NZ623656B2 - Anti-??tcr antibody - Google Patents
Anti-??tcr antibody Download PDFInfo
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- NZ623656B2 NZ623656B2 NZ623656A NZ62365612A NZ623656B2 NZ 623656 B2 NZ623656 B2 NZ 623656B2 NZ 623656 A NZ623656 A NZ 623656A NZ 62365612 A NZ62365612 A NZ 62365612A NZ 623656 B2 NZ623656 B2 NZ 623656B2
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- C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
- C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
- C07K16/2809—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily against the T-cell receptor (TcR)-CD3 complex
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- C07K16/2893—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against CD52
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Abstract
Disclosed is a humanised monoclonal antibody specific for the human ??TCR/CD3 complex which comprises a heavy chain variable region comprising an amino acid sequence selected from the group consisting of the sequences set forth as SEQ ID NOs: 7, 12, 13, 15 and 16, and a light chain variable region comprising the amino acid sequence set forth as SEQ ID NO: 14, wherein the sequences are as disclosed in the complete specification. Also disclosed is the use of said antibody in in the manufacture of a medicament for use in suppressing a T cell mediated response in a subject. omprising the amino acid sequence set forth as SEQ ID NO: 14, wherein the sequences are as disclosed in the complete specification. Also disclosed is the use of said antibody in in the manufacture of a medicament for use in suppressing a T cell mediated response in a subject.
Description
Anti-ocBTCR antibody
The present invention relates to an dy specific for the alpha beta T cell or
(dBTCR). I n particular, the invention relates to a humanized anti-ocBTCR antibody, which
is derived from the murine monoclonal antibody BMAOSt, and the use of said humanized
LA dy in immunosuppressive therapy.
Introduction
The use of immunosuppressive agents in autoimmune diseases and organ transplant
therapy is well documented; however the process is far from optimal. Toxicity,
opportunistic ions, cytokine storm and increased risk of cancer are prevalent in
patients d with these agents. The use of ics in this arena has improved t
outcome to some degree yet these side effects remain evident.
pose
The use of polyclonal antisera against lymphocytes is well known for the pur of
immunosuppression. However, antisera are labor—intensive to produce, show properties
which vary between batches, and the specificity which can be obtained using polyclonal
antisera is limited.
onal antibody production by hybridoma logy was first described by K6hler
and Milstein (Nature 256:495—497 (1975)). As compared to polyclonal ra,
monoclonal antibodies (mAbs) are more ic, and have more consistent properties.
mAbs have been most frequently and successfully used for immunosuppressive therapy
in clinical organ transplantation. However, most mAbs used as immunosuppressive
agents for treating autoimmune diseases and in transplant patients have a broad
immunosuppressive capacity, thus undesirably influencing functions of a wide um of
immune cells, presumably not all involved in graft rejection.
Mouse monoclonal antibodies against T cell surface receptor antigens were first produced
in 1979 using hybridoma technology (Kung et al. (1979) Science 206:347-349). Of the
three monoclonal antibodies discovered by Kung et a/., one antibody ated
muromonab—CD3 (OKT3) had d specificity to the CD3 receptor of the T cell, reacting
with more that 95% of peripheral mature T cells without affecting immature thymocytes.
Binding of OKT3 to the CD3 complex causes internalization of the CD3 receptor and loss
of CD3 positive cells from the periphery. Successful OKT3 treatment is associated with a
prompt decline in CD3 positive T cells from approximately 60% to less than 5%.
OKT3 has been extensively used for the treatment of patients oing acute allograft
rejection after kidney transplantation (Russell, P.S., Colvin, R.B., Cosimi, A (1984)
Annu. Rev. Med. 35:63 and Cosimi, A.B., Burton, R.C., Colvin, R B ef al. (1981)
Transplantation 32:535). Moreover, OKT3 and rabbit complement were used for purging
U‘i mature T cells from donor marrow to t acute graft versus host disease (GVHD) in
allogeneic bone marrow transplantation (Prentice, H.G., Blacklock, H.A., Janossy, G. etal.
(1982) Lancet 1:700 and Blacklock, H.A., Prentice, H.G., e, MJ. etal. (1983) Exp.
Hematol. 11:37). Whereas OKT3 ent seems to be effective in the prevention of
GVHD in allogeneic bone marrow transplantation for acute leukemia, a combined in
/ in
vifro vivo treatment with OKT3 failed to prevent GVHD during therapy for severe
combined immunodeficiency (Hayward, A. et al. (1982) Clin. Lab. Observ. 100:665).
ponses
Treatment of T cells with OKT3 s several res inconsistent with immune
suppression including T cell activation, production of immune mediators and T3-
modulation. The T3-antigen complex ized by CD3-mAbs (e.g., OKT3) is postulated
to play a crucial role during T cell activation. beta T lymphocytes recognize peptide—
MHC ligands by means of a multimeric protein ensemble termed the GB T cell antigen
receptor (TCR)-CD3 complex. This structure is composed of a variable 08 TCR dimer
which binds antigens and three invariant dimers (CD3ve, 55 and (C) which are involved in
TCR-CD3 surface transport, ization and signal transduction. The alpha beta T cell
receptor (dBTCR) is expressed on the majority (approx. 95%) of T cells and has a critical
role in T cell activation via engagement of antigen displayed on MHC. The remaining 5%
of cells are gamma delta T cell receptor (y8TCR) positive. The ySTCR positive cell
ponse
population plays an important role in the innate immune res in defense against
opportunistic infections of bacterial, viral and fungal . Gamma delta T cells do not
play a role in graft rejection in transplantation. Therefore, targeting the ocBTCR positive
cell population and sparing the y5TCR positive population should allow for significant
therapeutic efficacy whilst maintaining a baseline immune protection against opportunistic
infections.
The mouse lgG2b monoclonal dy BMA031 (Borst etal. (Nov. 1990) Hum. Immunol.
29(3):175-88; EP0403156) is specific for the common determinant on the TCR
alpha/beta/CD3 x, and does not bind to the gamma-delta TCR. BMA031 is highly
immunosuppressive and is e of inducing apoptosis of activated T cells via a
mechanism of activation—induced cell death (AICD) (Wesselborg et al. (May 1993) J.
l. 150(10): 345). In vitro it inhibits a mixed lymphocyte reaction and it has
shown preliminary clinical efficacy in prevention of graft rejection in a number of solid
organ transplant scenarios as well as the treatment of acute graft versus host e
) (Kurrle efal. (Feb 1989) Transplant Proc. 21 (1): 1017—1019). BMA031 does not
engage human Fc gamma receptors (FcyR) in the majority of the human population
(approximately 10 of human possess FcyRs which do bind to mouse lgG2b isotype). As
such the antibody does not cause T cell activation via cross-linking of the T cell receptor
and, therefore, it does not induce T cell activation or the associated cytokine release. In
this regard its profile is highly preferable over that of OKTS. However, BMA031 is a
murine dy and, as such, is not suitable for repeat dosing in human subjects in view
ponse
of the human anti-mouse antibody (HAMA) res elicited therein.
Several humanized versions of BMA031 have been described (see, E 0403156; also
Shearman eta/., (1991) J. l. 147:4366-4373). As noted in EP0403156, mere CDR
ng was not sful in retaining antigen binding. One clone with significant
framework modifications, EUCIV3, successfully bound to T cells; however, as noted in
EP0403156, binding to the ocBTCR is not as ive as the parent BMA031 antibody as
determined by flow cytometry competition assays. We have also shown that the ability of
EuClV3 to inhibit an in vitro immune response is significantly reduced as compared to
BMA031 (see, Fig. 2). In addition EuCIV3 was originally generated on a wild-type human
IgG1 or IgG4 backbone which still retains FcyR binding. These humanized antibodies
ore allowed for T cell activation, proliferation and the concomitant cytokine release
and as such were significantly different to the original properties of BMA031.
The modification of antibody glycosylation is known in the art. For example, it is known
that aglycosylated antibodies can have extensively modified functionality; see, Boyd et al.
(1996) Mol. l. 32:1311—1318. However, aglycosylated forms of humanized
BMA031, or derivatives with modified glycosylation patterns, have previously not been
descnbed.
There is a need in the art, therefore, for an anti-ocBTCR humanized antibody which
improves on the binding properties of EUCIV3 and advantageously retains the
immunosuppressive and non-T cell-activatory properties of BMA031.
Summary of the Invention
In a first aspect, there is provided a humanized monoclonal antibody which comprises the
CDRs of BMA031, and retains the g ty of BMA031 for its cognate antigen. In a
first embodiment, said humanized antibody comprises a heavy chain variable region
sing the CDRs set forth in SEQ ID NOs: 7, 12 or 13 and the human lGH3—23
framework set forth in SEQ ID NO: 17, wherein framework position 6 is a donor residue; in
an ative embodiment, framework position 18 is a donor residue. Optionally,
framework positions 49 and/ 69 are donor residues.
I n a second embodiment, the humanized monoclonal antibody comprises a heavy chain
U‘l variable region sing the CDRs set forth in SEQ ID NOs: 15 or 16 and the human
IGHV1-3*O1 ork set forth in SEQ ID NO: 18, wherein one or more of framework
positions 38, 44 and/or 48 is a donor e; in an alternative embodiment, framework
ons 44 and 48 are donor residues.
I n a third embodiment, the humanized monoclonal antibody comprises a light chain
variable region comprising the CDRs set forth in SEQ ID NO: 1 and the human IGKV3-
11*01 framework set forth in SEQ ID NO: 19, wherein framework positions 70 and/ 71
are donor es. Optionally, position 46 is a donor residue.
Examples of antibodies according to the first embodiment include antibodies which
comprise a heavy chain variable region selected from the heavy chains comprising the
sequences set forth in SEQ ID NO: 7, SEQ ID NO: 12 and SEQ ID NO: 13, and a light
chain variable region sequence comprising the ce as set forth in SEQ ID NO: 1
Examples of dies ing to the second embodiment include antibodies which
comprise a heavy chain variable region selected from the heavy chains comprising the
sequences set forth in SEQ ID NO: 15 and SEQ ID NO: 16, and a light chain variable
2O region sing the sequence as set forth in SEQ ID NO: 14. The humanized antibodies
according to the described embodiments are humanized versions of the BMA031. Their
primary structures differ from that of the humanized antibody EuCIV3, which has
decreased binding to the ocBTCR as ed to BMA031.
In the sequence listing, CDRs are ted by means of annotation or underlining.
Frameworks are all sequences outside of the CDRs, which are defined according to the
"Kabat" numbering system and extended, where applicable, by use of “IMGT” CDR
definition. If a framework residue is not indicated to be changed to match a donor
sequence, it will rily be understood to be an acceptor residue.
The humanized antibodies may comprise a constant region. In one embodiment, the
constant region is of human origin.
The humanized antibodies of the invention may be further modified by EC engineering.
Immunoglobulins are liable to cross—link Fcy receptors, which can lead to constitutive T
cell tion for anti—T cell antibodies. In order to avoid Fcy cross—linking, dies can be modified
to remove the Fc region, such as by the generation of Fab or F fragments; however, truncated
immunoglobulins lack beneficial effector functions and exhibit a lower serum half—life. Therefore, the
Fc region of the humanized antibody can be modified to prevent Fcy cross—linking. Exemplary
techniques include generation of sylated immunoglobulins, for instance by modification of the
Fc region by an N297Q mutation. Immunoglobulins which fail to bind Fcy are also described by
Armour et a/., (1999) Eur. J. Immunol. -2624. The modification effected to IgG1 is known as
the Aab modification, and consists in a combination of the Aa on, in which IgG residues are
substituted at positions 327, 330 and 881 and
, IgG2 residues substituted at positions 233-236, and
the Ab mutation, in which residue 236 is deleted. In another embodiment, the glycosylation pattern of
dies according to the invention can be modified.
I n one embodiment, the antibody comprised one or more of ons 8298N, T299A and Y3OOS.
I n embodiments, the dy comprises two or more of ons N297Q, S298N, T299A and
YSOOS. For example, there is provided a zed antibody comprising the multiple mutations
N297Q/8298N/Y3008, S298N/T299A/Y3008 or S298N/YSOOS.
I n a second aspect, there is provided a humanized monoclonal antibody which comprises the CDRs
of BMA031, and retains the T cell suppression properties of BMA031.
According to one embodiment, there is ed a humanized monoclonal antibody specific for the
human /CDS complex which comprises a heavy chain variable region comprising an amino
acid sequence selected from the group consisting of the sequences set forth as SEQ ID NOs: 7, 12,
13, 15 and 16, and a light chain variable region comprising the amino acid sequence set forth as SEQ
ID NO: 14.
In a third aspect, there is ed a nucleic acid encoding at least a heavy chain variable region of a
humanized monoclonal antibody according to the preceding aspects of the described embodiments.
The nucleic acid may encode variable and constant regions of the humanized antibody. Heavy and
light chains may be encoded on separate nucleic acids or on the same nucleic acid molecule.
According to a fourth aspect, there is provided a cell which expresses a nucleic acid according to the
preceding aspect. The cell is, for example, a cell adapted to express antibody molecules in culture.
The nucleic acid may include signal sequences and/or other sequences or modifications which are
required for, or which modulate, sion of the antibody molecule in the cell, and/or secretion of
the antibody molecule from the cell.
I n a further embodiment, a humanized antibody is provided as bed in the foregoing
aspects, for use in suppressing a T cell mediated response in a subject.
For example, the T cell mediated response can be involved in a condition selected from
tissue transplantation, including solid organ transplant and composite tissue transplant,
U‘i tissue grafting, le sclerosis and type 1 diabetes.
Moreover, another ment provides a method for treating a subject suffering from a
condition involving an aberrant T cell mediated response comprising administering to a
subject in need thereof a pharmaceutically effective dose of an antibody according to the
described embodiments.
Humanized non-activatory anti-ocBTCR antibodies which do not induce cytokine release
have thus been ted which are capable of selective modulation of the ocBTCR and of
inducing apoptosis of activated orBTCR positive T cells. These antibodies have been
generated for use as immunosuppressive agents in T cell mediated diseases. These
antibodies have been generated through humanization of a mouse anti—dBTCR antibody
BMA031 and by Fc—engineering of the humanized antibodies to prevent engagement of Fc
gamma receptors. The antibodies according to the bed embodiments retain the
binding affinity of BMA031, unlike the humanized ns of BMA031 available in the art.
Further, as shown in in vitro education assays, the immunosuppressive properties of
dies according to the described embodiments are or to those of BMA031.
Moreover, unlike the humanized BMA031 antibodies of the prior art, the antibodies
according to the described embodiments do not induce cytokine release in normal PBMC.
In accordance with a fifth aspect, there is provided an antibody comprising a modified Fc,
in which said modified Fc comprises a modified glycosylation pattern which reduces chR
or binding, comprising one or more of mutations 8298N, T299A and YBOOS.
In one ment, the antibody comprises two or more of ons N297Q, 8298N,
T299A and Y3OOS.
ln embodiments, the antibody comprises the multiple mutations 8298N/Y3OOS,
S298N/T299A/Y3008 or 8298N/Y3008.
For example, the antibody may be an antibody as bed in ing aspects of the
invention.
According to a sixth , there is provided a multispecific antibody comprising at least
a heavy chain of a first binding domain as described in the preceding aspects of the
invention, and a second binding domain specific for a tumor—specific antigen.
I n one embodiment, the first binding domain comprises a heavy chain according to the
U‘i second aspect of the invention.
The multispecific antibody can comprise many different conformations; in one
embodiment, it comprises an anti-TCR/CD3 scFv and an anti-tumor scFv.
In one embodiment, the multispecific antibody is bispecific.
Brief Description of the Figures
Fig. 1. BMA031 binds more strongly to ocBTCR compared to EuCIV3.
Competition of binding of eled BMAO31 antibody by BMA031 MolgG2b, BMA031
HulgG1 and EuCIV3 Hulth antibodies. EuCIV3 has a decreased potency ed to
Fig. 2. EuCIV3 is less potent than BMA031 in an in vitro education (IVE) assay.
Plot showing loss of performance of EuCIV3 humanized antibody in biological assay when
compared to parent BMA031 antibody. CD8+ T cells were treated with anti—dBTCR
antibodies at various concentrations (X—axis) and tured with autologous tic
cells pulsed with the CMV peptide 3 (pp65) for seven days.
Fig. 3. HEBE1 binds ocBTCR comparably to BMA031 in a competition assay.
Competition of binding of PE—labeled BMAO31 antibody by BMA031 Hulth, HEBE1
HulgG1 and EuCIV3 HulgG1 antibodies. EuCIV3 has a sed potency compared to
BMA031 and HEBE1.
Fig. 4. HEBE1 has similar potency to EuCIV3 in an in vitro education (IVE) assay.
The IVE assay was performed as described in respect of Fig. 2.
Fig. 5. Schematic showing iterative rounds of mutagenesis of anti-aBTCR variable
domains.
Framework in shaded box depicts the F region where certain mouse residues lie.
Shaded residues in first row of mutations are the mouse amino acids that are useful to
U‘i maintain off—rate. Shaded residues in second row of mutations are the mouse residues
surrounding the CDR regions which were retained during the final germlining process.
Fig. 6. Optimized humanized antibody has ed off-rate compared to BMA031.
The kinetics of antibody dissociation from ocBTCR on T cells was measured by flow
BE 1 .
cytometry. BMA031 had a better off-rate compared to EuC|V3 and H E By optimizing
E BE 1
the binding domain of H we were able to improve the off-rate of HEBE1.H10
compared to BMA031.
Fig. 7. zed humanized antibody has improved off-rate compared to .
The kinetics of antibody dissociation from ocBTCR on T cells was measured by flow
cytometry. By optimizing the binding domain of HEBE1 we were able to improve the off-
rate of HEBE1.H66 compared to BMA031.
Fig. 8. Optimized humanized dy has improved off-rate compared to BMA031
in both Aab and aglycosylated formats.
The cs of dy dissociation from dBTCR on T cells was measured by flow
cytometry.
Fig. 9. Optimization of HEBE1 leads to equivalent onality as BMA031.
IVE assay as described in Fig. 4. BMA031 inhibited education of CD8+ T cells, as they
were unable to lyse specific targets in a dose dependent manner. The parental humanized
antibody, HEBE1, was not as potent as BMA031 and was able to only inhibit ion at
the highest dose (similar results observed with a second non-improved humanized Ab,
HEBE1 H13). Further improvements were made to the zed dy, HEBE1 H10,
which had equivalent potency to BMA031 in this assay.
Fig. 10. IVE data with anti-aBTCR antibodies.
Both HEBE1 and GL1 BM series antibodies showed improvements in IVE results in
comparison with BMA031.
Fig. 11. Antigen positive cells from I assay as determined by antigen-specific
tetramer binding.
Cells which are antigen—positive (i.e., have been educated within IV assay) are able to
bind to an MHC—tetramer molecule. When the IV assay was conducted in the presence
U‘i of antibody which has been able to prevent the education of T cells to antigen, there were
fewer cells able to bind to the MHC-tetramer at the end of the assay.
Fig. 12. Proliferation of PBMCs in presence of anti-ocBTCR antibodies, OKT3 and
stimulatory beads.
The stimulatory activity of OKT3 was not seen in anti-dBTCR antibodies in this
comparison.
Fig. 13. ne release from PBMCs in presence of anti-ocBTCR antibodies.
Cytokine release profile of anti—orBTCR dies was similar to the profile demonstrated
by .
4. V E
Fig. 1 mma release from T cells in I assay.
CD8+ T cells were treated with anti—dBTCR antibodies at various concentrations (see Fig.
2, x—axis) and co—cultured with autologous dendritic cells pulsed with the CMV peptide
495—503 (pp65) for seven days in an in vitro education (IVE) assay. lFN—gamma release
was measured in this assay.
Fig. 15. Activation-induced apoptosis by anti-aBTCR antibodies.
Antigen stimulated CD8+ T cells were induced to apoptosis by g of anti-aBTCR
antibodies BMA031 and HEBE1 H66. The ability of HEBE1 H66 to induce apoptosis was
increased compared to BMA031.
Fig. 16. Isolation of glycosylation s and aglycosylated dies
sie-blue stained gel g expression and purification of glycosylation mutants
Fig. 17. Binding of dBTCR antibody mutants to human Fclella using Biacore.
Biacore was used to assess binding to recombinant human Fclella (V158 & F158).
Fig 18. Binding of dBTCR antibody mutants to human Fcle using Biacore.
Biacore was used to assess binding to recombinant human and Fcle.
Fig. 19. Cytokine release from PBMCs in ce of glycosylation mutant anti-
ocBTCR antibodies (day 2).
Cytokine release profile for TNFa, GM-CSF, IFNy and |L10 of anti-dBTCR antibodies was
similar to the profile demonstrated by BMA031 and H66 delta A
Fig. 20. Cytokine release from PBMCs in presence of glycosylation mutant anti-
ocBTCR antibodies (day 2).
Cytokine e profile for IL6, |L4 and IL2 of anti-dBTCR antibodies was similar to the
e trated by BMA031 and H66 delta AB.
Fig. 21. Cytokine release from PBMCs in presence of glycosylation mutant anti-
ocBTCFt antibodies (day 4).
Cytokine release profile for TNFa, GM-CSF, IFNy and |L10 of anti-dBTCR antibodies was
similar to the profile demonstrated by BMA031 and H66 delta A
Fig. 22. Cytokine release from PBMCs in presence of glycosylation mutant anti-
ocBTCR antibodies (day 4).
4 2
Cytokine release profile for lL6, |L and IL of anti—dBTCR antibodies was similar to the
e demonstrated by BMA031 and H66 delta AB.
Fig. 23: Binding profiles of TRACERS.
Binding profiles of bi-specific antibodies to both tumor target cells and human T cells
assessed by flow cytometery.
Fig. 24: Cytotoxic activity of different T cell recruitment arms.
A panel of humanised BMAO31 antibodies have been created and from this panel a
number of antibodies have been selected which y xic activity against tumor
antigen sing cell lines
Fig. 25: Cytokine release profile of different T cell recruitment arms.
A panel of TRACERs with different T cell recruitment arms show similar cytokine release
profiles. Large amounts of cytokines are ed following activation of T cells in the
presence of target cells whereas in the presence of only unstimulated human PBMC
observed cytokine levels are significantly lower.
Fig. 26: Binding of CD52 antibody mutants to human Fclella using Biacore.
Biacore was used to assess binding of modified anti—CD52 to inant human
U] Fclella . Anti-CD52 sing S298N/Y3008 mutations in the Fc domain were
used to assess the effector on of the modified molecule. A: binding to CD52 peptide.
B: binding to a (V158). C: control binding to mouse FcRn.
Detailed Description of the Invention
Unless otherwise , 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 methods or techniques of the t invention. Al publications
pose
cited herein are incorporated herein by nce in their entirety for the pur of
describing and disclosing the methodologies, reagents, and tools reported in the
publications that might be used in connection with the invention.
The methods and techniques of the present application are generally performed according
to conventional methods well known in the art and as described in various general and
more specific references that are cited and discussed throughout the present ication
unless otherwise indicated. See, e.g., Gennaro, A.R., ed. (1990) Remington's
Pharmaceutical es, 18th ed., Mack Publishing Co.; Hardman, J.G., Limbird, LE,
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.
l-lV, Blackwell Scientific Publications; Maniatis, T. et al., eds. (1989) Molecular Cloning: A
Laboratory , 2nd edition, Vols. l-lll, Cold Spring Harbor Laboratory Press; Ausubel,
FM. et al., eds. (1999) Short Protocols in Molecular Biology, 4th n, John Wiley &
Sons; Ream et al., eds. (1998) Molecular Biology Techniques: An Intensive Laboratory
Course, Academic Press; Newton, OR. and Graham, A., eds. (1997) PCB (Introduction to
Biotechniques Series), 2nd ed., er—Verlag.
A humanized monoclonal antibody, as referred to herein, is an antibody which is
ed of a human antibody framework, into which have been grafted complementarity
determining regions (CDRs) from a non—human antibody. Changes in the human acceptor
framework may also be made. Procedures for the design and production of humanized
antibodies are well known in the art, and have been described, for example, in y et
a/., U.S. Patent No. 4,816,567; Cabilly et a/., European Patent Application 0 125 023;
U‘l Boss et a/., U.S. Patent No. 4,816,397; Boss et al., European Patent Application 0 120
694; Neuberger, MS. ef al., WO 86/01533; ger, MS. et a/., European Patent
Application 0 194 276 B1; Winter, U.S. Patent No. 5,225,539; , European Patent
Application 0 239 400; Padlan, E.A. eta/., European Patent Application 0 519 596. Further
s on antibodies, humanized antibodies, human engineered antibodies, and methods
for their preparation can be found in Kontermann, R . and DUbel, S. eds. (2001, 2010)
Antibody Engineering, 2nd ed., Springer-Verlag, New York, N
The term "antibody", unless ted otherwise, is used to refer to entire antibodies as
well as antigen-binding fragments of such antibodies. For example, the term
asses
encomp four-chain lgG molecules, as well as antibody fragments.
As used herein, the term “antibody fragments” refers to portions of an intact full—length
antibody, for example, as further described below.
Antibodies may be of any class, such as lgG, lgA or lgM; and of any subclass, such as
lgG1 or lgG4. Different classes and subclasses of globulin have different
properties, which may be ageous in different applications. For example, lgG4
2O antibodies have d binding to Fc receptors.
icity, in the context of the antibodies described herein, means that the claimed
antibody be capable of selectively binding its defined cognate antigen, which is the
ocBTCRCDB complex. The antibodies of the invention bind the ocBTCR.CD3 complex
expressed on cells.
The human /CD3 complex is the T cell or complex presented on the surface
of T cells. See, Kuhns ef al., (2006) Immunity 24:133—139. This complex is targeted by
the murine monoclonal antibody BMA031 (see, European patent application EP 0 403
156; SEQ ID NOs: 1 and 2).
Naturally occurring immunoglobulins have a common core ure in which two identical
light chains (about 24 kD) and two identical heavy chains (about 55 or 70 kD) form a
tetramer. The amino—terminal portion of each chain is known as the variable (V) region
and can be distinguished from the more conserved constant (C) regions of the remainder
of each chain. Within the le region of the light chain (also called the VL domain) is a
C—terminal portion known as the J region. Within the variable region of the heavy chain
(also called the VH domain), there is a D region in addition to the J region. Most of the
amino acid ce variation in immunoglobulins is confined to three separate locations
in the V regions known as hypervariable regions or complementarity determining s
U‘l (CDRs) which are directly involved in antigen binding. Proceeding from the amino—
terminus, these regions are designated CDR1, CDR2 and CDR3, respectively. The CDRs
are held in place by more conserved framework regions (FRs). Proceeding from the
amino-terminus, these regions are designated FR1, FR2, FR3 and FR4, respectively. The
locations of CDB and F R regions and a numbering system have been defined by Kabat et
al. (Kabat, E et a/., ces of Proteins of Immunological Interest, Fifth Edition, US
Department of Health and Human Services, US. Government ng Office (1991), and
updates thereof which may be found online). In addition, CDR region boundaries have
been further defined by IMGT nomenclature.
Variable regions of antibodies according to the described ments may be found in
SEQ ID NOs: 5-7 and 12-16, and may be obtained by humanizing BMA031, that is, by
transferring the CDRs of BMA031 to a human ork. Two series of humanized
E BE 1
antibodies are described; the H series, comprising SEQ ID NOs: 5—7, 12 and 13, and
the GL1 BM series, comprising heavy chain le regions as shown in SEQ ID NOS: 8,
and 16. In both cases, the light chain variable region used is as shown in SEQ ID NO:
2O 14 (GL1 BM VK43).
The human frameworks used are IGH3-23 in the case of HEBE1, and 3*O1 and
IGKV3-11*O1 in the case of GL1 BM.
nt regions may be derived from any human antibody nt regions. Variable
region genes may be cloned into expression vectors in frame with constant region genes
to express heavy and light immunoglobulin chains. Such expression vectors can be
transfected into antibody producing host cells for antibody synthesis.
Human antibody variable and constant s may be derived from sequence databases.
For example, immunoglobulin sequences are available in the IMGT/LIGM database
(Giudicelli et a/., (2006) Nucleic Acids Res. 34 :(suppl. 1—D784) or VBase
(vbase.mrc—cpe.cam.ac.uk).
Aglycosylated antibodies can have extensively modified functionality; see, Boyd et al.
(1996) Mol. l. 32:1311-1318. A "delta ab" or Aab modification, referred to herein,
is an F0 modification as described in Armour et a/., (1999) Eur. J. Immunol. 292613—2624.
Techniques for modifying glycosylation of antibody Fc regions are known in the art, and
e chemical, enzymatic and mutational means, for example, mutation of the N297
position in the CH2 domain. Techniques for mutating antibody genes for producing
aglycosylated lgG les are described in Tao and Morrison (1989) J. Immunol.
95-2601.
"Nucleic acids" as referred to herein e D N molecules which encode the antibodies
U‘t of the invention. Preferred are expression vectors, which are suitable for expressing the
antibody genes in a host cell. sion vectors and host cells for antibody gene
expression are known in the art; see, for example, Morrow, K.J. c Engineering &
Biotechnology News (June 15, 2008) 28(12), and Backliwal, G. et al. (2008) Nucleic Acids
Res. 36(15):e96-e96.
1. Antibodies
The invention encompasses antigen-binding nts of the humanized anti-ocBTCR
antibodies. Fragments of the antibodies are capable of binding the ocBTCRCDB complex.
They encompass Fab, Fab’, F(ab’)2, and F(v) fragments, or the individual light or heavy
chain le regions or portion thereof. Fragments include, for example, Fab, Fab',
F(ab')2, Fv and scFv. These fragments lack the Fc portion of an intact antibody, clear
more rapidly from the circulation, and can have less non-specific tissue binding than an
intact antibody. These fragments can be produced from intact antibodies using well
known methods, for example by proteolytic cleavage with s such as papain (to
2O produce Fab fragments) or pepsin (to produce F(ab')2 fragments).
The antibodies and fragments also encomp single—chain antibody fragments (scFv)
that bind to the DB complex. An scFv comprises an antibody heavy chain
le 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 g site that binds ocBTCR. An scFv may comprise a VH region at the amino-
al 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 VH, 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 VL and VH regions
pair to form monovalent les (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 antibodies and nts also encompass domain antibody (dAb) fragments as
described in Ward, E.S. et al. (1989) Nature 341:544—546 which consist of a VH domain.
The antibodies and fragments also encompass heavy chain antibodies (HCAb). These
antibodies are reported to form antigen—binding regions using only heavy chain variable
region, in that these functional antibodies are dimers of heavy chains only (referred to as
U‘i "heavy—chain antibodies" or "HCAbs"). Accordingly, antibodies and fragments may be
heavy chain antibodies (HCAb) that specifically bind to the ocBTCR.CD3 complex.
The antibodies and fragments also encomp antibodies that are SMlPs or binding
domain immunoglobulin fusion proteins specific for ocBTCR.CD3 complex. These
constructs are -chain polypeptides comprising antigen-binding domains fused to
globulin domains necessary to carry out antibody effector functions (see, WO
17148).
The antibodies and fragments also encompass diabodies. These are bivalent antibodies
in which VH and VL domains are expressed on a single polypeptide chain, but using a
linker that is too short to allow for pairing between the two domains on the same chain.
This forces the domains to pair with mentary domains of another chain and thereby
creates two antigen-binding sites (see, for e. WO 61). Diabodies can be
bispecific or monospecific.
The antibody or antibody fragment thereof does not cross-react with any target other than
the qBTCRCDB complex.
The antibody or fragment thereof may be ed in order to increase its serum half—life,
for example, by adding molecules — such as PEG or other water soluble polymers,
including polysaccharide polymers to increase the half—life.
The antibodies and fragments thereof may be bispecific. For example, bispecific
antibodies may resemble single antibodies (or antibody nts) but have two different
antigen g sites (variable regions). Bispecific antibodies can be produced by various
methods — such as al techniques, "polydoma" techniques or recombinant DNA
techniques. Bispecific antibodies may have binding specificities for at least two different
epitopes, at least one of which is the dBTCRCDB complex. The other specificity may be
selected from any useful or desired specificities ing, for example, specificity for
human serum albumin for the extension of ife in vivo.
The use of bi-specific antibodies in the clinic for gy ations is now becoming
reality with the tri—functional Catumaxomab (Removmab®) ed for use in cases of
malignant ascites and the bi—specific antibody Blinatumomab now in phase II trials in
hematological malignancies. These molecules have in common a binding arm which binds
to T cells and a second arm which binds to the tumor target cell which s in T cell
mediated lysis of the tumor target. Also in common, these molecules recruit T cells via the
CD3 n located on the cell surface. An alternative to recruitment via CD3 is to make
use of the GB T cell receptor (dB TCR) which is also expressed on the surface of the cell.
U‘i Accordingly, antibodies according to the present ion can be used to develop anti—
tumor antibodies by combining a specificity for a tumor associated antigen with a
icity for the dB T cell receptor (dB TCR).
2. Antibody production
The amino acid sequences of the variable s of the antibodies described herein are
set forth in SEQ ID NOs: 5-7 and 12-16. Antibody production can be performed by any
technique known in the art, ing in transgenic organisms such as goats (see, Pollock
et al. (1999) J. Immunol. Methods 231:147-157), chickens (see, , K.J.J. (2000)
Genet. Eng. News 20:1-55), mice (see Pollock et al., supra) or plants (see, Doran, P
(2000) Curr. Opinion Biotechnol. 11:199—204, Ma. J.K—C. (1998) Nat. Med. 4:601—606,
Baez, J. et al. (2000) BioPharm. 13:50—54, Stoger, E. etal. (2000) Plant Mol. Biol. 42:583—
590). Antibodies may also be produced by chemical synthesis or by expression of genes
encoding the dies in host cells.
A polynucleotide encoding the antibody is isolated and inserted into a replicable construct
2O or vector such as a plasmid for further propagation or expression in a host cell. Constructs
or vectors (e.g., expression vectors) suitable for the expression of a humanized
immunoglobulin according to the described embodiments are available in the art. A
variety of vectors are available, including vectors which are maintained in single copy or
multiple copies in a host cell, or which become integrated into the host cell's
chromosome(s). The constructs or vectors can be introduced into a suitable host cell, and
cells which express a humanized immunoglobulin can be produced and maintained in
culture. A single vector or multiple vectors can be used for the expression of a humanized
immunoglobulin.
Polynucleotides encoding the antibody are readily isolated and sequenced using
conventional ures (e.g., oligonucleotide probes). Vectors that may be used include
plasmid, virus, phage, transposons, romosomes of which plasmids are a typical
embodiment. Generally such vectors r e a signal sequence, origin of
replication, one or more marker genes, an enhancer element, a promoter and transcription
termination sequences operably linked to the light and/or heavy chain cleotide so
as to facilitate sion. Polynucleotides encoding the light and heavy chains may be
inserted into separate vectors and introduced (e.g., by ormation, ection,
electroporation or transduction) into the same host cell concurrently or sequentially or, if
desired, both the heavy chain and light chain can be ed into the same vector prior to
such introduction.
U‘i A promoter can be provided for expression in a suitable host cell. Promoters can be
constitutive or ble. For example, a promoter can be operably linked to a c acid
encoding a humanized immunoglobulin or immunoglobulin chain, such that it s
expression of the encoded polypeptide. A y of suitable promoters for prokaryotic and
eukaryotic hosts are available. Prokaryotic promoters include lac, tac, T3, T promoters
for E. coli; 3-phosphoglycerate kinase or other glycolytic enzymes e.g., enolase,
glyceralderhyde 3-phosphate dehydrogenase, hexokinase, pyruvate decarboxylase,
phosphofructokinase, glucose 6 phosphate isomerase, 3-phosphoglycerate mutase and
glucokinase. Eukaryotic promoters include inducible yeast promoters such as l
dehydrogenase 2, isocytochrome C, acid atase, metallothionein and enzymes
responsible for nitrogen metabolism or maltose/galactose utilization; R polymerase I I
promoters including viral promoters such as polyoma, fowlpox and iruses (e.g.,
adenovirus 2), bovine papilloma virus, avian sarcoma virus, cytomegalovirus (in particular,
the immediate early gene promoter), irus, tis B virus, actin, rous sarcoma virus
(RSV) promoter and the early or late Simian virus 40 and non-viral promoters such as EF-
2O 1 alpha (Mizushima and Nagata (1990) Nucleic Acids Res. 18(17):5322). Those of skill in
the art will be able to select the appropriate promoter for expressing a humanized
antibody or portion thereof.
Where appropriate, e.g., for expression in cells of higher eukaroytes, additional er
elements can be included instead of or as well as those found located in the promoters
described above. Suitable mammalian enhancer sequences include enhancer elements
from globin, elastase, albumin, otein, metallothionine and insulin. Alternatively, one
may use an enhancer element from a eukaryotic cell virus such as SV4O enhancer,
cytomegalovirus early promoter enhancer, polyoma enhancer, baculoviral enhancer or
murine lgG2a locus (see, WO 04/009823). Whilst such ers are often d on the
vector at a site upstream to the promoter, they can also be located elsewhere e.g., within
the untranslated region or downstream of the polyadenylation signal. The choice and
positioning of enhancer may be based upon compatibility with the host cell used for
expression.
In addition, the vectors (9.9., expression vectors) may comprise a selectable marker for
selection of host cells carrying the vector, and, in the case of a replicable vector, an origin
of replication. Genes encoding products which confer antibiotic or drug resistance are
common able markers and may be used in prokaryotic (e.g., f3—lactamase gene
(ampicillin resistance), Tet gene (tetracycline resistance) and eukaryotic cells (9.9.,
neomycin (G418 or geneticin), gpt (mycophenolic acid), ampicillin, or hygromycin
U‘i resistance . Dihydrofolate reductase marker genes permit selection with
methotrexate in a variety of hosts. Genes encoding the gene product of auxotrophic
markers of the host (e.g., LEU2, URAB, HISS) are often used as selectable markers in
yeast. Use of viral (e.g., baculovirus) or phage vectors, and vectors which are capable of
integrating into the genome of the host cell, such as retroviral vectors, are also
plated.
I n eukaryotic systems, polyadenylation and termination signals are ly linked to
polynucleotide encoding the antibody of the invention. Such signals are typically placed 3'
of the o reading frame. In mammalian systems, non-limiting examples of
polyadenylation/termination signals include those derived from growth hormones,
elongation factor-1 alpha and viral (e.g., SV40) genes or retroviral long terminal repeats.
I n yeast systems, non—limiting examples of polydenylation/termination signals include
those derived from the phosphoglycerate kinase (PGK) and the alcohol dehydrogenase 1
(ADH) genes. In prokaryotic systems polyadenylation s are lly not required
and it is instead usual to employ r and more defined terminator sequences. The
2O choice of polyadenylation/termination sequences may be based upon compatibility with
the host cell used for expression. In addition to the above, other features that can be
employed to enhance yields e chromatin remodeling elements, introns and host cell
specific codon modification. The codon usage of the antibodies of the invention can be
modified to accommodate codon bias of the host cell such to augment ript and/or
product yield (9.9., Hoekema, A. et al. (1987) Mol. Cell Biol. 914-24). The choice of
codons may be based upon compatibility with the host cell used for expression.
The invention thus relates to isolated nucleic acid molecules that encode the humanized
immunoglobulins, or heavy or light chains, thereof. The invention also s to isolated
nucleic acid molecules that encode an antigen—binding portion of the immunoglobulins and
their chains.
The antibodies can be produced, for e, by the expression of one or more
recombinant nucleic acids encoding the antibody in a le host cell. The host cell can
be produced using any suitable method. For e, the expression constructs (e.g.,
one or more vectors, 9.9., a mammalian cell expression vector) described herein can be
introduced into a suitable host cell, and the resulting cell can be maintained (e.g., in
e, in an animal, in a plant) under ions suitable for expression of the
construct(s) or vector(s). Host cells can be prokaryotic, including bacterial cells such as E.
coli (e.g., strain DH5aTM) (lnvitrogen, Carlsbad, CA), PerC6 (Crucell, Leiden, NL), B.
subti/is and/ other suitable bacteria; eukaryotic cells, such as fungal or yeast cells (e.g.,
U‘i Pichia pastor/s, Aspergillus sp., Saccharomyces cerevisiae, Schizosaccharomyces
pombe, Neurospora crassa), or other lower otic cells, and cells of higher
eukaryotes such as those from insects (e.g., Drosophi/a der 82 cells, St9 insect
cells) (WO 94/126087 (O'Connor)), BTl-TN-SB1-4 (High FiveT'V') insect cells (lnvitrogen),
mammals (e.g., COS cells, such as COS-1 (ATCC Accession No. CRL-1650) and COS-7
(ATCC Accession No. CRL-1651), CHO (e.g., ATCC Accession No. CRL-9096), CHO
DG44 (Urlaub, G. and Chasin, L (1980) Proc. Natl. Acad. Sci. USA, 77(7):4216-4220),
293 (ATCC Accession No. CRL-1573), HeLa (ATCC ion No. COL-2), CVl (ATCC
Accession No. COL-70), WOP (Dailey, L., et al. (1985) J. Virol., 54:739-749), 3T3, 293T
(Pear, W.S., et al. (1993) Proc. Natl. Acad. Sci. USA, 90:8392—8396), NSO cells, SP2/
cells, HuT 78 cells, and the like, or plants (e.g., tobacco, lemna (duckweed), and algae).
See, for example, Ausubel, F M et al., eds. Current Protocols in Molecular Biology,
Greene Publishing Associates and John Wiley & Sons Inc. (1993). I n some embodiments,
the host cell is not part of a multicellular organism (e.g., plant or animal), e.g., it is an
isolated host cell or is part of a cell e.
2O Host cells may be cultured in spinner flasks, shake flasks, roller bottles, wave rs
(e.g., System 1000 from otechcom) or hollow fibre systems but it is preferred for
large scale production that stirred tank reactors or bag reactors (e.g., Wave h,
Somerset, New Jersey USA) are used particularly for suspension cultures. Stirred tank
rs can be adapted for aeration using e.g., spargers, baffles or low shear impellers.
For bubble columns and airlift rs, direct aeration with air or oxygen bubbles maybe
used. Where the host cells are cultured in a serum-free culture medium, the medium can
be supplemented with a cell protective agent such as pluronic F—68 to help prevent cell
damage as a result of the aeration process. Depending on the host cell characteristics,
microcarriers maybe used as growth substrates for age dependent cell lines, or the
cells maybe adapted to suspension culture. The culturing of host cells, particularly
vertebrate host cells, may utilize a variety of operational modes such as batch, fed—batch,
repeated batch processing (see, Drapeau et al. (1994) Cytotechnology 15:103—109),
ed batch process or perfusion culture. gh recombinantly transformed
mammalian host cells may be cultured in serum—containing media such media comprising
fetal calf serum (FCS), it is preferred that such host cells are cultured in serum—free media
such as disclosed in Keen et al. (1995) Cytotechno/ogy —163, or commercially
available media such as ProCHO—CDM or UltraCHOT'V' (Cambrex NJ, USA),
supplemented where necessary with an energy source such as glucose and synthetic
growth factors such as recombinant insulin. The serum—free culturing of host cells may
require that those cells are adapted to grow in serum—free ions. One tion
U‘l approach is to culture such host cells in serum containing media and repeatedly exchange
80% of the culture medium for the serum-free media so that the host cells learn to adapt
in serum-free conditions (see, e.g., Scharfenberg, K. etal. (1995) Animal Cell Technology:
pments Towards the 21st y (Beuvery, E.C. et al., eds), pp.619-623, Kluwer
Academic publishers).
Antibodies according to the described embodiments may be secreted into the medium
and red and purified therefrom using a variety of techniques to provide a degree of
purification suitable for the intended use. For example, the use of therapeutic antibodies
for the treatment of human patients typically mandates at least 95 purity as ined
% %
by reducing SDS-PAGE, more typically 98 or 99 purity, when compared to the culture
media comprising the therapeutic antibodies. In the first instance, cell debris from the
e media can be removed using centrifugation followed by a clarification step of the
supernatant using e.g., iltration, ultrafiltration and/ depth filtration. Alternatively,
the antibody can be harvested by microfiltration, ultrafiltration or depth filtration without
prior centrifugation. A variety of other techniques such as is and gel ophoresis
2O and chromatographic techniques such as hydroxyapatite (HA), affinity tography
(optionally involving an affinity tagging system such as polyhistidine) and/or hydrophobic
interaction Chromatography (HIC) (see, US 5,429,746) are available. In one embodiment,
the antibodies, following various clarification steps, are ed using Protein A or G
affinity chromatography followed by further chromatography steps such as ion exchange
and/or HA chromatography, anion or cation exchange, size ion chromatography
and ammonium sulphate precipitation. Various virus removal steps may also be employed
(e.g., nanofiltration using, e.g., a DV—20 filter). Following these various steps, a purified
preparation comprising at least 10 mg/ml or greater, e.g., 100 mg/ml or greater of the
antibody of the invention is provided and, therefore, forms another embodiment of the
invention. Concentration to 100 mg/ml or greater can be generated by ultracentrifugation.
Such preparations are ntially free of aggregated forms of antibodies of the
ion.
Bacterial systems are particularly suited for the expression of antibody fragments. Such
fragments are zed intracellularly or within the periplasm. Insoluble asmic
proteins can be extracted and refolded to form active proteins according to methods
known to those skilled in the art, see, Sanchez etal. (1999) J. Biotechnol. 72:13—20; Cupit,
P M etal. (1999) Lett. Appl. Microbiol. 29:273—277.
The t invention also relates to cells sing a nucleic acid, e.g., a vector, of the
invention (e.g., an expression vector). For example, a c acid (i.e., one or more
U‘i nucleic acids) encoding the heavy and light chains of a humanized globulin
ing to the described embodiments, or a uct (i.e., one or more constructs, e.g.,
one or more vectors) comprising such nucleic ), can be uced into a suitable
host cell by a method appropriate to the host cell selected (e.g., transformation,
transfection, electroporation, infection), with the nucleic acid(s) being, or becoming,
operably linked to one or more expression control elements (9.9., in a vector, in a
construct created by processes in the cell, integrated into the host cell genome). Host
cells can be maintained under conditions suitable for expression (9.9., in the presence of
inducer, suitable media supplemented with appropriate salts, growth factors, antibiotic,
nutritional supplements, etc), whereby the encoded polypeptide(s) are ed. I f
desired, the encoded humanized antibody can be isolated, for example, from the host
k . asses
cells, culture medium, or mil This s encomp sion in a host cell (9.9., a
mammary gland cell) of a transgenic animal or plant (e.g., tobacco) (see, e.g., WO
92/03918).
3. Therapeutic Applications
Suppression of T cell activity is desirable in a number of situations in which
immunosuppression is warranted, and/or an autoimmune condition occurs. Accordingly,
targeting of the dBTCR.CD3 complex is indicated in the treatment of es involving an
inappropriate or red immune response, such as inflammation, autoimmunity, and
other conditions involving such mechanisms. In one embodiment, such disease or
disorder is an mune and/or inflammatory disease. Examples of such autoimmune
and/or inflammatory diseases are ic Lupus Erythematosus (SLE), Rheumatoid
Arthritis (RA) and inflammatory bowel disease (IBD) (including ulcerative colitis (U0) and
Crohn's disease (CD)), multiple sclerosis (MS), scleroderma and type 1 diabetes (T1D),
and other diseases and disorders, such as PV (pemphigus vulgaris), psoriasis, atopic
dermatitis, celiac disease, Chronic Obstructive Lung disease, Hashimoto's thyroiditis,
Graves' disease (thyroid), Sjogren’s me, Guillain—barré syndrome, Goodpasture's
syndrome, Addison's disease, Wegener's granulomatosis, primary biliary sclerosis,
sclerosing cholangitis, autoimmune hepatitis, polymyalgia rheumatica, Raynaud's
enon, temporal arteritis, giant cell arteritis, autoimmune hemolytic anemia,
pernicious anemia, teritis nodosa, behcet‘s disease, primary bilary sis, uveitis,
myocarditis, rheumatic fever, ankylosing spondylitis, glomerulenephritis, sarcoidosis,
dermatomyositis, myasthenia gravis, polymyositis, alopecia areata, and vitilgo.
BD .
I n one embodiment, such disease or disorder is SLE, R A or I In one embodiment,
U‘i such disease or disorder is MS.
In another embodiment, the antibodies according to the described embodiments are used
to aid transplantation by immunosuppressing the subject. Such use alleviates graft-
versus-host e. For a description of existing treatments for versus-host
disease, see , e.g., Svennilson, Bone Marrow Transplantation (2005) —867, and
references cited therein. Advantageously, the antibodies of the invention may be used in
combination with other available therapies.
With regard to the treatment of autoimmune diseases, combination therapy may include
administration of an antibody of the present invention together with a medicament, which
together with the antibody comprises an effective amount for ting or ng such
autoimmune diseases. Where said autoimmune disease is Type 1 es, the
combination therapy may encomp one or more of an agent that promotes the growth
of atic beta—cells or enhances beta—cell transplantation, such as beta cell growth or
survival factors or immunomodulatory dies. Where said mune disease is
rheumatoid arthritis, said combination therapy may encompass one or more of
rexate, an anti—TNF—B antibody, a TNF—B receptor—lg fusion protein, an anti—IL—15 or
anti—lL—21 antibody, a non—steroidal anti—inflammatory drug (NSAID), or a disease—
modifying anti—rheumatic drug (DMARD). For example, the additional agent may be a
biological agent such as an anti-TNF agent (e.g., Enbrel®, imab (Remicade®) and
adalimumab a®) or rituximab (Rituxan®). Where said autoimmune disease is
hematopoietic transplant rejection, poietic growth factor(s) (such as erythropoietin,
G—CSF, GM—CSF, IL—3, IL—11, thrombopoietin, etc.) or antimicrobial(s) (such as antibiotic,
antiviral, antifungal drugs) may be administered. Where said autoimmune disease is
psoriasis, the additional agent may be one or more of tar and derivatives thereof,
phototherapy, corticosteroids, Cyclosporine A, vitamin D analogs, methotrexate, p38
mitogen—activated protein kinase (MAPK) inhibitors, as well as biologic agents such as
anti—TNF— agents and Rituxan® . Where said autoimmune disease is an inflammatory
bowel disease (IBD) such as, for example, Crohn's Disease or ulcerative colitis, the
additional agent may be one or more of aminosalicylates, corticosteroids,
immunomodulators, antibiotics, or biologic agents such as Remicade® and Humira®.
The combination treatment may be carried out in any way as deemed ary or
convenient by the person skilled in the art and for the purpose of this specification, no
limitations with regard to the order, amount, repetition or relative amount of the
compounds to be used in combination is contemplated. Accordingly, the antibodies
U‘i according to the described embodiments may be formulated into pharmaceutical
compositions for use in therapy.
4. Pharmaceutical Compositions
In a preferred embodiment, there is provided a pharmaceutical composition comprising an
antibody ing to the invention, or a ligand or ligands identifiable by an assay method
as defined in the previous aspect of the invention. Ligands may be immunoglobulins,
peptides, nucleic acids or small molecules, as discussed herein. They are referred to, in
the ing discussion, as “compounds”.
A ceutical composition according to the invention is a ition of matter
comprising a compound or compounds capable of modulating T cell activity as an active
ingredient. The nd is in the form of any pharmaceutically acceptable salt, or e.g.,
where appropriate, an analog, free base form, tautomer, enantiomer racemate, or
combination thereof. The active ients of a pharmaceutical composition comprising
the active ingredient according to the invention are contemplated to exhibit therapeutic
activity, for example, in the treatment of graft-versus-host disease, when administered in
2O amount which depends on the particular case.
In another ment, one or more compounds of the ion may be used in
combination with any art recognized compound known to be suitable for ng the
particular indication in treating any of the aforementioned conditions. Accordingly, one or
more compounds of the invention may be combined with one or more art ized
compounds known to be suitable for treating the foregoing indications such that a
convenient, single composition can be administered to the subject. Dosage regima may
be ed to provide the optimum therapeutic response.
For example, several divided doses may be administered daily or the dose may be
proportionally reduced as indicated by the exigencies of the therapeutic ion.
The active ingredient may be administered in a convenient manner such as by the oral,
intravenous (where water soluble), intramuscular, subcutaneous, intranasal, intradermal
or suppository routes or implanting (9.9., using slow release molecules).
Depending on the route of administration, the active ingredient may be required to be
coated in a material to protect said ients from the action of enzymes, acids and
other natural conditions which may inactivate said ingredient.
I n order to administer the active ient by means other than eral administration,
U‘l it will be coated by, or administered with, a material to prevent its inactivation. For
example, the active ingredient may be administered in an adjuvant, co-administered with
enzyme inhibitors or in liposomes. nt is used in its broadest sense and includes any
immune stimulating compound such as interferon. Adjuvants contemplated herein include
resorcinols, non-ionic tants such as yethylene oleyl ether and n-hexadecyl
polyethylene ether. Enzyme inhibitors include atic trypsin.
Liposomes include water-in-oil-in-water CGF emulsions as well as conventional
liposomes.
The active ingredient may also be administered parenterally or intraperitoneally.
sions can also be prepared in ol, liquid hylene glycols, and mixtures
thereof and in oils. Under ordinary conditions of storage and use, these preparations
contain a preservative to prevent the growth of microorganisms.
The pharmaceutical forms suitable for injectable use include sterile aqueous solutions
(where water soluble) or dispersions and sterile powders for the extemporaneous
preparation of sterile injectable solutions or dispersion. In all cases the form must be
sterile and must be fluid to the extent that easy syringability exists. It must be stable under
the conditions of manufacture and storage and must be preserved against the
contaminating action of microorganisms such as bacteria and fungi. The carrier can be a
solvent or dispersion medium containing, for example, water, ethanol, polyol (for example,
glycerol, ene glycol, and liquid polyethylene glycol, and the like), suitable mixtures
thereof, and vegetable oils. The proper fluidity can be maintained, for example, by the use
of a g such as lecithin, by the maintenance of the required particle size in the case
of dispersion and by the use of superfactants.
The prevention of the action of microorganisms can be brought about by various
antibacterial and ngal agents, for example, parabens, chlorobutanol, phenol, sorbic
acid, thirmerosal, and the like. In certain cases, it may be preferable to include isotonic
agents, for example, sugars or sodium chloride. Prolonged absorption of the injectable
compositions can be brought about by the use in the compositions of agents delaying
absorption, for example, aluminium monostearate and gelatin.
Sterile able solutions are prepared by incorporating the active ingredient in
the required amount in the appropriate solvent with several of the other ingredients
U‘i enumerated above, as required, followed by ed sterilization. Generally, dispersions
are prepared by incorporating the sterilized active ingredient into a sterile vehicle which
contains the basic dispersion medium and the required other ingredients from those
enumerated above. In the case of sterile powders for the preparation of sterile injectable
solutions, the preferred methods of preparation are vacuum drying and the freeze-drying
que which yield a powder of the active ingredient plus any additional desired
ingredient from usly sterile-filtered solution thereof.
Various other als may be t as coatings or to otherwise modify the physical
form of the dosage unit. Of , any material used in preparing any dosage unit form
should be pharmaceutically pure and substantially non-toxic in the amounts employed. In
addition, the active ingredient may be incorporated into sustained—release
ations and formulations.
As used herein "pharmaceutically acceptable r and/or diluent" includes any
and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic
and tion delaying agents and the like. The use of such media and agents for
2O pharmaceutical active substances is well known in the art. Except insofar as any
conventional media or agent is incompatible with the active ingredient, use thereof in the
therapeutic compositions is contemplated. Supplementary active ingredients can also be
incorporated into the compositions.
It is especially advantageous to formulate parenteral compositions in dosage unit form for
ease of administration and mity of dosage. Dosage unit form as used herein refers
to physically discrete units suited as unitary dosages for the mammalian subjects to be
treated; each unit containing a predetermined quantity of active material calculated to
produce the desired therapeutic effect in association with the required pharmaceutical
carrier. The specification for the novel dosage unit forms of the invention are dictated by
and ly dependent on (a) the unique characteristics of the active al and the
particular therapeutic effect to be achieved, and (b) the limitations inherent in the art of
nding such as active material for the treatment of disease in living subjects having
a diseased condition in which bodily health is impaired.
The principal active ingredients are compounded for convenient and effective
administration in ive amounts with a suitable pharmaceutically acceptable r in
dosage unit form. In the case of compositions containing supplementary active
ingredients, the dosages are determined by reference to the usual dose and manner of
U‘l administration of the said ingredients.
In order to facilitate delivery of peptide compounds, including antibodies, to cells, peptides
may be ed in order to improve their ability to cross a cell membrane. For example,
US 5,149,782 discloses the use of fusogenic peptides, ion-channel forming peptides,
membrane peptides, hain fatty acids and other membrane blending agents to
increase n transport across the cell membrane. These and other methods are also
37016
described in WO 97/ and US 5,108,921, incorporated herein by reference.
I n a further aspect there is provided the active ingredient of the invention as hereinbefore
defined for use in the treatment of disease either alone or in combination with art
recognized compounds known to be suitable for treating the particular indication.
Consequently there is provided the use of an active ient of the invention for the
manufacture of a medicament for the treatment of disease associated with an aberrant
ponse.
immune res
Moreover, there is provided a method for treating a condition ated with
an aberrant immune response, comprising administering to a subject a therapeutically
2O ive amount of a ligand identifiable using an assay method as described above.
The invention is further described, for the purposes of illustration only, in the ing
examples.
Comparative Example 1
Binding and biological activity of EuCIV3 is decreased compared with BMA031
Using flow cytometry, we have shown that EuCIVS is inferior to BMA031 in T cell binding
(Fig. 1). In this competition assay, T cells were incubated on ice in the presence of a fixed
concentration of directly Phycoerythrin—labeled MolgG2b—BMAO31 (murine competitor)
and an increasing concentration of anti—dBTCR antibodies. After 20 minutes incubation,
the cells were washed and surface bound directly Phycoerythrin—labeled MolgG2b—
BMA031 was detected by flow cytometry. The BMA031 HulgG1 ic antibody
competes much more effectively than EuClV3.
In order to assess its ability to inhibit T cell ty in vivo, CD8+ T cells were treated with
anti—dBTCR antibodies at s concentrations (see, Fig. 2, x—axis) and co—cultured with
autologous dendritic cells (DCs) pulsed with the CMV peptide 495—503 (pp65) for seven
days in an in vitro education (IVE) assay.
Normal donor aphaeresis products from + individuals were obtained from
HemaCare Corp., Van Nuys, CA). PBMC were isolated by centrifugation over Ficoll (GE
U‘i Healthcare, Piscataway, NJ). CD8+ T cells were isolated using magnetic beads
(lnvitrogen, Carlsbad, California) ing to the manufacturer’s ctions. To
generate autologous immature tic cells, PBMC were resuspended in RPMI 1640/5%
human AB serum (Sigma), plated in triple flasks (Corning) and ted for more than 2
hours at 37°C/5%C02. The adherent monocytes were then rinsed with PBS and cultured
P M I / 5% B
for 6 days in R 1640 human A serum supplemented with GM-CSF (lmmunex,
-4 /DC
Seattle, WA) and lL (PeproTech, Rocky Hill, NJ). Prior to establishing the T cell co-
cultures, the DCs were pulsed with peptides (10ug/ml) for 4 hours and then matured.
Mature dendritic cells were generated by the addition of 50ng/ml TNF-alpha, 25ng/ml lL-
1B, l lL-6, 500ng/ml PGE-2 (PeproTech, Rocky Hill, NJ) and culturing the dendritic
cells for an additional 24 hours. The e-pulsed DCs were then added to the
previously isolated CD8+ T cells at a T:DC ratio of 10:1. Cultures were immediately fed
with lL-2 (100 lU/ml) added to the cultures. The cultures were supplemented with lL-2
(100 lU/ml) on day 4. The bulk cultures were assayed for peptide reactivity in a chromium
release assay on day 7.
2O The graph in Fig. 2 shows lysis data from the chromium release assay, where untreated T
cells were successfully educated against pp65 e and able to lyse specific targets at
>50%. BMA031 inhibited education of these T cells, as they were unable to lyse specific
targets in a dose—dependent manner. Humanized dy EuClV3 was less potent than
BMAO31 and was only able to inhibit education at the highest dose.
Example 2
Fc Engineering of BMA031 chimeric antibodies
In vitro profile
We have assessed the in vitro profile of BMA031 in a panel of assays. Table 1 shows the
in vitro profile of BMA031. BMA031 is compared to OKT3 in these assays.
In the PBMC eration assay, human PBMC were cultured with increasing
concentrations of therapeutic antibody for 72 hours, 3H-thymidine was added and cells
were harvested 18 hours later.
For the T cell depletion/Cytokine Release assay, human PBMC were ed with
increasing concentrations of therapeutic antibody and were analyzed daily for cell counts
and viability (Vi—Cell, n Coulter) out to day 7. Cell supernatants were also
harvested, stored at 2000 and analyzed on an 8—plex cytokine panel (Bio—Rad).
BMA031 does not induce: (i) PBMC proliferation; (ii) T cell depletion; (iii) CD25
expression; or (iv) cytokine e. In contrast, OKT3 does induce all of the
U‘l aforementioned effects. BMA031 and OKT3 are e of blocking the education of
CD8+ cells to peptide in an in vitro education (IVE) assay and are also e of blocking
a mixed cyte reaction (MLR). BMA031 also induces apoptosis of activated T cells
(activation-induced cell death; AICD).
Unlike BMA031, a chimeric version of BMA031 (HulgG1), with wild-type human IgG1
constant region, had an in vitro profile comparable to OKT3 (Table 1). We postulated that
FcyR involvement was critical for this change of in vitro profile for HulgG1 BMA031
ed to BMA031 MolgGZb. Therefore we made F(ab’)2 fragments of BMAO31
HulgG1 and found these to recover the profile of BMA031 MolgG2b. By Fc engineering
we incorporated modifications that removed FcyR binding in mutations known as “delta
ab” (Armour ef al. (1999) Eur. J. Immunol., 292613—2624) and by generating an
aglycosylated form of HulgG4 ). HulgG1 delta ab and HulgG4 agly anti-dBTCR
antibodies had the same in vitro profile as BMAO31 MolgG2b (Table 1).
Normal PBMC Antigen Activated T-cells
TSTCR uflTCR FcyR PBMC CD25 Cylokine MLR Apoptosis/ IVE
Depletion
binding binding binding Proliferation Expression Inhibition Inhibition
OKT3
BMAO31 b
BMA031 HulgG1
BMA031 F(ab)2
BMA031 Aab HulgG1
HEBE1 Aab HulgG1
HEBE1 |gG4 agly
Table 1
Example 3
Construction of humanized antibodies with improved g
We have generated two series of humanized versions of BMAO31 called HEBE1 series
(IGH3—23) and GL1BM series (IGHV1—3*01 & |GKV3—11*01; see, VBase, vbase.mrc—
cpe.cam.ac.uk). Initial ng of BMA031 heavy chain CDR regions onto IGH3—23
ork regions (see, SEQ ID NOs: 5 and 6) improved the binding of the antibody to
the ocBTCR as shown by a competition assay (Fig. 3); see, e 2. r, this
improvement did not translate into a functional improvement in the antibody as shown by
an IV assay (Fig. 4).
Example 4
Optimization of humanized antibodies
The strategy for optimization of the humanized antibodies was based u nesis
and functional ing. Optimization was started with block changes of amino acid
residues in one of each of the four framework regions of the variable domains, from
mouse to human. Key framework regions were fied in each of the GL1BM HC,
BE 1
GL1 B M LC and H E HC series. Following this identification, individual residues within
those framework regions were mutated to human germline residues from the original
mouse ce. Framework residues for which identity with the mouse sequence was
found to be important to retaining the binding properties of the antibody were retained as
mouse residues. Otherwise, framework residues were changed to match the human
germline amino acid sequence. This was continued across the sequence until the
minimal number of mouse residues, to retain the original binding properties of the
antibody, were identified. See Fig. 5. We have demonstrated that several of the
antibodies from these series have an improved binding compared to BMA031 as
2O determined by antibody off-rate from T cells (Figs. 6, 7 and 8).
For off—rate assays, 105 human T cells were incubated for 30—60 minutes at room
temperature in 100uL full growth media containing 2ug/mL of the antibodies expressed as
HulgG1—Aab. The cells were then , resuspended in 50uL full growth media and
20ug/mL of HEBE1 F(ab’)2 was added in order to t the ing of the dissociated
candidate antibodies. At the end of this time course assay, the cells were fixed and the
level of remaining HulgG1—Aab antibody bound to the cell surface was ed by flow
cytometry via a PE labeled goat anti-HulgG secondary antibody.
We have also demonstrated that the antibodies are active in preventing the immune
response in an IVE (Figs. 9, 10 and 11) and a MLR assay. In the IVE assay, er
binding was used as a quantitative measurement for the IVE. The percentage of cells
which were antigen specific was determined by staining the T cells with a directly labeled
tetramer that is specific for the educating peptide. Briefly, day 7 CD8+ T cells from the IVE
were stained with tetramer by standard flow cytometry staining ols and analyzed on
B D FACSCaIibur. I n addition, the humanized antibodies demonstrated comparable levels
of erative potential on PBMCs and cytokine release as compared to BMA031 (Figs.
12 and 13).
The antibodies also showed an ability to inhibit the release of IFNytrom T cells in an IV
U‘l assay (Fig. 14). In addition we have shown that a number of these antibodies have an
improved ability to elicit activation-induced cell death (AICD) of ted ocBTCR-positive
T cells compared to BMA031 (Fig. 15). In the AICD assay, antigen-specific CD8+ T cells
were cultured with therapeutic antibody. At 24 hours, 48 hours and 72 hours cells were
stained for apoptosis markers Annexin-V and 7-AAD. Cells were also stained with
tetramer to track sis with effects on antigen-specific T cells.
In conclusion, we have made significant improvement over previous attempts to humanize
BMA031. The discovery of antibodies with an improved off-rate compared to BMA031 is
an unexpected finding via this process. This ement in binding correlates with an
improvement in potency to suppress an immune response as demonstrated in the IVE
assay (Figs. 10 and 11). The specificity of the antibodies for ocBTCR, the decreased
genicity by humanization, the specific apoptosis of activated T cells and the lack
of T cell activation u antibody binding make these antibodies excellent candidates for
poses.
therapeutic pur
Example 5
Generation of Fc mutants for reduced effector function.
ered Fc variants was designed and generated where a glycosylation site is
introduced at amino acid Ser 298 position, next to the naturally—occurring Asn297 site. The
ylation at Asn297 was either kept or knocked out by mutations. Mutations and
glycosylation results are set forth in Table 2.
# Mutation Predicted result Expected t
1 N297Q No glycosylation Agly Control
No glycosylation Agly Control,
2 T299A unknown effector
function
No glycosylation at 297 but Reduced effector
N297Q/8298N/Y3008
3 engineered glycosylation SiteI I I functionI
(NSY)
at 298
No glycosylation at 297 but Reduced effector
8298N/T299A/Y3008
4 engineered glycosylation SiteI I I functionI
(STY)
at 298
Two potential glycosylation Positive control for
sites at 297 & 298; Double reduced effector Y3008
S298N/ (SY)
glycosylation?I Mixed I functionI
glycosylation?
Table 2
ons were made on the heavy chain of (18 T—cell receptor antibody clone #66 by
H E BE 1
Quikchange using a pENTRiLICiIgG1 template. The V domain of H Aab |ng
#66 was amplified with LIC primers, and cloned into mutated or wild type
pENTR_L|C_IgG1 by LIC to create a full—length Ab mutants or wild type. The subcloning
was verified with Dralll/Xhol double digest, producing ~125O bp insert in the successful
clones. Those full—length s were then cloned into an expression vector,
pCEP4(—E+|)Dest, via Gateway cloning. The ons were then confirmed by DNA
sequencing.
Two constructs, HEBE1 Agly lgG4 and HEBE1 Aab lng in pCEP4, were used as
controls in H EK293 transfection.
The mutants, wt and controls (Agly and Aab) were transfected into —EBNA cells in
triple—flask for expression. Proteins were ed from 160 ml of conditioned media (GM)
with 1 ml HiTrap protein A columns (GE) on hannel peristaltic pump. Five
micrograms of each atant were analyzed on 4—20 Tris—Glycine reducing and non—
reducing SDS—PAGE (see Figure 16). The heavy chain of the aglycosylated mutants
(N297Q, T299A, and Agly l, is lower (arrow in black), consistent with the loss of the
s in these antibody. The heavy chains of the engineered glycosylated antibodies
U‘l (NSY, STY, SY, Aab, and wt control, arrows in red), however, migrate the same way as
the ype control. This result is consistent with the ed outcome of engineered
glycosylation site at 298 positions. SEC-HPLC analysis indicated that all mutants are
expressed as monomers.
G/ycosy/ation analysis by LC-MS.
The ered H66 lgG1 Fc variants were partially reduced with 20mM D T at 37°C for
min. The samples were analyzed by capillary LC/ on an Agilent 1100 capillary
HPLC system coupling with a QSTAR qq TOF hybrid system (Applied Biosystem).
Bayesian protein truct with ne correction and er modeling in Analyst
T299A/ Y3008
08 1.1 ed Bisoystem) was used for data analysis. For mutant S298N/
H66 antibody lead, one glycosylation site was ed at amino acid 298 with bi—
antennary and tri— antennary complex—type glycans detected as the major species, as well
as GOF, G1 F and (32F.
Binding of (1,8 TCR antibody mutants to human FcyR/lla and Fcle using Biacore.
Biacore was used to assess binding to recombinant human Fclella (V158 & F158) and
2O Fcle. All 4 flowcells of a CM5 chip were immobilized with anti-HPC4 antibody via the
standard amine coupling procedure provided by Biacore. The anti—HPC4 antibody was
diluted to 50ug/mL in 10mM sodium acetate pH 5.0 for the coupling reaction and injected
for 25 min at 5uL/min. Approximately 12,000 RU of antibody was immobilized to the chip
surface. Recombinant human Fclella-V158 and Fclella-F158 were diluted to 0.6ug/mL
in binding buffer, HBS—P with 1mM CaClg, and injected to flowcells 2 and 4, respectively,
for 3 min at 5uL/min to capture 300 — 400 RU receptor to the anti-HPC4 chip. In order to
distinguish between the low binders, three times more la was captured to the
PC4 surface than usually used in this assay. Flowcells 1 and 3 were used as
reference controls. Each antibody was diluted to 200nM in binding buffer and injected
over all 4 flowcells for 4 min, followed by 5 min dissociation in butter. The surfaces were
regenerated with 10mM EDTA in HBS—EP butter for 3 min at 20uL/min.
The results are shown in Figure 17.
Biacore was also used to e the chRI binding. Anti—tetra His antibody was buffer
exchanged into 10mM sodium acetate pH 4.0 using a Zeba ing column and diluted
to 25ug/mL in the acetate buffer for amine coupling. Two flowcells of a CM5 chip were
immobilized with ~9000 RU of the anti—Tetra—His antibody after 20 min injection at
U‘i 5uL/min. r to the us experiment, ten times more Fcle was captured to the
anti-tetra-His surface in order to compare weak binders. Recombinant human chRl was
diluted foug/mL in HBS—EP binding buffer and injected to flowcell 2 for 1 min at 5uL/min
to capture ~1000 RU receptor to the anti-tetra-His chip. A single concentration of
antibody, 100nM, was injected for 3 min at 30uL/min over the captured receptor and
control surface. Dissociation was monitored for 3 min. The e was regeneration with
two 30 sec injections of 10mM glycine pH 2.5 at 20uL/min.
The results are shown in Figure 18.
The result suggests very little binding of the glycoengineered mutants to chRllla or
T299A/ YSOOS
Fcle. H66 8298N/ in particular has almost completely abolished binding to
both receptors. This mutant was chosen as the lead for ed characterization.
Stability characterization using Circular Dichroism (CD).
The stability of the S298N/T299A/Y3008 antibody mutant was monitored by a Far—UV CD
thermo melting experiment where the CD signal at 216nm and 222nm was monitored as
temperature increases that eventually leads to the unfolding of the antibody. The CD
2O spectra were collected on a Jasco 815 ophotometer at a protein concentration of
imately 0.5 mg/mL in PBS buffer in a quartz cuvette (Hellma, Inc) with a path length
of 10 mm. Temperature was controlled by a thermoelectric peltier (Jasco model
AWCtOO) and was ramped at a rate of 1°C/min from 25-89 °C. CD signal and HT voltage
were both collected. Data was obtained from 210-260 nm with data intervals of 0.5 nm
and at temperature intervals of 1 °C. The scanning speed was 50 nm/min and a data
pitch of 0.5 nm. A bandwidth of 2.5 nm was used with a sensitivity setting of medium. 4
ate scans were performed for each sample. The result suggest that both delta AB
H66 and the T299A/Y3008 H66 mutant show similar thermal behavior and have
the same onset temperature for degradation around 630. In other word, the mutant is as
stable as the delta AB format.
See Figure 18.
Example 6
Functional is of Fc—engineered mutants
PBMC proliferation and cytokine release assays were conducted as set forth in Example
2. Normal donor PBMC were thawed and treated under the following conditions (all in
media containing complement):
U] - Untreated
- , molgG2b10ug/ml
- OKT3, molgG2a10ug/ml
0 H66, hulgG1 deltaAB10ug/ml, 1ug/ml and 0.1ug/ml
T299A/ Y3008
c H66, hulgG1 S298N/ 10ug/ml, 1ug/ml and 0.1ug/ml
Cytokines were ted at day 2 (D2) and day 4 (D4) for Bioplex Analysis (IL2, lL4, IL6,
lL8, lLlO, GM-CSF, IFNg, TNFa). Cells were stained at D4 for CD4, CD8, CD25 and
abTCR expression.
The results, shown in Figures 19-22, demonstrate that H66 8298N/T299A/Y3008
behaved similarly to the H66 deltaAB in all cell based , showing minimal T-cell
activation by CD25 expression; binding to abTCR, gh with slightly different kinetics
to deltaAB; minimal cytokine release at both D2 and D4 time points; the mutant was in fact
superior to deltaAB at D4 in respect of several of the cytokines.
The 8298N/T299A/Y3008 mutant thus eliminated effector function as effectively as the
deltaAB mutation.
Example 7
Bispecific antibodies
Bi—specific molecules were constructed comprised of two single chain antibodies (scFv)
linked together via a short amino acid linker whereby one arm is capable of binding a
tumor target and the other e of binding T cells via the 06 TCR. The ific
le is referred to herein as a TRACER (T cell Receptor Activated Cytotoxic
EnableR).
The following humanized anti—dBTCR scFv constructs were made:
GL1BMASXVK1
GL1BMASXVK27
GL1BMASVH11XVK1
GL1BMASVH15XVK1
U] SVH28XVK43
GL1BMASVH31XVK43
The sequences of the heavy and light chains are set forth in SEQ ID nos 14-16 and 20-24
Characterization of these molecules comprised an assessment of binding to tumor target
and T cells, in vitro cytotoxic activity and cytokine release profile in the presence and
absence of tumor target cells.
The binding profile assessed by flow cytometery shows that anti— dB TCR bi—specific
dies are able to bind both the tumor target cell line and T cells. See Figure 23.
I n v/fro cytotoxic activity measured by flow cytometery shows that T cells recruited via
anti— dB TCR cific antibody are capable of inducing T cell mediated lysis. See
Figure 24
Analysis of the cytokine release profile has shown that u binding of both arms of the
cific antibody there is a high level of TH1/ cytokine release from the T cells
which is not seen in the absence of target cells. Taken together this mechanism of action
shows a similar profile to that of the CD3 based bispecifics described in the literature.
Example 8: Preparation and terization of an engineered Fc variant in anti-
CD52 antibody.
In order to test the generality of the applicability of the Fc mutations described herein,
glycosylation mutant 8298N/Y3008 was also prepared in an anti-CD52 antibody (clone
203) to see whether the or function modulation with the loss of chRlll binding
applies to a different antibody backbone. S298N/Y3OOS 203 t DNA was prepared
by quick change mutagenesis. The protein was purified from conditioned media after
HEK293 ent ection. Anti—CD52 203 wild—type antibody was produced in parallel
as a control. Biacore was used to characterize the antigen—binding, Fclell, and binding
properties of the purified antibodies (see Figure 26).
Y3008
The S298N/ 203 variant binds to CD52 peptide tightly and the binding sensorgram
is undistinguishable with the wild—type control, suggesting that this on on the Fc
U‘i domain does not affect its antigen binding (Figure 26A).
To assay Fc or function, Fclell receptor (Val158) was used in binding studies. The
mutant and wild-type control antibody were d to 200nM and injected to HPC4-tag
ed Fclella. Fclell binding is almost undetectable for the S298N/Y3008 mutant,
which indicates loss of effector function with this variant (Figure 268). The mutant also
binds to FcRn receptor with the same affinity as the wild-type antibody control so we
expect no change in its circulation half-life or other pharmacokinetic properties. (see
YBOOS
Figure 260). We conclude that the S298N/ mutation is able to antibodies in
general, to reduce or eliminate undesired Fc effector function, for e through
engagement of human Fcy receptors.
Claims (12)
1. A humanized onal antibody specific for the human ocBTCR/CDB complex which comprises a heavy chain variable region comprising an amino acid sequence selected from the group ting of the sequences set forth as SEQ ID NOs: 7, 12, 13, 15 and 16, and a light chain variable region comprising the amino acid sequence set forth as SEQ ID NO: 1
2. The humanized monoclonal antibody according to claim 1, wherein the heavy Chain variable region is ed from the group consisting of the amino acid ces set forth as SEQ ID NO: 7, SEQ ID NO: 12 and SEQ ID NO: 13.
3. The humanized monoclonal antibody according to claim 1, wherein the heavy chain variable region is selected from the group consisting of the amino acid sequences set forth as SEQ ID NO: 15 and SEQ ID NO: 16.
4. The humanized monoclonal antibody according to any one of the preceding claims, further comprising a constant region of human origin.
5. The humanized monoclonal antibody according to claim 4, further comprising an F0 modification which reduces Fcy receptor binding.
6. The humanized monoclonal antibody according to claim 5, which comprises an aglycosylayted Fc region or a delta ab modification.
7. The zed monoclonal dy according to claim 5, which comprises a modified glycosylation pattern.
8. A nucleic acid encoding at least a heavy chain variable region and a light chain variable region of a humanized monoclonal antibody ing to any one of claims 1 to 7.
9. An isolated cell which expressesethe nucleic acid according to claim 8, wherein cells capable of generating humans and transformed cells in a human host, are excluded.
10. A zed antibody according to any one of claims 1 to 7 for use in suppressing a T cell mediated response in a subject.
11. Use of a humanized antibody ing to any one of claims 1 to 7 in the cture of a medicament for use in suppressing a T cell mediated response in a subject.
12. A humanized dy according to claim 10 or the use according to claim 11, wherein the T ponse cell mediated res is involved in a condition selected from tissue transplantation including solid organ transplant and composite tissue transplant, tissue grafting, multiple sis and type 1 diabetes. GENZYME CORPORATION A T ER M A RK P A T E N T A N D T R A D E W MARKS ATTORNEYS P38649NZOO :03: gmo_>_ 5:: 6:80 _m_ Fmo<_>_m_ m>_03m
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
NZ717726A NZ717726B2 (en) | 2011-09-12 | 2012-09-12 | Anti-??tcr antibody |
Applications Claiming Priority (3)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US201161533510P | 2011-09-12 | 2011-09-12 | |
US61/533,510 | 2011-09-12 | ||
PCT/EP2012/003819 WO2013037484A2 (en) | 2011-09-12 | 2012-09-12 | Anti-aplhabetatcr antibody |
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NZ623656A NZ623656A (en) | 2016-04-29 |
NZ623656B2 true NZ623656B2 (en) | 2016-08-02 |
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