Respiratory syncytial virus
infection
KBRV-201 Kenta Biotech Ltd Respiratory syncytial virus
infection; Viral respiratory
tract infection
CAL-203 Calmune Corp Respiratory syncytial virus
infection
HNK-20 Acambis Inc Respiratory syncytial virus
infection
EGX-220 EvoGenix Pty Ltd Respiratory syncytial virus
infection
human monoclonal antibody (SARS Humabs LLC SARS coronavirus infection
infection), Humabs
SARS coronavirus antibody, Medarex Inc SARS coronavirus infection
Medarex/University of Massachusetts
anti-SARS antibodies, Verenium Verenium Corp SARS coronavirus infection
immune globulin (SARS), Cangene Cangene Corp SARS coronavirus infection
hyperimmune globulins (SARS), Advantek Advantek Biologies SARS coronavirus infection
Ltd
ACY-1 1 Acceptys Inc Vaccinia virus infection;
Variola virus infection
Veronate BioResearch Bacterial infection; Candida
Ireland infection; Staphylococcus
aureus infection;
Staphylococcus infection
human monoclonal antibodies Crucell NV Bacterial infection
(Enterococcus/Staphylococcus infection),
Crucell/Medlmmune
KBSA-301 Kenta Biotech Ltd Staphylococcus aureus
infection
KBSA-302 Kenta Biotech Ltd Staphylococcus aureus
infection
Saurestat Strox Staphylococcus aureus
Biopharmaceuticals infection
LLC
SX5 Strox Staphylococcus aureus
Biopharmaceuticals infection
LLC
SX8 Strox Staphylococcus aureus
Biopharmaceuticals infection
LLC
anti-staphylococcus heteropolymer (HP) Medlmmune LLC Bacterial infection
monoclonal antibody (infection),
Medlmmune
Staphguard Excelimmune Inc Staphylococcus aureus
infection
Aurograb NeuTec Pharma Staphylococcus aureus
pic infection
SA-IGIV Inhibitex Inc Staphylococcus aureus
infection
SE-MAb, Inhibitex/BioResearch Inhibitex Inc Staphylococcus infection
antibacterial antibodies, Wyeth Haptogen Ltd Bacterial infection; MRSA
Pharmaceuticals/DaeWoong infection
XTL-Staph-MAb XTL Staphylococcus aureus
Biopharmaceuticals infection
Ltd
anti-S agalactiae humanized monoclonal Intercell AG Streptococcus agalactiae
antibodies, Intercell infection
hemolytic disease therapy, AMRAD Zenyth Hemolytic anemia; Hemolytic
Therapeutics Ltd streptococcus infection
antibody therapy (Streptococcus ACE Biosciences Streptococcus pneumoniae
pneumoniae infection), ACE A S infection
Biosciences/Crucell
anti-S pneumoniae humanized Intercell AG Streptococcus pneumoniae
monoclonal antibodies, Intercell/Kyowa infection
Hakko Kirin
CaroRx Planet Dental caries; Streptococcus
Biotechnology Inc mutans infection
Streptococcus mutans therapy, EPIcyte EPIcyte Dental caries; Streptococcus
Pharmaceutical Inc mutans infection
Streptococcus pneumoniae mAb, Cytovax Cytovax Streptococcus pneumoniae
Biotechnologies Inc infection
anti-S pyogenes humanized monoclonal Intercell AG Group A Streptococcus
antibodies, Merck & Co infection
anti-tick-borne encephalitis virus State Research Flavivirus infection
monoclonal antibodies, SRC VB Vector Center of Virology
and Biotechnology
VECTOR
anti-smallpox monoclonal antibodies, Kyowa Hakko Kirin Variola virus infection
Kyowa Hakko Kirin Co Ltd
SYM-002 Symphogen A S Vaccinia virus infection
human monoclonal antibody (smallpox), US Army Medical Variola virus infection
USAMRIID/BioFactura Research Institute
of Infectious
Diseases
monoclonal antibody cocktail (smallpox), MacroGenics Inc Variola virus infection
MacroGenics
VariZIG Cangene Corp Neuralgia; Varicella zoster
virus infection
monoclonal antibodies (Varicella zoster), Affitech A S Varicella zoster virus
Affitech infection
monoclonal antibody (varicella), Scotgen Scotgen Varicella zoster virus
Biopharmaceuticals infection
Inc
varicella zoster virus MAbs, Teijin Teijin Ltd Varicella zoster vims
infection
anti-VZV MAb (humanized), PDL Novartis AG Varicella zoster virus
infection
Varicellon CSL Behring Varicella zoster virus
infection
Western equine encephalitis virus vaccine ViRexx Medical Western equine encephalitis
(antibody, Chimigen), Paladin Corp virus infection
WNV-HP Elusys Viral infection; West Nile
Therapeutics Inc virus infection
Omr-lgG-am OMRIX Immune deficiency; West
Biopharmaceuticals Nile virus infection
SA
immune globulin (West Nile virus), Cangene Corp West Nile virus infection
Cangene
CR-4374 Crucell NV West Nile virus infection
anti-plague antibodies, Verenium Verenium Corp Yersinia pestis infection
MGAWN-1 Washington West Nile virus infection
University in St
Louis
6. Administration of Compounds
Generally, the compounds according to the invention will be utilised in purified form
together with pharmacologically appropriate carriers. Typically, these carriers include
aqueous or alcoholic/aqueous solutions, emulsions or suspensions, any including saline
and/or buffered media. Parenteral vehicles include sodium chloride solution, Ringer's
dextrose, dextrose and sodium chloride and lactated Ringer's. Suitable physiologicallyacceptable
adjuvants, if necessary to keep a polypeptide complex in suspension, may be
chosen from thickeners such as carboxymethylcellulose, polyvinylpyrrolidone, gelatin and
alginates.
Intravenous vehicles include fluid and nutrient replenishers and electrolyte replenishes,
such as those based on Ringer's dextrose. Preservatives and other additives, such as
antimicrobials, antioxidants, chelating agents and inert gases, may also be present (Mack
(1982) Remington's Pharmaceutical Sciences, 16th Edition).
The compounds of the present invention may be used as separately administered
compositions or in conjunction with other agents. These can include further antibodies,
antibody fragments and conjugates, and various immunotherapeutic drugs, such as
cylcosporine, methotrexate, adriamycin or cisplatinum, and immunotoxins.
Pharmaceutical compositions can include "cocktails" of various cytotoxic or other agents
in conjunction with the selected antibodies, receptors or binding proteins thereof of the
present invention, or even combinations of selected polypeptides according to the
present invention having different specificities, such as polypeptides selected using
different target ligands, whether or not they are pooled prior to administration.
The route of administration of pharmaceutical compositions according to the invention
may be any of those commonly known to those of ordinary skill in the art. For therapy,
including without limitation immunotherapy, the selected antibodies, receptors or binding
proteins thereof of the invention can be administered to any patient in accordance with
standard techniques. The administration can be by any appropriate mode, including
parenterally, intravenously, intramuscularly, intraperitoneally, transderm al^, via the
pulmona ry route, or also, appropriately, by direct infusion with a catheter. The dosage
and frequency of administration will depend on the age, sex and condition of the patient,
concurrent administration of other drugs, counterindications and other parameters to be
taken into account by the clinician.
The compounds of this invention can be lyophilised for storage and reconstituted in a
suitable carrier prio r to use. This technique has been shown to be effective and art-known
lyophilisation and reconstitution techniques can be employed. It will be appreciated by
those skilled in the art that lyophilisation and reconstitution can lead to varying degrees of
activity loss and that use levels may have to be adjusted upward to compensate.
The compositions containing the present peptide ligands or a cocktail thereof can be
administered for prophylactic and/or therapeutic treatments. In certain therapeutic
applications, an adequate amount to accomplish at least partial inhibition, suppression,
modulation, killing, or some other measurable parameter, of a population of selected cells
is defined as a "therapeutically-effective dose". Amounts needed to achieve this dosage
will depend upon the severity of the disease and the general state of the patient's own
immune system, but generally range from 0.005 to 5.0 g of selected peptide ligand per
kilogram of body weight, with doses of 0.05 to 2.0 mg/kg/dose being more commonly
used. For prophylactic applications, compositions containing the present peptide ligands
or cocktails thereof may also be administered in similar or slightly lower dosages.
A composition containing a compound according to the present invention may be utilised
in prophylactic and therapeutic settings to aid in the alteration, inactivation, killing or
removal of a select target cell population in a mammal. In addition, the selected
repertoires of polypeptides described herein may be used extracorporeal^ or in vitro
selectively to kill, deplete or otherwise effectively remove a target cell population from a
heterogeneous collection of cells. Blood from a mammal may be combined
extracorporeal^ with the selected peptide ligands whereby the undesired cells are killed
or otherwise removed from the blood for return to the mammal in accordance with
standard techniques.
The invention is further described in the following examples.
Materials & Methods
Cells lines
HEK293T, HeLa, TE671 , QT35 and HT1080 were maintained in Dulbecco's modification
of Eagle's medium (DMEM) supplemented with 10% fetal bovine serum and 100 lU/ml
penicillin and 100 g/ml streptomycin at 37 °C in a humid incubator. 293F cells
(Invitrogen, Paisley, UK) were grown in serum-free Freestyle medium (Invitrogen) in an
orbital shaker at 50 rpm at 37 °C. Where appropriate, cells were selected in 1 mg/ml
G418 (Invitrogen) or 2 m g ml puromycin (Sigma-Aldrich, Poole, UK).
Virus production
Coxsackievirus was produced as described 18 with modification. Plasmid eGFP-CVB3
encoding strain pH3 with an eGFP sequence and a cleavage sequence at the N-terminus
of the viral polypeptide was transfected into a 10 cm dish of HEK293T cells using
Superfect (Qiagen, Crawley, UK) according to manufacturer's instruction. After 48 h, cells
were mechanically dislodged from the dish, freeze-thawed three times to release virions,
and supernatant clarified at 1,000 g before filtration at 0.45 m h Viral stock was expanded
in HeLa cells for 48 h, virus particles harvested by freeze-thaw and filtration as above.
Aliquots were frozen at -80 °C until required. Titres were typically in the range of 10s to
10 7 lU/ml. Adenovirus Ad5-GFP 19 was grown in transcomplementation cell line 293F for
72 h, before three rounds of freeze-thaw to release virus particles and filtration at 0.45
m h . Virus stock was purified by two rounds of ultracentrifugation banding on a caesium
chloride gradient, dialysed into PBS/10% glycerol and frozen at -80 °C until required.
Titres of purified virus were typically 08 to 09 lU/ml.
Generation of stable knockdown and over-expressing cell lines
Human TRIM21 DNA was cloned into pDONAI (Takara, Saint-Germain-en-Laye, France)
as a Notl/Sall restriction fragment to generate pDON-T21. DNA encoding a small hairpin
(sh) RNA directed to human TRIM21 sequence GCAGCACGCTTGACAATGA was
cloned into pSIREN Retro-Q (Clontech) to produce pSIREN-shT21 . Control shRNA
directed to luciferase was encoded by pSIREN-shLuc. Retroviral transduction particles
were produced by transfection of 4 x 106 HEK293T cells with 5 m g of pDON-T21,
pSIREN-shT21 , empty pDONAI or pSIREN-Luc along with 5 m g each of MLV gag-pol
expression plasmid pCMVi and VSV-G expression plasmid pMDG20 . Supernatant was
harvested after 72 h and filtered at 0.45 m and used to transduce HeLa cells. Stably
transduced cells were selected with G41 8 (pDON-T21 , pDONAI) or puromycin (pSIRENshT21
, pSIREN-shLuc). Levels of TRI 21 protein were monitored by western blotting
(sc-25351 , Santa Cruz).
Transient siRNA knockdown
Cells were plated at 1 x 10 cells per well in six-well plates and allowed to adhere
overnight. 150 pmol each of small interfering (si) RNA oligonucleotides T21 siRNA1
(UCAUUGUCAAGCGUGCUGC, Dharmacon , Lafayette, CO, USA) and T21 siRNA2
(UGGCAUG GAGGCACCUGAAGGUGG; Invitrogen) or 300 pmol control oligo
(Invitrogen) were transfected into cells using Oligofectamine (Invitrogen). Cells were
washed after 3 h and incubated for 72 h before infection. Where indicated , 1000 U IFN-a
(PBL InterferonSou rce, Edison, NJ , USA) was added 48 h after knockdown.
Virus neutralisation assays
For both Ad5-GFP and eGFP-CVB3 infections, target HeLa cells were seeded at 1 x 105
cells per well in 2 ml complete D E in six-well plates the day before infection. Where
stated, cells were incubated with 000 U IFN-a. 5 x 104 infectious units (IU) AdV5-GFP
were incubated with antibody in a 10 m I volume for 30 min at room temperature before
addition to cells. Cells were incubated for 48 h before washing, trypsinisation and fixing in
4% paraformaldehyde. For coxsackievirus, 2 x 104 IU were incubated with antibody in a
200 m I incubation for 30 min at room temperature. Infected cells were fixed 8 h after
infection to preclude spreading infection. For both viruses, GFP positive cells were
enumerated by flow cytometry (FACSCalibur, BD Biosciences, San Jose, CA, USA).
Antibodies used in VNAs were pooled human serum IgG and Ig (090707 and 09071 3;
Athens Research and Technology, Athens, GA, USA), purified 9C1 2 anti-adenovirus 5
hexon mouse IgG (hybridoma obtained from the Developmental Studies Hybridoma
Bank, University of Iowa, IA, USA), goat anti-adenovirus polyclonal antibody (01 51-9004,
Abd Serotec, Oxford , UK and AB1 056, Millipore, Watford , UK).
Immunofluorescence
2.5 x 10 HeLa cells were seeded onto coverslips in 24-well plates and allowed to adhere
overnight. Cells were washed twice in DMEM before infection. 5 x 104 IU AdV5-GFP were
incubated with polyclonal or monoclonal anti-hexon adenovirus antibody (eg. 500 ng of
mouse monoclonal IgG in a 20 m I volume for 30 in at room temperature before addition
of 230 mI D EM). Cells were infected with 250 mI of this mixture for 30 min at 37 °C. Cells
were washed three times with PBS, fixed with 4% paraformaldehyde, permeabilised with
0.5% Triton X-1 00 in PBS and blocked with PBS-BSA (5% bovine serum albumin, 0.1%
Tween in PBS) for 1 h. Immunostaining for TRIM21 was performed with a rabbit 50 kDa
Ro/SSA primary antibody 20960 (Santa Cruz Biotechnology, Inc. , Santa Cruz, U.S.A.)
and for ubiquitin with a goat primary 6085 (Santa Cruz Biotechnology, Inc. , Santa Cruz,
U.S.A.) at 1 in 200 dilution in PBS-BSA. AlexaFluor-conjugated secondary antibodies
( Invitrogen) were used to detect primary antibodies at 1 in 200 dilution. Streptavidin
coated 0.25 m h latex beads (Sigma-Aldrich) were incubated with rabbit anti-streptavidin
polyclonal serum S6390 (Sigma-Aldrich) overnight at 4 °C. Beads were washed three
times with PBS and transfected into cells using Oligofectamine. Cells were washed with
PBS 3 h after transfection and fixed as above. Immunostaining for TRIM21 was
performed with immune serum raised in mouse against recombinant TRIM21 RBCC and
for conjugated ubiquitin as above both at 1 in 200 dilution in PBS-BSA. AlexaFluorconjugated
secondary antibodies (Invitrogen) were used to detect primary antibodies at 1
in 500 dilution. Confocal images were taken using a Zeiss 63X lens on a Jena LSM 7 10
microscope (Carl Zeiss Microimaging GmbH, Germany).
Fate-of-capsid assay
HeLa cells were plated at 2 x 105 cells per well in a 6 well plate in 2ml DMEM and left
overnight to attach. A proportion of the wells were treated with 8mM MG1 32 (Boston
Biochem) for 4 h. Untreated cells were exposed to an equivalent quantity of DMSO for
the duration of the treatment. 4 x 10 IU Ad5-GFP were mixed with 6 g 9C1 2
monoclonal antibody and incubated at room temp for 30 min then added onto the cells in
1 ml complete media. Infections were incubated at 37 °C for 1 hr before removing
infection mixtures and replacing with DMEM . Cells were harvested at indicated time
points post initial infection and boiled in 100 mI 1 x LDS sample buffer with reducing agent
(Invitrogen). Virus was detected with goat anti-hexon Ad5 ( 1 :1000 , AB1 056, Millipore)
and HRP conjugated anti goat IgG ( 1 :5000, sc-2056, Santa Cruz) . Antibody was
detected with donkey anti - mouse IgG ( 1 :500, AP1 92 Millipore) and protein A-HRP
( 1 :2000, 6 10438, BD Biosciences). TRIM21 was detected with TRI M21 RBCC immune
sera ( 1 :2000) and protein A HRP to avoid cross-reaction to the mouse antibody on the
gel.
Immunoblotting
Cells from a single well of a 6 well plate were scraped off, resuspended and heated at
98°C for 5 min in 100 i 1 x LDS sample buffer with reducing agent (Invitrogen). Equal
volumes were loaded onto a 4-12% NuPAGE gel and electrophoresed in 1 x MOPS
buffer (Invitrogen). Proteins were transferred onto Protran nitrocellulose membrane
(Whatman) and immunoblotted with the indicated antibodies. In all cases blots were
incubated with antibody in PBS containing 5% milk, 0.1% Tween and washed with PBSTween.
Visualisation was carried out using ECL Plus Western Blotting Detection System
(GE Healthcare). Westerns were stripped for re-probing as per manufacturers
instructions with 1 x Re-Blot Plus Strong Solution (2504, Millipore). Loading control blots
were carried out with rabbit polyclonal b-actin ( 1 :1000, #4967, Cell Signalling).
Fluorescence titration
Full-length and ARING-Box recombinant TRIM21 was expressed as MBP-fusion proteins
in E.coli and purified using amylose resin and size-exclusion chromatography. The BP
tag was removed via tev protease cleavage and cleaved TRIM21 was dialysed into
20mM Tris pH8, 100mM NaCI, 1mM DTT. Steady-state fluorescence titration experiments
were performed at 20°C using a Cary Eclipse fluorescence spectrophotometer (Varian)
with excitation at 296nm and emission at 335nm, using 15nm slit-widths and a PMT
voltage of 850. The quenching in intrinsic TRI 21 tryptophan fluorescence upon titration
of IgG was measured with an averaging time of 5s. Each titration was fit using
Kaleidagraph (Synergy Software) to the quadratic expression F = F R + f ((- (lo - TR0 +
d) ± ((( lo - TRo + d)2 ) + (4KdTR0)) ))/2; where F is the observed fluorescence, FTR
is the molar TRIM21 fluorescence, f is the molar change in fluorescence, (TR0) is the
total TRIM21 concentration, (l0) is the total antibody concentration, and K is the
dissociation constant.
Fluorescence anisotropy
The PRYSPRY domain of TRIM21 was expressed and purified as previously described 4,5 .
The protein was labelled with Alexa Fluor 488 5-SDP ester (Invitrogen) and dialysed into
50mM Tris pH 8 with 200mM NaCI. Anisotropy experiments were performed using a Cary
Eclipse fluorescence spectrophotometer (Varian) with excitation at 488nm and emission
at 530nm, using 10nm slit-widths and a PMT voltage of 600. IgM (Athens Research and
Technology, Athens, GA, USA) was titrated into 50nM PRYSPRY and the polarised
fluorescence averaged over 5s. The dissociation constant (Kd) was determined by fitting
the change in anisotropy to the quadratic expression given above using Kaleidagraph
(Synergy Software).
SEC MALS
SEC MALS was performed using a Wyatt Heleos II 18 angle light scattering instrument
coupled to a Wyatt Optilab rEX online refractive index detector. Samples were prepared
as described above and resolved on a Superdex S-200 analytical gel filtration column
running at 0.5 ml/min before passing through the light scattering and refractive index
detectors in a standard SEC MALS format. Protein concentration was determined from
the refractive index based on 0.186 ARI for 1 mg/ml, and combined with the observed
scattered intensity to calculate absolute molecular mass using Wyatt's ASTRA analysis
software. The major species in TRIM21 has a mass of 107 kDa averaged across the
indicated region of the peak. The predicted mass of monomeric TRI M21 is 54kDa,
making TRIM21 a dimer in solution and not a trimer as previously reported. SEC MALS of
IgG gives the expected mass of 154 kDa with small (<1 0%) levels of dimer mass 325
kDa, which is typical for IgG. TRIM21 -lgG complex resolves as multiple peaks,
corresponding to excess IgG with mass and elution volume as previously and a peak with
mass ~280kDa . The 280kDa peak is consistent with a 1:1 complex of TRIM21 :lgG,
where each protein is a homodimer.
Complementation neutralisation assay using exogenous TRIM21
HeLa cells were seeded at 1 x 105 cells per well in 2 ml complete DMEM in six-well plates
the day before infection. 5 x 104 IU AdV5-GFP were incubated with 4 g of goat antiadenovirus
polyclonal antibody (AB1 056, Millipore, Watford, UK) for 15 min before
addition of 200 g of appropriate recombinant TRIM21 protein, 100 mI total volume, and
incubation for a further 15 min at room temperature. Media on the cells were exchanged
for this mixture made up to 1 ml with complete DMEM. Cells were incubated at 37 °C in a
humid incubator for 48 h and then treated as in a virus neutralisation assay (see above) .
In vitro ubiquitination assays
In vitro assays were carried out largely as described 2 1 . Reactions were carried out in 1 x
Ubiquitination buffer (50mM Tris-HCI pH7.4, 2.5mM MgCI2, 0.5mM DTT) with the addition
of 2mM ATP, 300ng His-Uba1 , 300ng His-UbcH5c, 1 g ubiquitin (Sigma) and 50ng MBPTRIM21
or MBP-TRIM21 ARing-Box as indicated. Human Uba1 and UbcH5c were
expressed in bacteria and purified using Ni-NTA resin (Qiagen) as described 2 1. Antibody
adenovirus mixtures were made by incubating 5 x 104 IU AdV5-GFP per 150ng goat
polyclonal anti-hexon (Millipore) for 30 min, where 1m I mix contains 3.6 x 104 IU and
106ng antibody. Increasing amounts were added into the reaction mixture as indicated.
Controls with either just Ad5 or anti-hexon antibody contained 1.25 x 10s IU and 150ng
antibody respectively. Reaction mixtures were incubated at 37 °C for 1 h then stopped by
addition of LDS sample buffer and heating to 98°C for 5 min. Samples were run on a gel
and Western blotted for TRI 21 ( 1 :500, sc-25351, Santa Cruz), Ad5 hexon (donkey antigoat
IgG HRP 1:5000 sc-2056, Santa Cruz) or ubiquitin (1:1000, FK-2, Enzo Life
sciences) as indicated.
Example 1: Antibodies are internalised
It is assumed that antibodies do not routinely enter the cytosol during viral infection. To
test this, we pre-incubated adenovirus (a model human virus that causes respiratory
disease) with antibody and added the virions to cultured HeLa cells. Adenovirus was
chosen as it is a non-enveloped virus and its capsid is naturally exposed to serum
antibody prior to cellular infection. After 30 minutes of infection the cells were fixed and a
fluorescent anti-lgG antibody was added to detect antibody-coated virions. As can be
seen in Figure 1A, antibody-coated virions successfully infect cells. Similar results were
obtained using polyclonal anti-hexon antibodies and human serum IgG. Adenovirus
enters the cell by binding to the CAR receptor and becoming endocytosed. We found that
addition of antibody does not prevent this process and that antibody remains attached to
virus post-entry.
Example 2: TRIM21 mediates intracellular viral neutralisation
To address whether antibody-coated virus is accessible to cytosolic TRIM21, we costained
for TRIM21 . As shown in Figure 1A, TRIM21 is efficiently recruited to antibodycoated
viral particles.
Next, we tested the effect of TRIM21 recruitment to virions by quantifying the levels of
adenovirus infection. We used a virus that carries a GFP gene so that infection efficiency
could be determined by flow cytometry analysis. A standard viral neutralisation assay was
performed on HeLa cells pre-treated with control siRNA, TRI 21 siRNA, interferon-a
(IFNa) or IFNa and TRIM21 siRNA (Figure 1B). To take account of toxicity and variable
cell death between these different conditions, we measured the decrease in infection due
to the addition of antibody. In the absence of antibody, adenovirus infected -50% of cells.
The percentage of infected cells decreased rapidly with increasing antibody concentration
such that at 400 ng/m I antibody, infection was reduced by >50-fold (Figure B). However,
in cells depleted of TRIM21 , addition of 400 ng/m I antibody had a minimal effect on
infection (~3-fold).
During an immune response, FN activates the transcription of antiviral genes. We found
that TRIM21 is IFNa regulated and that the modest levels of endogenou s TRI 21 protein
are greatly increased by IFNa (Figure 1C). Consistent with this result, pre-incubation of
cells with IFNa increased the effect of antibody neutralisation such that at 400 ng/m I
antibody, infection decreased >230-fold. IFNa has pleiotropic effects but without addition
of antibody we observed little impact on adenovirus infection. To show that IFNa/antibody
neutralisation synergy is TRI 21-dependent, we specifically depleted the TRIM21 levels
that are up-regulated by IFNa, leading to >95% recovery of infectivity (Figure 1B). In all
experiments, antibody neutralisation of viral infection directly correlated with TRI 21
levels (Figure 1C). For example, cells expressing the most TRIM21 were almost 2 orders
of magnitude more resistant to adenovirus infection than those expressing the least.
Example 3: Variation of conditions
To demonstrate that TRIM21 /antibody intracellular neutralisation is not adenovirusspecific,
we tested its effect on coxsackievirus B3 infection. Coxsackievirus B3 is a
picornavirus, of the same genus as poliovirus, and is a leading cause of aseptic
meningitis. A replication -competent strain bearing a GFP-reporter gene was used to
infect HeLa cells pre-treated with combinations of TRIM21 siRNA and IFNa as described
above. Infection time was limited to < 16hrs to prevent spreading infection. Endogenous
levels of TRIM21 were insufficient to mediate a significant block (infection increases 2-
fold upon TRIM21 depletion), however, treatment with IFNa gave an almost total block to
infection in the presence of 15 g/ml antibody (Figure 1D). Depletion of TRIM21 in IFNatreated
cells recovered infection levels to those of untreated cells, demonstrating that this
is a TRIM21 -dependent effect.
We confirmed the robustness of this phenotype by examining the effect of different siRNA
sequences, cell types and types of antibody. As can be seen in Figure 2A, different
TRIM21 siRNA's with different target sequences reversed antibody neutralisation of
adenovirus infection by knocking-down TRI 21 levels. Next, we tested a range of cell
lines includ ing HeLa, HT1 080 and TE671 . A stable TRI 21 knockdown line was
established in each case using an shRNA vector based on the sequence of siRNA 2. In
all cells, TRIM21 mediated antibody neutralisation of adenovirus (Figure 2B) . Finally, we
tested the effect on adenovirus neutralisation of two different anti-Ad5 polyclonal
antibodies (Abd Serotec and Millipore) and an anti-Ad5 hexon monoclonal (9C1 2). In
every case, neutralisation of adenovirus was enhanced by TRI M21 upregulation and
reversed by TRIM21 KD (Figure 2C).
It is commonly thought that antibodies neutralise virus by blocking receptor binding and
preventing cell entry. However, >90% of the antibody neutralisation of adenovirus we
observe is mediated by TRIM21 (for example, at 200 ng/m I antibody there are 0.27%
infected cells in HeLa controls versus 10% in cells depleted of TRI M21 ) . To test which of
these two mechanisms dominates in a polyclonal response, we looked at the
neutralisation of adenovirus by pooled human serum IgG. We found that the majority of
the neutralisation affect (within the concentration range tested) was mediated by TRIM21
(Figure 2D). TRIM21 binds to IgG via the Fc domain, therefore antibody fragments
lacking the Fc should no longer be capable of neutralising virus as effectively. To confirm
that it is TRIM21 and not receptor blocking that is the primary source of viral
neutralisation we treated serum IgG with pepsin. Pepsin cleavage of IgG removes the Fc
and generates Fab2 fragments, which are still bivalent and capable of cross-linking
antigen. Moreover, Fab2 fragments bind antigen with the same affinity as IgG. We found
that Fab2 fragments were no longer able to neutralise adenovirus infection efficiently
(Figure 2D). Furthermore, when using Fab2, IFNa treatment or TRIM21 KD no longer
affected adenovirus infection.
Example 4: Interaction with IgM and IgA
The foregoing exampled demonstrate that in order for antibodies to mediate intracellular
viral neutralisation they must contain an Fc-fragment and TRIM21 must be present.
During the early stages of infection, in which innate immunity is critical, IgM rather than
IgG antibodies dominate the antibody repertoire. We tested whether TRI M21 interacts
with IgM and if so the importance of TRI M21 in IgM viral neutralisation. To investigate
TRIM21 :lgM binding, we labelled the TRIM21 PRYSPRY domain with an Alexa 488
fluorophore and measured its fluorescence anisotropy upon titration of IgM (Figure 2E).
The resulting titration curve was fit (Materials & Methods) to give an affinity (K ) of 16.8
mM ± 1.5 mM. The in vivo affinity of TRI M21 to IgM is likely to be significantly higher
however as full-length TRIM21 is a multimer. Complement C 1q, which binds IgM with
nanomolar affinity, has undetectable affinity when measured a s a monomer 6.
Next, we tested the effect of serum IgM on adenovirus infection. We found that pooled
human serum IgM and TRIM21 operate synergistically to neutralise adenovirus infection
(Figure 2F). Furthermore, as with IgG, the neutralisation of virus by IgM required
TRIM21. This suggests that TRIM21 works alongside innate immunity and is protective in
the early stages of a humoral immune response.
The same effect was seen in respect of IgA (Figure 2G). IgA is important as it is the
major isotype in the mucosa, which is often the first point of contact with a virus. An
infection experiment using serum secreted IgA shows that TRIM21 can use IgA to
neutralize virus. Anti-TRIM21 siRNA prevents viral neutralisation, whilst IFN-a
potentiates it.
Example 5: Mechanism of TRIM21 immunity
The previous examples demonstrate that there is an intracellular immune response
mediated by TRIM21 and antibodies that is capable of preventing viral infection. Next, we
examined the mechanism by which this intracellular neutralisation occurs. We
investigated the mechanism in three ways. First, we determined how TRIM21 targets
antibody and the thermodynamics of interaction. Second, we examined what events
subsequent to targeting are required for neutralisation. Third, we asked how virus is
neutralised.
TRIM21 is a multi-domain protein consisting of RING, B Box, coiled-coil and PRYSPRY
domains. We tested the role of these domains in IgG binding using multi-angle light
scattering (MALS) and fluorescence titration spectroscopy. Analysis of the MALS data
reveals that recombinant full-length TRIM21 forms a stable dimer and not a trimer as
previously reported 7 (Figure 3A). Furthermore, when mixed with IgG, TRIM21 forms a
stoichiometric complex consisting of 1 antibody and 1 TRIM21 (Figure 3A). Deletion of
the RING domain alone resulted in a destabilised recombinant protein, however deletion
of both RING and B Box did not affect TRIM21 stability or its ability to dimerise (data not
shown). Fluorescence titration spectroscopy revealed that full-length TRIM21 and ARINGBox
bound to IgG with a similar dissociation constant (K ) of < 1nM (Figure 3B&C). As
the monomeric PRYSPRY domain binds with ~150nM affinity 4,5 , this indicates that the
coiled-coil domain is required for TRIM21 dimerisation and the simultaneous engagement
of both IgG heavy chains. The sub-nM affinity of TRIM21 for IgG makes TRIM21 the
highest affinity antibody receptor in the human body. The evolution of such a high affinity
interaction explains how TRI 21 efficiently targets virus.
Next, we looked at what happens to the virus after TRIM21 is recruited and the role of the
RING and B box domains. As RING domains often display E3 ubiquitin ligase activity, we
hypothesised that TRIM21 may target bound virus for degradation via ubiquitination. Cells
possess two pathways for degradation of ubiquitinated material - the proteasome and
autophagy. To explore the role of these pathways in TRIM21 neutralisation of virus we
performed viral infection experiments in the presence of G132 (a proteasome inhibitor)
and 3-methyladenine (3-MA; an autophagy inhibitor). The autophagy inhibitor had no
affect on infectivity, however G132 significantly reversed TRIM21 neutralisation of
infectivity (Figure 3D). Titration experiments showed a direct correlation between
increasing levels of MG132 and reduced neutralisation (Figure 3E). The ability of MG132
to reverse neutralisation was dependent upon the presence of antibody and TRIM21.
Moreover, addition of MG132 could not recover infection in cells depleted of TRIM21,
showing that TRIM21 and proteasome function are essential components on the
neutralisation pathway (Figure 3F).
To determine whether ubiquitination is essential to target virus to the proteasome, we
tested the ability of full-length TRIM21 and ARING-Box recombinant proteins to neutralise
infection. We incubated protein with antibody-coated virions and allowed the virus to
infect cells depleted of TRIM21 . As can be seen in Figure 4A, deletion of the RING and B
Box domains prevents TRIM21 neutralisation of virus. We attempted to repeat these
experiments in cells over-expressing TRIM21, however this led to loss of function. Loss of
function could be partially reversed with interferon suggesting that over-expression
titrates essential co-factors rather than generates inactive protein (data not shown). To
confirm that the neutralisation we observe with recombinant proteins correlates with
ubiquitin ligase activity, we compared the ability of full-length and ARING-Box proteins to
auto-ubiquitinate. Whilst deletion of the RING and B Box domains has no effect on IgG
binding, it abolishes ubiquitination (Figure 4B). Thus both TRIM21 ubiquitination activity
and proteasomal function are required for viral neutralisation. To demonstrate that it is the
intracellular TRI 2 -associated viral particle that is ubiquitinated, we examined infected
cells by confocal microscopy and stained for ubiquitin. As can be seen in Figure 4C,
virions co-localised with TRIM21 are also positive for ubiquitin.
Whilst E3 ubiquitin ligases are known to auto-ubiquitinate, it is the transfer of ubiquitin to
substrate that is thought to be important for proteasomal targeting. However, proteasomal
targeting via auto-ubiquitination would allow TRIM21 to neutralise any virus and prevent
evolution of viral mutants that escape ubiquitin-conjugation. Consistent with this
mechanism, whilst we found that TRIM21 efficiently forms ubiquitin chains on itself we
found no detectable ubiquitination of either IgG or virus in our in vitro ubiquitination assay
(Figure 4D). This correlates with the extremely high affinity with which TRI 2 1 has
evolved to bind antibody. If TRI 2 1 were transferring ubiquitin to antibody or virus
through normal enzymatic turnover then a high affinity would translate as a highly
inefficient K ,
To determine what happens to virus after TRIM21 -mediated targeting to the proteasome,
we performed a fate-of-capsid timecourse experiment. We compared the levels of hexon
protein (viral capsid) in infected HeLa cells to those in cells depleted of TRIM21 . By 2
hours post-infection there was markedly less hexon in HeLa com pared to TRI M21
depleted cells (Figure 4E). This indicates both that TRIM21 mediates degradation of virus
and that it is a rapid process. Addition of MG1 32 prevented the decline in hexon levels,
confirming that virus is being physically degraded in a proteasome-dependent manner.
As proteasomal targeting by TRI 2 1 requires virus to be antibody-bound, we also looked
at the antibody levels in infected cells. We found that the destruction of virus is paralleled
by disposal of antibody (Figure 4E). In contrast, we saw little affect of MG 32 on TRIM21
levels suggesting that only a fraction of the total pool of cellular TRI 2 1 is degraded or
that TRI M21 is recycled (Figure 4E).
The combination of antibody targeting and TRIM21 auto-ubiquitination mean that no
direct viral interactions are required for neutralisation. This means that TRIM21 -mediated
immunity should be broadly effective against most intracellular pathogens. To test this,
we transfected cells with streptavidin latex beads coated in anti-streptavidin antibody.
TRIM21 is efficiently recruited to the antibody-bound beads (Figure 5). Furthermore,
TRIM21 associated beads are positive for ubiquitin. Thus, TRIM21 does not require any
direct pathogen interaction for binding or ubiquitination, should be effective against a
broad spectrum of pathogens and be difficult to evade.
Example 6: Activity in cell culture
Embryonic fibroblast cells were prepared from either wild-type or TRIM2 1 KO C57BL/6
mice (22) and challenged with GFP-adenovirus in the presence of 9C12, a monoclonal
anti-hexon antibody (available from DSHB , Iowa); hexon is the major coat protein of
adenovirus. 9C12 potently prevented infection of cells from wild-type mice but had
almost no affect preventing infection of cells from knock-out mice. Almost all the cells
from the knock-out were infected even in the presence of saturating concentrations of
antibody. See Figure 6. This shows that TRIM21 provides very potent protection against
viral infection and that it is important for the ability of antibodies to neutralize, as a
potently neutralizing antibody becomes non-neutralizing in the absence of TRIM21.
Example 7 : Activity in wild-type and knock-out mice
Virus preparation
A 3T3 mouse fibroblast cell line was infected with mouse adenovirus type 1 (MAV-1)
reference strain purchased from American Type Culture Collection (ATCC). Four days
later infected cells and supernatant were collected. Virus was released from the cells by 3
repeated freeze-thaw cycles. Cell supernatant and pellet free cell lysate were pooled
together and MAV-1 particles were purified by twice by equilibrium centrifugation in
continuous CsCI gradients. Virus was quantified by measuring A260 value which
corresponded to 1.8x10 3 pfu/ml. Virus infectivity was measured by end point dilution
assay and tissue culture 50% infectious dose value (TCID50), calculated by the Reed
and Munch method, was 8.4x108/ ml or 5.8x1 08 pfu/ml.
Experimental infections
For LD50 determinations, six-week-old C57BL6 mice were infected by intraperitoneal
(i.p.) injection (four animals per dose) of 10-fold serially diluted doses of MAV-1 in 100 ul
of PBS and observed up to twice daily for morbidity and mortality. Infection of wild type
mice with 4x1 05 pfu resulted in 75% mortality rate (Fig. 8). Therefore for all further
experiment in C57BU6 wild type and TRIM21 knockouts mice we choose a subclinical
4x1 04 pfu dose of MAV-1.
Viral titres
To test involvement of TRIM21 in immunity to infection we challenged 6 WT and 6 KO
naive mice with 4x104 pfu dose of MAV-1. Unless mice exceed moderate symptoms they
were culled on day 9 p.i. Spleen and brain were collected from culled animals and both
virus and genomic DNA were prepared. Genomic DNA was used in RT-PCR with
specific hexon primers to quantitate viral levels. Virus was titrated by TCID50 to
determine viraemia in each animal. The experiment was designed such that the ability of
TRIM21 to augment the primary immune response (IgM) is the principle determinant of
survival and/or viraemia.
To determine the role of TRIM21 in protective immunity (IgG) the experiment was
performed on MAV-1 challenged mice that have neutralizing antibody to the virus.
Therefore, mice were challenged with a subclinical dose of MAV-1, and rechallenged
after 9 days with a clinical dose of virus.
In both experiments, it is observed that TRIM21 KO mice show increased viral load
and/or mortality as a result of MAV-1 infection we conclude that the presence of
TRIM21 is important for mediating the antiviral effects of antibody treatment in mouse,
which are known to be effective against adenovirus infection (16).
Example 8: Antibody-TRIM21 Fusion
We have tested a monoclonal anti-hexon mouse antibody (9C12) and found it to possess
potent neutralization activity against adenoviral infection (see Example 6). cDNA was
prepared from the 9C12 hybridoma cells and light and heavy chains were amplified by
PCR using the following primers:
LIGHT CHAIN
Kappa chain into CMV promoter of pBudCE4.1 :
FORWARD:
VL1S acgt GTCGAC ccaccATG GAG ACA GAC ACA CTC CTG CTA T
VL2S acgtGTCGACccaccATG GAT TTT CAA GTG CAG ATT TTC AG
VL3S acgt GTCGAC ccaccATG GAG WCA CAK WCT CAG GTC TTT RTA
VL4S acgt GTCGAC ccaccATG KCC CCW CT CAG YTY CT GT
VL5S acgt GTCGAC ccaccATG AAG TTG CCT GTT AGG CTG TTG
REVERSE
CLX c tg c agaCTAACACTCATTCCTGTTGAAGC
HEAVY CHAIN
Heavy chain gamma-1 into EF-1a promoter of pBudCE4.1 (Figure 7):
FORWARD
VH1N aataGCGGCCGCcaccATGGRATGSAGCTGKGTMATSCTCTT
VH2N aataGCGGCCGCcaccATGRACTTCGGGYTGAGCTKGGTTTT
VH3N aataGCGGCCGCcaccATGGCTGTCTTGGGGCTGCTCTTCT
VH4N aataGCGGCCGCcacCATGATRGTGTTRAGTCTTYTGTRCCTG
REVERSE
CHKpn catgGGTACCTCATTTACCAGGAGAGTGGGAG
Where: r=a, g; y=c, t ; m=a, c ; k=g, t ; s=c, g; w=a, t ; v=a, c, g; n=a, c, g, t .
Amplified DNA was then sequenced to give the following light and heavy chain
sequences:
Heavy Chain: SEQ ID NO 13
Light Chain: SEQ ID NO 14
The corresponding amino acid sequence was then reverse translated into a codon
optimised DNA sequence for expression in pBudCE4.1 ( Figure 7). Each chain was
cloned with an N-terminal secretory signal to ensure secretion of the antibody protein.
The optimised sequences are as follows:
Heavy Chain: SEQ ID NO 15
Light Chain: SEQ ID NO 16
The resulting recombinant 9C12 expression vector was then used as the starting point to
clone fusion proteins, such that the C-terminus of the heavy chain (end of the CH3) was
joined via a short linker to the beginning of TRIM21. Three variants were cloned
representing fusion of either full-length TRI 21, the RING, B Box and Coiled-coil
domains, or the RING and B Box. In one form, the resulting fusion sequences were:
9C12-Full TRIM21 fusion: SEQ D NO 17
9C12-RBCC fusion: SEQ ID NO 18
9C12-RB fusion: SEQ ID NO 19
In each case these heavy chain fusions were expressed together with an unmodified light
chain in the multi-chain expression vector pBud (see Fig. 7). Expression of these
constructs can be performed in cell lines such as the suspension cell line 293 F.
Antibody is secreted into the medium. In order to purify antibody from media,
supernatant is applied to a Protein A affinity resin and then eluted in low pH buffer. After
elution, the purified protein is returned to a physiological saline buffer. The purity of
purified protein is assayed by SDS PAGE.
The following experiments are then used to test the efficacy of the chimeric proteins:
GFP adenovirus is pre-incubated with the chimeric proteins at a range of concentrations.
The adenovirus-chimera mixture is added to cultured cells at a viral titre designed to yield
an MOI of ~0.5. Infected cells are incubated for ~24 hours and the infection efficiency
determined by FACS analysis by counting the number of GFP positive cells. Cell lines
that can be used to test efficacy include adenovirus-permissive cell lines such as 293,
HeLa and MEF. To further demonstrate the efficacy of the chimeras, these experiments
can be carried out under conditions of endogenous TRIM21 depletion by siRNA or
shRNA or in cells where TRIM21 has been genetically knocked-out.
Example 9
The above example pertains to molecules in which the activities of virus binding and
TRIM21 function are combined in a single polypeptide. If this single polypeptide requires
another polypeptide chain to be functional (for instance a light chain) this must be
included prior to incubation with virus, usually during expression. In the next example we
describe a molecule that can bind antibodies and has TRIM21 activity in the absence of
endogenous T IM21 protein. Thus instead of having a single molecule with both virus
binding and TRIM21 function, the two activities are separated into two discrete molecules
- one with the ability to bind a pathogen (as exemplified by an antibody) and a second
with the ability to bind the first and cause the pathogen to be neutralized (as exemplified
by TRIM21). We have already described previously the addition of TRIM21 exogenously
and shown that this has antiviral activity. This example describes the fusion of the
antibody binding domain of Protein A (pA) to the catalytic domains of TRIM21. Three
examples are given, using either the RING, B Box and coiled-coil domains, the RING and
B Box domains or the RING domain. A further example is a modification in which the
Protein A domain is found at the C-terminus. Further constructs are envisaged in which
pA is replaced with another antibody binding domain (eg Protein G, selected peptide
ligands) and/or in which the catalytic domains are replaced (eg with those of another
TRIM protein) to preserve ubiquitin-proteasome recruitment.
pA-RBCC SEQ: ID NO 20
PA-RB: SEQ ID NO 2 1
PA-R: SEQ ID NO 22
RBCC-Pa: SEQ ID NO 23
These sequences were cloned into a bacterial expression construct with affinity tags to
allow efficient purification (His and MBP tags). The proteins were expressed overnight at
25°C, the cells lysed and protein purified on affinity resin followed by gel filtration.
Antiviral efficacy was tested by incubating the proteins at a range of concentrations with
an antiviral antibody (eg 9c12) and adding the mixture to virus (eg GFP adenovirus)
before infecting cultured cells at an MOI of -0.5. Permissive cell lines must be used (eg
for adenovirus - HeLa, 293, MEFs). Infection efficiency is then determined by FACS, by
counting the number of GFP positive cells.
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Claims
1. A compound comprising:
(a) a ligand which binds, directly or indirectly, specifically to an antigen of a pathogen,
provided that said ligand is not the PRYSPRY domain of TRI M21 ; and
(b) a RING domain and/or an inducer of TRIM21 expression.
2. A compound according to claim 1, wherein the ligand binds directly to the antigen
and is selected from the group consisting of at least part of an immunoglobulin molecule,
a peptide and/or nucleic acid aptamer, and a structured polypeptide ligand .
3. A compound according to claim 2, wherein the immunoglobulin molecule is
selected from the group consisting of an IgG, IgA, Ig , IgE, IgD, F(ab') , Fab, Fv, scFv,
dAb, V HH, IgNAR, a modified TCR, and multivalent combinations thereof.
4. A compound according to claim 3, wherein the immunoglobulin molecule
comprises at least one of a V H domain and a V L domain.
5. A compound according to any preceding claim, wherein the antigen is specific to a
virus.
6. A compound according to claim 1, wherein the ligand binds indirectly to the
antigen, and is selected from the group consisting of Protein A, Protein G, Protein L, an
anti-immunoglobulin peptide and an anti-immunoglobulin antibody.
7. A compound according to any preceding claim, wherein the RING domain
possesses E3 ligase activity.
8. A compound according to any preceding claim, wherein the RING domain is
derived from a TRIM polypeptide.
9. A compound according to claim 8, wherein the TRIM polypeptide is selected from
the group consisting of TRI M5a, TRIM 19, TRI M21 and TRI M28.
10. A compound according to any preceding claim , which comprises two or more
RING domains.
11. A compound according to any preceding claim , further comprising
polypeptide B-box domain and/or a TRIM polypeptide coiled-coil domain.
12. A compound according to claim 11 , comprising a TRIM polypeptide, wherein the
B30.2 domain has been replaced with at least one of a VH domain and a VL domain.
13. A compound according to any one of claims 1 to 12, wherein the inducer of
TRIM21 expression is interferon or an interferon inducer.
14. A compound according to claim 13, wherein the interferon inducer is selected from
the group consisting of a viral or bacterial antigen , a polyanion, a TLR agonist and a small
molecule interferon inducer.
15. A compound according to claim 13 or claim 14, wherein the interferon or interferon
inducer is bound to the compound by means of a labile linker.
16. A method for treating a pathogenic infection, comprising administering to a
subject a compound according to any preceding claim.
17. Use of a compound according to any one of claims 1 to 15, for treating a
pathogenic infection.
18. A method for treating an infection in a subject, comprising co-administering to the
subject an antibody specific for an antigen of a pathogen causing said infection, and a
polypeptide comprising a ligand which binds to said antibody, and a RING domain.
19. Use of an antibody specific for an antigen of a pathogen causing an infection in a
subject, and a polypeptide comprising a ligand which binds to said antibody and a RING
domain, for the treatment of said infection.
20. A method for treating an infection in a subject suffering from such an infection,
comprising administering to the subject a therapeutically effective amount of a
polypeptide comprising a polypeptide comprising a ligand which binds, indirectly, to an
antigen of a pathogen and a RING domain.
2 1. Use of a polypeptide comprising a polypeptide comprising a ligand which binds,
indirectly, to an antigen of a pathogen and a RING domain for the treatment of an
infectious disease in a subject.
22. A method according to claim 18 or 20, or the use according to claim 19 or 2 1,
wherein the ligand is selected from the group consisting of the TRIM21 PRYSPRY
domain, Protein A, Protein G, Protein L, an anti-immunoglobulin peptide and an anti¬
immunoglobulin antibody.
23. A method or use according to any one of claims 18 to 22, wherein the polypeptide
further comprises a TRIM polypeptide coiled coil domain and/or a TRIM polypeptide BBox
domain.
24. A method or use according to claim 23, wherein the polypeptide is human
TRIM21.