U.S. patent application number 16/502699 was filed with the patent office on 2019-11-28 for molecules with specificity for cd45 and cd79.
The applicant listed for this patent is UCB BIOPHARMA SPRL. Invention is credited to HELENE MARGARET FINNEY, STEPHEN EDWARD RAPECKI, KERRY LOUISE TYSON, MICHAEL JOHN WRIGHT.
Application Number | 20190359713 16/502699 |
Document ID | / |
Family ID | 51454240 |
Filed Date | 2019-11-28 |
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United States Patent
Application |
20190359713 |
Kind Code |
A1 |
FINNEY; HELENE MARGARET ; et
al. |
November 28, 2019 |
MOLECULES WITH SPECIFICITY FOR CD45 AND CD79
Abstract
The present disclosure relates to a multispecific molecule
comprising a binding domain specific to the antigen CD45 and a
binding domain specific to the antigen CD79a and/or CD79b,
compositions comprising same and use of each in treatment, for
example treatment of autoimmune disease.
Inventors: |
FINNEY; HELENE MARGARET;
(SLOUGH, GB) ; RAPECKI; STEPHEN EDWARD; (SLOUGH,
GB) ; WRIGHT; MICHAEL JOHN; (SLOUGH, GB) ;
TYSON; KERRY LOUISE; (SLOUGH, GB) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
UCB BIOPHARMA SPRL |
Brussels |
|
BE |
|
|
Family ID: |
51454240 |
Appl. No.: |
16/502699 |
Filed: |
July 3, 2019 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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15326499 |
Jan 16, 2017 |
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PCT/EP2015/066368 |
Jul 16, 2015 |
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16502699 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C07K 2317/24 20130101;
C07K 2317/55 20130101; C07K 16/2803 20130101; C07K 2317/31
20130101; C07K 2317/76 20130101; C07K 2317/622 20130101; A61P 37/06
20180101; C07K 16/468 20130101; A61P 35/00 20180101; C07K 16/18
20130101; A61K 2039/505 20130101; C07K 16/289 20130101 |
International
Class: |
C07K 16/28 20060101
C07K016/28; C07K 16/46 20060101 C07K016/46; C07K 16/18 20060101
C07K016/18 |
Foreign Application Data
Date |
Code |
Application Number |
Jul 16, 2014 |
GB |
1412659.3 |
Claims
1-33. (canceled)
34. An antibody comprising a binding domain specific to antigen
CD45 and a binding domain specific to antigen CD79b, wherein the
binding domain specific to the antigen CD45 comprises: 3 heavy
chain CDRs comprising SEQ ID NO: 128 for CDRH1, SEQ ID NO: 129 for
CDRH2 and SEQ ID NO: 130 for CDRH3 and 3 light chain CDRs
comprising SEQ ID NO: 125 for CDRL1, SEQ ID NO: 126 for CDRL2 and
SEQ ID NO: 127 for CDRL3; wherein the binding domain specific to
the antigen CD79b comprises: 3 heavy chain CDRs comprising SEQ ID
NO: 88 for CDRH1, SEQ ID NO: 89 for CDRH2 and SEQ ID NO: 90 for
CDRH3 and 3 light chain CDRs comprising SEQ ID NO: 85 for CDRL1,
SEQ ID NO: 86 for CDRL2 and SEQ ID NO: 87, or a derivative thereof,
for CDRL3 wherein, in at least one CDR (a) at least one cysteine
residue has been substituted with another amino acid, (b) at least
one aspartic acid isomerisation site has been removed by
substituting at least one amino acid, (c) at least one asparagine
deamidation site has been removed by substituting at least one
amino acid, (d) at least one glycosylation site has been removed by
substituting at least one amino acid, or (e) a combination thereof.
Description
FIELD OF INVENTION
[0001] The present disclosure relates to a molecule which is at
least bispecific to the antigens CD45 and CD79, a formulation
comprising said molecule and use of any one of the same in
treatment. The present disclosure also extends to methods of
preparing said molecules and said formulations. In an independent
aspect the disclosure also extends to novel antibody sequences and
fragments described herein.
BACKGROUND OF INVENTION
[0002] Biological mechanisms in vivo are extremely complicated
cascades of signals, which are difficult to deconvolute and
understand. An example of such signalling is that required to
activate B-cells. The B cell antigen receptor (BCR) is composed of
membrane immunoglobulin (mIg) molecules and associated
Ig.alpha./Ig.beta. (CD79a/CD79b) heterodimers (.alpha./.beta.). The
mIg subunits bind antigen, resulting in receptor aggregation, while
the .alpha./.beta. subunits transduce signals to the cell interior.
BCR aggregation rapidly activates the Src family kinases Lyn, Blk,
and Fyn as well as the Syk and Btk tyrosine kinases. This initiates
the formation of a `signalosome` composed of the BCR, the
aforementioned tyrosine kinases, adaptor proteins such as CD19 and
BLNK, and signaling enzymes such as PLC.gamma.2, PI3K, and Vav.
[0003] Signals emanating from the signalosome activate multiple
signaling cascades that involve kinases, GTPases, and transcription
factors. This results in changes in cell metabolism, gene
expression, and cytoskeletal organization. The complexity of BCR
signaling permits many distinct outcomes, including survival,
tolerance (anergy) or apoptosis, proliferation, and differentiation
into antibody-producing cells or memory B cells. The outcome of the
response is determined by the maturation state of the cell, the
nature of the antigen, the magnitude and duration of BCR signaling,
and signals from other receptors such as CD40, the IL-21 receptor,
and BAFF-R.
[0004] Many other transmembrane proteins, some of which are
receptors, modulate specific elements of BCR signaling. A few of
these, including CD45, CD19, CD22, PIR-B, and Fc.gamma.RIIB1
(CD32). The magnitude and duration of BCR signaling are limited by
negative feedback loops including those involving the
Lyn/CD22/SHP-1 pathway, the Cbp/Csk pathway, SHIP, Cbl, Dok-1,
Dok-3, Fc.gamma.RIIB1, PIR-B, and internalization of the BCR.
[0005] In vivo, B cells are often activated by antigen-presenting
cells that capture antigens and display them on their cell surface.
Activation of B cells by such membrane-associated antigens requires
BCR-induced cytoskeletal reorganization.
[0006] Autoreactive B cells are responsible for the production of
pathogenic autoantibodies which can either directly or indirectly
cause or exacerbate autoimmune conditions. Depletion of CD20
positive B cells has been used to successfully treat a number of
autoimmune conditions and thus established conclusively that B
cells play an important role in causing or maintaining a number of
autoimmune diseases. Although B cell depletion has been a
successful therapeutic option evidence also exists that control of
B cell growth and activation status can also be an effective way to
modulate B cell function. Alternative strategies that do not
deplete B cells and offer the flexibility of controlling B cells
without long term suppression of B cell immunity which has been
shown to be associated with some side effects would therefore be
desirable. In addition not all B cell responses or activities are
harmful and evidence suggests that maintenance of regulatory B cell
populations can be protective. Such an approach should be effective
in diseases which have abnormal B cell function caused by
inappropriate or excessive BcR signalling. Examples include, but
are not limited to, inflammation, autoimmunity and cancer. Of
particular interest are diseases that either have a direct
requirement for BcR signalling or require inhibition or stimulation
of humoral immune responses.
[0007] Bispecific antibodies are widely expected to play a major
role in the next generation of biotherapeutics (D. Holmes, Nature
Rev Drug Disc November 2011: 10; 798). They have the potential to
deliver superior, long term, broad efficacy in a greater proportion
of patients. This can be achieved by either co-engaging different
antigens simultaneously within a common disease pathway, thereby
reducing redundancy; or by targeting antigens from independent
pathways to provide an additive or synergistic effect.
[0008] To date strategies to inhibit B cell function without
deleting the B cell have focused on exploiting the natural
mechanism of regulation by CD32b (FcgRIIB). These include
bispecific antibodies to CD79b/CD32b (Veri et al., Arthritis &
Rheumatism 2010 62 1933-1943), CD19/CD32b (Karnell et al., J.
Immunol 2014 192 1430-1490) and an antibody to CD19 with an Fc with
enhanced CD32b binding (Chu et al., Arthritis & Rheumatology
2014 66 1153-1164).
[0009] Bispecific antibodies are widely expected to play a major
role in the next generation of biotherapeutics (D. Holmes, Nature
Rev Drug Disc November 2011:10; 798). They have the potential to
deliver superior, long term, broad efficacy in a greater proportion
of patients. This can be achieved by either co-engaging different
antigens simultaneously within a common disease pathway, thereby
reducing redundancy; or by targeting antigens from independent
pathways to provide an additive or synergistic effect.
[0010] To date strategies to inhibit B cell function without
deleting the B cell have focused on exploiting the natural
mechanism of regulation by CD32b (FcgRIIB). These include
bispecific antibodies to CD79b/CD32b (Arthritis & Rheumatism
2010 62 1933-1943), CD19/CD32b (J. Immunol 2014 192 1430-1490) and
an antibody to CD19 with an Fc with enhanced CD32b binding
(Arthritis & Rheumatology 2014 66 1153-1164).
[0011] Co-ligation of Fc gamma receptor IIb (CD32b) with the B cell
receptor occurs to naturally regulate signalling, in particular
when antigen is bound to antibody in small immune complexes. CD32b
then recruits the phosphatases SHP-1 and SHIP-1 which antagonise
BcR activation. Although this natural regulatory mechanism can
control B cell function, disruption of CD32b function caused by
variation in the protein sequence of CD32b can lead to autoimmune
disease and this receptor can be down regulated in autoimmune
disease--e.g. as in the case of SLE. Alternative ways of blocking B
cell activity are thus desirable as they offer alternative,
non-natural, ways of regulating BcR function. These alternative
mechanisms are likely to be particularly important when natural
mechanisms are dis-functional in the given disease.
[0012] Bispecific antibodies facilitate access to novel biology
such as: [0013] 1) cross-linking receptors on a cell, if
appropriate, [0014] 2) inducing cell mediated effects, [0015] 3)
localizing a cytokine to a cell to regulate signaling or locally
block cytokine function, [0016] 4) engaging multiple epitopes
simultaneously to generate "new activity", increase function or
specificity, which may not be exhibited by a single monoclonal
antibody or indeed mixtures of un-linked antibodies
(poly-monoclonals), including mixtures directed to different
antigens.
[0017] Under normal physiological conditions upon antigen binding
CD45 is excluded from the BcR complex. The present inventors have
surprisingly found that using a bispecific antibody to couple the
BcR (CD79) to the molecule CD45, BCR signalling can be inhibited.
Thus by physically linking the BcR with CD45 through use of a
bispecific antibody the inventors have found that activation in B
cells can be inhibited.
[0018] The present inventors have therefore identified a
synergistic function for molecules which are at least bispecific
for CD45 and CD79. This function seems to be detectable primarily
when binding regions with the combination of specificities are
provided in a bispecific (multispecific) format, as opposed to
simply being provided as a mixture of, for example monoclonal
antibodies or binding fragments thereof.
[0019] The multispecific molecules of the invention are therefore
useful in controlling aberrant B cell functions associated with
certain diseases such as autoimmunity and cancer.
SUMMARY OF THE DISCLOSURE
[0020] Thus provided is a multispecific molecule comprising a
binding domain specific to the antigen CD45 and a binding domain
specific to the antigen CD79.
[0021] The combination according to the present disclosure in a
bispecific format shows interesting biological activity in
functional in vitro assays, for example inhibition of B cell
signalling as measured by any one of the following: inhibition of
phosphorylation of Akt 5473, inhibition of phosphorylation of P38
and PLC.gamma.2 Y759 inhibition of IkB, in addition to the
inhibition of expression of CD86, CD71 and/or CD40 on B cells. The
same level of activity is not apparent for individual components
alone or the components provided in admixture. However, the
activity is apparent when a bispecific construct with specificity
for CD45 and CD79 is provided.
[0022] The inhibition of certain B cell functions observed in these
assays is indicative that a multispecific molecule of the
invention, comprising a binding domain specific to CD45 and a
binding domain specific to CD79, may be used to alter B cell
function and, for example to provide a therapeutic alternative to
depletion of B cells.
[0023] B cell receptor signalling is a critical function of the B
cell and a requirement for antigen specific activation of B cells.
BcR signalling is critical from early stages of B cell development
through to the activation and development of memory B cell
responses. The B cell receptor is composed of a surface
immunoglobulin (Ig) molecule which associates with heterodimeric
complex of CD79a and CD79b. When surface Ig recognises antigen it
is thought that this results in a clustering of the CD79a/b complex
which results in downstream activation of the immediate signalling
cascade, which includes Src family kinases as well as Syk and Btk
tyrosine kinases. This signalling complex then can recruit adaptor
proteins such as CD19 and BLNK and results in activation of
PLC.gamma.2 and PI3K which in turn can activate further downstream
pathways such as those that control B cell growth, survival and
differentiation. This signalling complex can be further regulated
by other second signals via signalling through BAFF-R, IL-21R and
CD40 and can also be regulated by other signalling molecules such
as CD19, CD21, CD83, CD22, CD32b and CD45 amongst others. Upon
recognition of antigen by the BcR one of the first responses
activated is the up-regulation of surface receptors such as the
co-stimulatory molecules CD80 and CD86. These molecules bind to
corresponding receptors on T cells which deliver further survival
and activation signals that allow survival and expansion of T cells
that recognise antigen in the context of MHC class II. This
response is further amplified by the ability of B cells to present
antigen in the context of MHC class II back to the T cell, which
releases factors such as IL-2 and IL-21. These cytokines in turn
expand B cell number greatly.
[0024] Furthermore, inhibition of B cell receptor signalling can
lead to inhibition of downstream functions. One such outcome would
be the inhibition of co-stimulatory molecules such as CD86 (or
reduced expression of the same) which will lead to the inhibition
of T cell function, survival and differentiation.
[0025] Thus inhibition of B cell receptor signalling can be
beneficial in controlling aberrant B cell functions associated with
autoimmunity and cancer. B cell receptor signalling is required for
B cell proliferation, differentiation, antigen presentation and
cytokine release in autoimmune disease. Thus inhibiting BcR
activity can regulate B cell functions such as immunoglobulin
secretion, T cell activation and control inappropriate B cell
activity associated with, for example autoimmune conditions. In
addition there are some B cell leukaemias and lymphomas that
require B cell receptor signalling for survival and growth which
may be controlled by inhibitors of B cell receptor activation.
[0026] In one embodiment the binding domain or binding domains of
the multi-specific molecules of the present invention each
independently comprise one or two (such as two) antibody variable
domains specific to a relevant antigen (such as CD45 or CD79 or a
further antigen if the molecule is at least trispecific).
[0027] CD79 as used herein refers to the complex composed of CD79a
and CD79b. Accordingly, antibodies which bind CD79 may bind to
CD79a and/or CD79b. Binds to CD79a and/or CD79b as employed herein
refers to: specific to CD79a, specific to CD79b, specific to both
CD79a and b (i.e. recognises an epitope on CD79a and also
recognises an epitope on CD79b--i.e. pan specific) or is specific
to the complex of CD79a and CD79b (i.e. recognises an epitope
formed from the interaction of CD79a and CD79b in the complex
form).
[0028] In one embodiment an antibody or binding fragment thereof
employed in the molecules of the present disclosure is specific to
CD79a.
[0029] In one embodiment an antibody or binding fragment thereof
employed in the molecules of the present disclosure is specific to
CD79b.
[0030] In one embodiment an antibody or binding fragment thereof
employed in the molecules of the present disclosure is specific to
CD79 complex, i.e. it recognises an epitope present in the complex
and is specific thereto, for example an epitope comprising an
interaction between CD79a and CD79b.
[0031] In one embodiment even where the binding domain is specific
to CD79a or CD79b it will be appreciated that the binding domain
will still bind to CD79a or CD79b when in the complex form.
[0032] Where there are two variable regions in a binding domain or
in each binding domain then the two variable regions will generally
work co-operatively to provide specificity for the relevant
antigen, for example they are a cognate pair or affinity matured to
provide adequate affinity such that the domain is specific to a
particular antigen. Typically they are a heavy and light chain
variable region pair (VH/VL pair).
[0033] In one embodiment the molecule of the present disclosure is
bispecific.
[0034] In one embodiment the molecule of the present disclosure is
trispecific, for example where the third binding domain is specific
to serum albumin, for example human serum albumin.
[0035] In one embodiment the molecule of the present disclosure is
monospecific for CD79 and CD45 i.e. the molecule only comprises one
binding domain which binds CD79 and one binding domain which binds
CD45.
[0036] In one embodiment the multispecific molecule of the present
disclosure is a single chain.
[0037] In one embodiment the multispecific molecule of the present
disclosure comprises a heavy chain and also a light chain. In one
example, as employed herein a heavy and light chain pairing is not
referred to as a dimer, particularly where in one embodiment the
molecule of the present disclosure does not comprise multimers,
such as dimers of the antibody, unit/fragment or components.
[0038] In one aspect, there is provided a multi-specific antibody
molecule comprising or consisting of: [0039] a) a polypeptide chain
of formula (I):
[0039] V.sub.H--CH.sub.1--X--(V.sub.1).sub.p; [0040] b) a
polypeptide chain of formula (II):
[0040] V.sub.L--C.sub.L--Y--(V.sub.2).sub.q; [0041] wherein: [0042]
V.sub.H represents a heavy chain variable domain; [0043] CH.sub.1
represents a domain of a heavy chain constant region, for example
domain 1 thereof; [0044] X represents a bond or linker, for example
an amino acid linker; [0045] Y represents a bond or linker, for
example an amino acid linker; [0046] V.sub.1 represents a dab,
scFv, dsscFv or dsFv; [0047] V.sub.L represents a variable domain,
for example a light chain variable domain; [0048] C.sub.L
represents a domain from a constant region, for example a light
chain constant region domain, such as Ckappa; [0049] V.sub.2
represents a dab, scFv, dsscFv or dsFv; [0050] p is 0 or 1; [0051]
q is 0 or 1; and when p is 1 q is 0 or 1 and when q is 1 p is 0 or
1 i.e. p and q do not both represent 0.
[0052] In one embodiment the molecule comprises no more than one
binding site for CD45 and no more than one binding site for
CD79.
[0053] The above format is particularly useful for screening
combinations of variable regions, for example in longer term assays
and for therapeutic use.
[0054] In one embodiment q is 0 and p is 1.
[0055] In one embodiment q is 1 and p is 1.
[0056] In one embodiment V.sub.1 is a dab and V.sub.2 is a dab and
together they form a single binding domain of a co-operative pair
of variable regions, such as a cognate V.sub.H/V.sub.L pair.
[0057] In one embodiment V.sub.H and V.sub.L are specific to, CD79,
for example CD79a or CD79b.
[0058] In one embodiment the V.sub.1 is specific to, CD79, for
example CD79a.
[0059] In one embodiment the V.sub.2 is specific to, CD79, for
example CD79a.
[0060] In one embodiment the V.sub.1 is specific to, CD79, for
example CD79b.
[0061] In one embodiment the V.sub.2 is specific to, CD79, for
example CD79b.
[0062] In one embodiment the V.sub.1 and V.sub.2 together (eg as
one binding domain) are specific to, CD79, for example CD79a or
CD79b.
[0063] In one embodiment V.sub.H and V.sub.L are specific to,
CD45.
[0064] In one embodiment the V.sub.1 is specific to, CD45.
[0065] In one embodiment the V.sub.2 is specific to, CD45.
[0066] In one embodiment the V.sub.1 and V.sub.2 together (eg as
one binding domain) are specific to, CD45.
[0067] In one embodiment the molecule of the present disclosure is
or comprises a fusion protein.
[0068] In one embodiment there is provided a multispecific molecule
according to the present disclosure, which is a bispecific protein
complex having the formula A-X:Y-B wherein: [0069] A-X is a first
fusion protein; [0070] Y-B is a second fusion protein; [0071] X:Y
is a heterodimeric-tether; [0072] A comprises a first binding
domain specific to CD45, CD79a, CD79b or a complexed of CD79a and
b; [0073] B comprises a second binding domain specific to CD45,
CD79a, CD79b or a complexed of CD79a and b; [0074] X is a first
binding partner of a binding pair; [0075] Y is a second binding
partner of the binding pair; and [0076] : is an interaction (such
as a binding interaction) between X and Y, and [0077] wherein at
least one of A or B is specific to CD45 and the other is specific
to CD79a, CD79b or a complexed form thereof.
[0078] The above format is a convenient because it provides a rapid
and efficient way of assembling bispecific formats that, for
example can be subjected to in vitro testing in functional assays.
This may facilitate the choice of a preferred pair of variable
regions, which may subsequently be incorporated into an
alternative, therapeutic multispecific antibody format.
[0079] Whilst not wishing to be bound by theory different
permutations of variable regions specific to CD45 combined with a
range of variable regions specific to CD79 may give access to
different nuances in biological function.
[0080] The invention also provides novel CD45 antibodies, for
example for use in the multispecific molecules of the present
invention or for incorporation into any other suitable antibody
format. The invention also provides novel CD79 antibodies, for
example for use in the multispecific molecules of the present
invention or for incorporation into any other suitable antibody
format.
BRIEF DESCRIPTION OF THE FIGURES
[0081] FIG. 1 is a bar chart of the relative potency of inhibition
of phosphorylated Akt for bispecific and bivalent combinations of
antibodies with specificity for CD45 and CD79b.
[0082] FIG. 2 is a bar chart of the relative potency of inhibition
of phosphorylated PLCg2 for bispecific and bivalent combinations of
antibodies with specificity for CD45 and CD79b.
[0083] FIG. 3 is a graph showing the titration of the effect of the
bispecific combination of CD45 and CD79b on CD86 expression on
anti-IgM stimulated B cells.
[0084] FIG. 4 is a graph of inhibition of phosphorylated PLCg2 for
bispecific proteins with specificity for CD45 and CD79b with
different V regions
[0085] FIG. 5 is an extract from Chan and Carter Reviews Immunology
vol 10, May 2010, 301 showing certain antibody formats.
[0086] FIG. 6 shows data for the antigen grid cross specificities.
Antigen 2=CD79b and antigen 4=CD45. Values are percentage
inhibition (negative value for activation) of phosphorlylation of
Syk & represent the mean of multiple V region combinations
evaluated.
[0087] FIG. 7 shows data for the antigen grid cross specificities.
Antigen 2=CD79b and antigen 4=CD45. Values are percentage
inhibition (negative value for activation) of PLC.gamma.2 &
represent the mean of multiple V region combinations evaluated.
[0088] FIG. 8 shows data for the antigen grid cross specificities.
Antigen 2=CD79b and antigen 4=CD45Values are percentage inhibition
(negative value for activation) of AKT & represent the mean of
multiple V region combinations evaluated.
[0089] FIG. 9 shows the percentage inhibition of the
phosphorlylation of Syk, PLC.gamma.2 & AKT for each V-region
combination for CD79b specificity in Fab-X combined with CD45
specificity in Fab-Y.
[0090] FIG. 10 shows the percentage inhibition of the
phosphorlylation of Syk, PLC.gamma.2 & AKT for each V-region
combination for CD79b specificity in Fab-Y combined with CD45
specificity in Fab-X
[0091] FIGS. 11 & 12 shows inhibition of PLC.gamma.2 (+/-SD) by
purified CD79b-CD45 (transiently expressed) on IgM stimulated
B-cells from donor 129 & 130
[0092] FIGS. 13 & 14 shows inhibition of p38 (+/-SD) by
purified CD79b-CD45 (transiently expressed) on IgM stimulated
B-cells from donor 129 & 130
[0093] FIGS. 15 & 16 shows inhibition of Akt (+/-SD) by
purified CD79b-CD45 (transiently expressed) on IgM stimulated
B-cells from donor 129 & 130
[0094] FIG. 17 shows the inhibition of tetanus toxoid IgG
production from PBMCs cultured with different multispecific
molecules
DETAILED DESCRIPTION OF THE DISCLOSURE
[0095] "Multispecific molecule" as employed herein refers to a
molecule with the ability to specifically bind at least two
distinct antigens, for example different antigens. In one
embodiment the multispecific molecule is a bispecific, trispecific
or tetraspecific molecule, in particular a bispecific molecule or
trispecific molecule.
[0096] Thus in one aspect the disclosure extends to a molecule of a
suitable format specific to at least CD45 and CD79a and to use of
antibodies/fragments or combinations thereof specific to CD45 and
CD79a in a multispecific molecule, such as a bispecific format or
trispecific format.
[0097] Thus in one aspect the disclosure extends to a molecule of a
suitable format specific to at least CD45 and CD79b and to use of
antibodies/fragments or combinations thereof specific to CD45 and
CD79b in a multispecific molecule, such as a bispecific format or
trispecific format.
[0098] Thus in one aspect the disclosure extends to a molecule of a
suitable format specific to at least CD45 and CD79a/b complex and
to use of antibodies/fragments or combinations thereof specific to
CD45 and CD79a/b complex in a multispecific molecule, such as a
bispecific format or trispecific format.
[0099] In one embodiment the molecule of the present disclosure is
trispecific, for example where the third binding domain is capable
of extending the half-life of the molecule, for example by binding
a serum carrier protein.
[0100] A variety of proteins exist in plasma and include
thyroxine-binding protein, transthyretin, .alpha.1-acid
glycoprotein, transferrin, fibrinogen and albumin, or a fragment of
any thereof (Bartalena & Robbins, 1993, Clinics in Lab. Med.
13:583-598; Bree et al., 1986, Clin. Pharmacokin. 11:336-342;
Gitlin et al. 1964, J. Clin. Invest. 10:1938-1951; Peters, 1985,
Adv Protein Chem. 37:161-245; Waldeman & Strober, 1969, Progr.
Allergy, 13:1-110. In on example the third binding domain is
specific to serum albumin, for example human serum albumin.
Multispecific Molecule Formats
[0101] Examples of suitable multispecific molecules are known in
the art, for example as disclosed in the review "The coming of Age
of Engineered Multivalent Antibodies, Nunez-Prado et al Drug
Discovery Today Vol 20 Number 5 Mar. 2015, page 588-594, D. Holmes,
Nature Rev Drug Disc November 2011:10; 798, Chan and Carter, Nature
Reviews Immunology vol 10, May 2010, 301 incorporated herein by
reference.
[0102] In one embodiment multispecific formats include those known
in the art and those described herein, such as wherein the molecule
format is selected from the group comprising or consisting of:
[0103] tandem sdAb, tandem sdAb-sdAb (three sdAbs); [0104]
(scFv).sub.2 (also referred to as tandem scFv), scFv-dsFv,
dsscFv-dsFv (dsFv).sub.2; [0105] diabody, dsdiabody, didsdiabody,
[0106] scdiabody, dsscdiabody, didsscdiabody; [0107] Dart antibody
i.e, VL.sub.1 linker VH.sub.2 linker and VH.sub.1 linker VL.sub.2
wherein the C-terminus of VH.sub.1 and VH.sub.2 are joined by a
disulfide bond; [0108] BITE, dsBiTE, didsBiTE; [0109] Di-diabody
(see Nunez-Prado et al in particular molecule number 25 in FIG. 1
therein), dsdi-diabody, didsdi-diabody; [0110] triabody,
dstriabody, didstriabody, tridstriabody; [0111] tetrabodies,
dstetrabody, didstetrabody, tridstetrabody, tetradstetrabody;
[0112] tandab (see Nunez-Prado et al in particular molecule number
22 in FIG. 1 therein); dstandab, didstandab, tridstandab,
tetradstandab; [0113] [sc(Fv).sub.2].sub.2, (see Nunez-Prado et al
in particular molecule number 22 in FIG. 1 therein),
ds[sc(Fv).sub.2].sub.2, dids[sc(Fv).sub.2].sub.2,
trids[sc(Fv).sub.2].sub.2, tetrads[sc(Fv).sub.2].sub.2, [0114]
Pentabody (see Nunez-Prado et al in particular molecule number 27
in FIG. 1 therein); [0115] Fab-scFv (also referred to as a bibody),
Fab'scFv, FabdsscFv (or BYbe), Fab'dsscFv; [0116] tribody,
dstribody, didstribody (also referred to as FabdidsscFv or TrYbe or
Fab-(dsscFv).sub.2), Fab'didsscFv; [0117] Fabdab, FabFv, Fab'dab,
Fab'Fv; [0118] Fab single linker Fv (also referred to herein as
FabdsFv as disclosed in WO2014/096390), Fab' single linker Fv (also
referred to herein as Fab'dsFv); [0119] FabscFv single linker Fv,
Fab'scFv single linker Fv; [0120] FabdsscFv single linker Fv,
Fab'dsscFv single linker Fv; [0121] FvFabFv, FvFab'Fv, dsFvFabFv,
dsFvFab'Fv, FvFabdsFv, FvFab'dsFv, dsFvFabdsFv, dsFvFab'dsFv,
[0122] FabFvFv, Fab'FvFv, FabdsFvFv, Fab' dsFvFv, FabFvdsFv,
Fab'FvdsFv, FabdsFvdsFv, Fab' dsFvdsFv, [0123] diFab, diFab'
including a chemically conjugated diFab', [0124] (FabscFv).sub.2,
(Fab).sub.2scFvdsFv, (Fab).sub.2dsscFvdsFv, (FabdscFv).sub.2,
[0125] (Fab'scFv).sub.2, (Fab').sub.2scFvdsFv,
(Fab').sub.2dsscFvdsFv, (Fab'dscFv).sub.2, [0126] V.sub.HHC.sub.K
(see Nunez-Prado et al in particular molecule number 6 in FIG. 1
therein); [0127] minibody, dsminibody, didsminibody, [0128] a
miniantibody (ZIP) [see Nunez-Prado et al in particular molecule
number 7 in FIG. 1 therein], dsminiantibody (ZIP) and
didsminiantibody (ZIP); [0129] tribi-minibody [see Nunez-Prado et
al in particular molecule number 15 in FIG. 1 therein]
dstribi-minibody, didstribi-minibody, tridstribi-minibody; [0130]
diabody-CH.sub.3, dsdiabody-CH.sub.3, didsdiabody-CH.sub.3,
scdiabody-CH.sub.3, dsscdiabody-CH.sub.3, didsscdiabody-CH.sub.3,
[0131] tandemscFv-CH.sub.3, tandemdsscFv-CH.sub.3,
tandemdidsscFv-CH.sub.3, tandemtridsscFv-CH.sub.3,
tandemtetradsscFv-CH.sub.3, [0132] scFv-Fc (also referred to herein
as a (scFvCH.sub.2CH.sub.3).sub.2) as described in WO2008/012543
and a single chain version thereof, dsscFvscFv-Fc, dsscFv-Fc (also
referred to herein as (dsscFvCH.sub.2CH.sub.3).sub.2),
scFv-dsFv-Fc, dsscFv-dsFv-Fc, dsFv-Fc (also referred to herein a
(dsFvCH.sub.2CH.sub.3).sub.2), [0133] scorpion molecule (Trubion)
i.e. a binding domain, linker --CH.sub.2CH.sub.3 binding domain as
described in U.S. Pat. No. 8,409,577; [0134] SMIP (Trubion) i.e.
(scFv-CH.sub.2CH.sub.3).sub.2; [0135] (dsFvCH.sub.2CH.sub.3).sub.2,
tandem scFv-Fc, tandem dsscFvscFv-Fc, tandem dsscFv-Fc, [0136]
scFv-Fc-scFv, dsscFv-Fc-scFv, scFv-Fc-dsscFv, [0137] diabody-Fc,
dsdiabody-Fc, didsdiabody-Fc, triabody-Fc, dstriabody-Fc,
didstriabody-Fc, tridstriabody-Fc, tetrabody-Fc, dstetrabody-Fc,
didstetrabody-Fc, tridstetrabody-Fc, tetradstetrabody-Fc,
dstetrabody-Fc, didstetrabody-Fc, tridstetrabody-Fc,
tetradstetrabody-Fc, scdiabody-Fc, dsscdiabody, didsscdiabody;
[0138] bi or trifunctional antibody, for example with different
heavy chain variable regions and common light chains for example
Merus bispecific antibody format (BICLONICS) with common light
chains of a fixed sequence and different heavy chains (including
different CDRs) and engineered CH.sub.3 domain to drive the
dimerization o the different heavy chains, [0139] Duobody (i.e.
wherein one full length chain in the antibody has different
specificity to the other full length chain in the antibody); [0140]
a full-length antibody wherein Fab arm exchange has been employed
to create a bispecific format; [0141] bi or tri functional
antibody, wherein a full-length antibody has common heavy chain and
different light chains also referred to as kappa/lambda body` or
`.kappa./.lamda.-body, see for example WO2012/023053 incorporated
herein by reference; [0142] Ig-scFv one, two, three or four from
the C terminus of heavy or light chain, scFv-Ig one, two, three or
four from the N terminus of heavy or light chain, single linker
Ig-Fv, Ig-dsscFv one, two, three or four from the C terminus of
heavy or light chain (with one, two, three or four disulfide
bonds); [0143] Ig-dsscFv one, two, three or four from the N
terminus of heavy or light chain (with one, two, three or four
disulfide bonds), [0144] Ig single linker Fv (see
PCT/EP2015/064450), [0145] Ig-dab, dab-Ig, scFv-Ig, V-Ig, Ig-V,
[0146] scFabFvFc, scFabdsFvFc (single linker version scFavFv),
(FabFvFc).sub.2, (FabdsFvFc).sub.2, scFab'FvFc, scFab' dsFvFc,
(Fab'FvFc).sub.2, (Fab' dsFvFc).sub.2 and [0147] DVDIg, which are
discussed in more detail below.
[0148] In one embodiment multispecific molecule formats include
those known in the art and those described herein, such as wherein
the molecule format is selected from the group comprising or
consisting of: diabody, scdiabody, triabody, tribody, tetrabodies,
tandem scFv, FabFv, Fab'Fv, FabdsFv, Fab-scFv, Fab-dsscFv,
Fab-(dsscFv).sub.2, diFab, diFab', tandem scFv-Fc, scFv-Fc-scFv,
scdiabody-Fc, scdiabody-CH.sub.3, Ig-scFv, scFv-Ig, V-Ig, Ig-V,
Duobody and DVDIg, which are discussed in more detail below.
[0149] In one embodiment the multispecific antibody molecule of the
present disclosure does not comprise an Fc domain i.e. does not
comprise a CH2 and CH3 domain, for example the molecule is selected
from the group comprising a tandem scFv, scFv-dsFv, dsscFv-dsFv
didsFv, diabody, dsdiabody, didsdiabody, scdiabody (also referred
to as an (scFv)2), dsscdiabody, triabody, dstriabody, didstriabody,
tridstriabody, tetrabodies, dstetrabody, didstetrabody,
tridstetrabody, tetradstetrabody, tribody, dstribody, didstribody,
Fabdab, FabFv, Fab'dab, Fab'Fv, Fab single linker Fv (as disclosed
in WO2014/096390), Fab' single linker Fv, FabdsFv, Fab'dsFv,
Fab-scFv (also referred to as a bibody), Fab'scFv, FabdsscFv,
Fab'dsscFv, FabdidsscFv, Fab'didsscFv, FabscFv single linker Fv,
Fab'scFv single linker Fv, FabdsscFvs single linker Fv, Fab'dsscFv
single linker Fv, FvFabFv, FvFab'Fv, dsFvFabFv, dsFvFab'Fv,
FvFabdsFv, FvFab'dsFv, dsFvFabdsFv, dsFvFab'dsFv, FabFvFv,
Fab'FvFv, FabdsFvFv, Fab'dsFvFv, FabFvdsFv, Fab'FvdsFv,
FabdsFvdsFv, Fab'dsFvdsFv, diFab, diFab' including a chemically
conjugated diFab', (FabscFv).sub.2, (Fab).sub.2scFvdsFv,
(Fab).sub.2dsscFvdsFv, (FabdscFv).sub.2, minibody, dsminibody,
didsminibody, diabody-CH.sub.3, dsdiabody-CH.sub.3,
didsdiabody-CH.sub.3, scdiabody-CH.sub.3, dsscdiabody-CH.sub.3,
didsscdiabody-CH.sub.3, tandemscFv-CH.sub.3, tandemdsscFv-CH.sub.3,
tandemdidsscFv-CH.sub.3, tandemtridsscFv-CH.sub.3 and
tandemtetradsscFv-CH.sub.3.
[0150] In one embodiment the molecule of the present disclosure
does not comprise an Fc domain.
[0151] In one embodiment the molecule of the present disclosure
comprises an altered Fc domain as described herein below.
[0152] Fc domain as employed herein generally refers to
--(CH.sub.2CH.sub.3).sub.2, unless the context clearly indicates
otherwise.
[0153] In one embodiment the molecule of the present disclosure
does not comprise a --CH.sub.2CH.sub.3 fragment.
[0154] In one embodiment the molecule of the present disclosure
does not comprise a CH.sub.2 domain.
[0155] In one embodiment the molecule of the present disclosure
does not comprise a CH.sub.3 domain.
[0156] Molecule as employed herein is used in the biochemistry
sense to refer to a group of atoms that form an organic, in
particular proteinaceous mass, which includes a complex suitable
for handling as a single entity under appropriate conditions once
the complex has been formed, for example a complex formed by two or
more polypeptide chains.
[0157] Molecule and construct are used interchangeably herein,
unless the context indicates otherwise. Although, construct may be
employed more often to refer to a polynucleotide molecule and
molecule may be employed more often to refer an entity primarily
comprising an amino acid sequence.
[0158] Specificity (or specific) as employed herein refers to where
the partners in the interaction only recognise each other or have
significantly higher affinity for each other in comparison to
non-partners, for example at least 2, 3, 4, 5, 6, 7, 8, 9, 10 times
higher affinity, than for example a background level of binding or
binding to another unrelated protein.
[0159] A `binding domain` as employed herein refers to a binding
region, typically a polypeptide, capable of binding a target
antigen, for example with sufficient affinity to characterise the
domain as specific for the antigen.
[0160] Any suitable binding domains may be used in the
multispecific molecules of the present invention. These may be
derived from any suitable source.
[0161] In one embodiment a biocompatible framework structure is
used in a binding domain of the molecules of the present disclosure
and such structures are based on protein scaffolds or skeletons
other than immunoglobulin domains. For example, those based on
fibronectin, ankyrin, lipocalin, neocarzinostain, cytochrome b, CP1
zinc finger, PST1, coiled coil, LACI-D1, Z domain and tendramisat
domains may be used (See for example, Nygren and Uhlen, 1997,
Current Opinion in Structural Biology, 7, 463-469).
[0162] The term `multi-specific molecules` as used herein may also
include binding agents based on biological scaffolds including
Adnectins, Affibodies, Darpins, Phylomers, Avimers, Aptamers,
Anticalins, Tetranectins, Microbodies, Affilins and Kunitz
domains.
[0163] The multispecific molecule of the present invention is
typically a multispecific antibody molecule, ie. at least one or
more of the binding domains of the multispecific molecule are
derived from an antibody or fragment thereof.
[0164] Where the binding domain is derived from an antibody, a
"binding domain or site" as employed herein is the part of the
antibody that contacts the antigen. In one embodiment the binding
domain contains at least one variable domain or a derivative
thereof, for example a pair of variable domains or derivatives
thereof, such as a cognate pair of variable domains or a derivative
thereof. Typically this is a VH/VL pair.
[0165] Variable regions (also referred to herein as variable
domains) generally comprise 3 CDRs and a suitable framework. In one
embodiment the binding domain comprises two variable regions, a
light chain variable region and a heavy chain variable region and
together these elements contribute to the specificity of the
binding interaction of the antibody or binding fragment.
[0166] A "cognate pair" as employed herein refers to a heavy and
light chain pair of variable domains (or a derivative thereof, such
as a humanised version thereof) isolated from a host as a
pre-formed couple. This definition does not include variable
domains isolated from a library, wherein the original pairing from
a host is not retained. Cognate pairs may be advantageous because
they are often affinity matured in the host and therefore may have
higher affinity for the antigen to which they are specific, than a
combination of variable domain pairs selected from a library, such
as phage library.
[0167] A "derivative of a naturally occurring domain" as employed
herein is intended to refer to where one, two, three, four or five
amino acids in a naturally occurring sequence have been replaced or
deleted, for example to optimize the properties of the domain such
as by eliminating undesirable properties but wherein the
characterizing feature(s) of the domain is/are retained. Examples
of modifications are those to remove glycosylation sites, GPI
anchors, or solvent exposed lysines. These modifications can be
achieved by replacing the relevant amino acid residues with a
conservative amino acid substitution.
[0168] Other modification in the CDRs may, for example include
replacing one or more cysteines with, for example a serine residue.
Asn can be the substrate for deamination and this propensity can be
reduced by replacing Asn and/or a neighbouring amino acid with an
alternative amino acid, such as a conservative substitution. The
amino acid Asp in the CDRs may be subject to isomerization. The
latter can be minimized by replacing Asp and/or a neighboring amino
acid with an alternative amino acid, for example a conservative
substitution.
[0169] Humanised versions of a variable region are also a
derivative thereof, in the context of the present specification.
Humanisation may include the replacement of a non-human framework
for a human framework and optionally the back-mutation of one or
more residues to "donor residues". Donor residues as employed
herein refers to residues found in the original variable region
isolated from the host, in particular replacing a given amino acid
in the human framework with the amino acid in the corresponding
location in the donor framework.
[0170] In one embodiment, the binding domain or each binding domain
is part of (included or incorporated in) an antibody or an antibody
fragment.
[0171] In one embodiment the binding domains in the molecules of
the present disclosure are in immunoglobulin/antibody
molecules.
[0172] As used herein "antibody molecule" includes antibodies and
binding fragments thereof.
[0173] In one embodiment the term "antibody" as used herein refers
to an immunoglobulin molecule capable of specific binding to a
target antigen, such as a carbohydrate, polynucleotide, lipid,
polypeptide, peptide etc., via at least one antigen recognition
site (also referred to as a binding site or binding domain herein),
located in the variable region of the immunoglobulin molecule.
"Antibody fragments" as employed herein refer to antibody binding
fragments including but not limited to Fab, modified Fab, Fab',
modified Fab', F(ab')2, Fv, single domain antibodies, scFv, Fv, bi,
tri or tetra-valent antibodies, Bis-scFv, diabodies, triabodies,
tetrabodies and epitope-binding fragments of any of the above (see
for example Holliger and Hudson, 2005, Nature Biotech.
23(9):1126-1136; Adair and Lawson, 2005, Drug Design
Reviews--Online 2(3), 209-217).
[0174] A "binding fragment" as employed herein refers to a fragment
capable of binding a target peptide or antigen with sufficient
affinity to characterise the fragment as specific for the peptide
or antigen
[0175] The methods for creating and manufacturing these antibody
fragments are well known in the art (see for example Verma et al.,
1998, Journal of Immunological Methods, 216:165-181). Other
antibody fragments for use in the present disclosure include the
Fab and Fab' fragments described in WO05/003169, WO05/003170 and
WO05/003171. Multi-valent antibodies may comprise multiple
specificities e.g. bispecific or may be monospecific (see for
example WO92/22853, WO05/113605, WO2009/040562 and
WO2010/035012).
[0176] The term "Fab fragment" as used herein refers to an antibody
fragment comprising a light chain fragment comprising a VL
(variable light) domain and a constant domain of a light chain
(C.sub.L), and a V.sub.H (variable heavy) domain and a first
constant domain (CH.sub.1) of a heavy chain.
[0177] The Fv refers to two variable domains, for example
co-operative variable domains, such as a cognate pair or affinity
matured variable domains, i.e. a V.sub.H and V.sub.L pair.
[0178] Co-operative variable domains as employed herein are
variable domains that complement each other and/or both contribute
to antigen binding to render the Fv (V.sub.H/V.sub.L pair) specific
for the antigen in question. [0179] "Single domain antibody" (also
referred to herein as a dab and sdAb) as used herein refers to an
antibody fragment consisting of a single monomeric variable
antibody domain. Examples of single domain antibodies include
V.sub.H or V.sub.L or V.sub.HH. [0180] Tandem-sdAb as employed
herein refers to two domain antibodies connected by a linker, for
example a peptide linker, in particular where the domain antibodies
have specificity for different antigens. [0181] Tandem-sdAb-sdAb as
employed herein refers to three domain antibodies connected in
series by two linkers, for example peptide linkers, in particular
where the domain antibodies have specificity for different
antigens. [0182] dsFv as employed herein refers to an Fv with an
intra-variable disulfide bond. The dsFv may be a component of a
larger molecule, for example the one of the variable domains may be
linked, for example via an amino acid linker to another antibody
fragment/component. [0183] (dsFv).sub.2 as employed herein refers
to a dsFv with one domain linked, for example via a peptide linker
or a disulfide bond (for example between, the C-terminus of two
V.sub.H's) to a domain in a second dsFv, the format resembles a
(scFv).sub.2 described below but each pair of variable regions
comprise a intra-variable region disulfide bond. [0184] Component
as employed herein refers to a building block or portion of a
multispecific molecule of the present disclosure, in particular
where the component is an antibody fragment such as scFv, Fab or
other fragment, in particular as described herein. [0185]
Single-chain Fv or abbreviated as "scFv", as used herein refers to
an antibody fragment that comprises V.sub.H and V.sub.L antibody
domains linked (for example by a peptide linker) to form a single
polypeptide chain. The constant regions of the heavy and light
chain are omitted in this format. [0186] dsscFv as employed herein
refers to scFv with an intra-variable region disulfide bond. [0187]
Tandem scFv (also referred to herein as a discFv or (scFv).sub.2))
as employed herein refers to two scFvs linked via a single linker
such that there is a single inter-Fv linker, for example as shown
in FIG. 5b. [0188] Tandem dsscFv (also referred to herein as a
scFvdsscFv or dsscFvscFv) as employed herein refers to two scFvs
linked via a single linker such that there is a single inter-Fv
linker, for example as shown in FIG. 5b, and wherein one of the
scFv has an intravariable region disulfide bond. [0189] Tandem
didsscFv (also referred to herein as a didsscFv) as employed herein
refers to two scFvs linked via a single linker such that there is a
single inter-Fv linker, for example as shown in FIG. 5b, and
wherein each scFv comprises an intravariable region disulfide bond.
[0190] scFv-dsFv as employed herein is a scFv linked, for example
by a peptide linker, to an Fv domain which is comprised of two
variable domains linked via a disulfide bond to form a dsFv. In
this format the VH or VL of the scFv may be linked to the VH or VL
of the dsFv. [0191] dsscFv-dsFv as employed herein is a dsscFv
linked, for example by a peptide linker, to an Fv domain which is
comprised of two variable domains linked via a disulfide bond to
form a dsFv. In this format the VH or VL of the dsscFv may be
linked to the VH or VL of the dsFv. [0192] Diabody as employed
herein refers to two Fv pairs V.sub.H/V.sub.L and a further
V.sub.H/V.sub.L pair which have two inter-Fv linkers, such that the
V.sub.H of a first Fv is linked to the V.sub.L of the second Fv and
the V.sub.L of the first Fv is linked to the V.sub.H of the second
Fv. [0193] dsDiabody as employed herein refers to a diabody
comprising an intra-variable region disulfide bond. [0194]
didsDiabody as employed herein refers to a diabody comprising two
intra-variable region disulfide bonds, i.e. one ds between each
pair of variable regions. [0195] Sc-diabody as employed herein
refers a diabody comprising an intra-Fv linker, such that the
molecule comprises three linkers and forms two normal scFvs, for
example VH.sub.1linkerVL.sub.1 linker VH.sub.2 linker VL.sub.2
[0196] dssc-diabody as employed herein refers to a sc-diabody with
an intra-variable region disulfide bond. [0197] didssc-diabody as
employed herein refers to a sc-diabody with an intra-variable
region disulfide bond between each pair of variable regions. [0198]
Dart as employed herein refers to VL.sub.1 linker VH.sub.2 linker
and VH.sub.1 linker VL.sub.2 wherein the C-terminous of VH.sub.1
and VH.sub.2 are joined by a disulfide bond Paul A. Moore et al
Blood, 2011; 117(17):4542-4551. [0199] BITE as employed herein
refers to a molecule comprising two pairs of variable domains in
the following format; a domain from pair 1 (eg VH.sub.1) connected
via a linker to a domain from pair 2 (eg VH.sub.2 or VL.sub.2) said
second domain connected by a linker to the further domain from pair
1 (eg VL.sub.1) in turn connected to the remaining domain from pair
two (i.e VL.sub.2 or VH.sub.2). [0200] Di-diabody see Nunez-Prado
et al in particular molecule number 25 in FIG. 1 therein. [0201]
Dsdi-diabody as employed herein is a di-diabody with an
intra-variable region disulfide bond. [0202] Didsdi-diabody as
employed herein is a di-diabody with an intra-variable region
disulfide bond between each pair of variable regions. [0203]
Triabody as employed herein refers to a format similar to the
diabody comprising three Fvs and three inter-Fv linkers. [0204]
dstriabody as employed herein refers to a triabody comprising an
intra-variable region disulfide bond between one of the variable
domain pairs. [0205] Didstriabody as employed herein refers to a
triabody comprising two intra-variable region disulfide bonds, i.e.
one ds between each of two variable domain pairs. [0206]
Tridstriabody as employed herein refers to a triabody comprising
three intra-variable region disulfide bonds i.e. one ds between
each pair of variable regions. [0207] Tetrabody as employed herein
refers to a format similar to the diabody comprising four Fvs and
four inter-Fv linkers. [0208] dstetrabody as employed herein refers
to a tetrabody comprising an intra-variable region disulfide bond
between one of the variable domain pairs. [0209] Didstetrabody as
employed herein refers to a tetrabody comprising two intra-variable
region disulfide bonds, i.e. one ds between each of two variable
domain pairs. [0210] Tridstetrabody as employed herein refers to a
tetrabody comprising three intra-variable region disulfide bonds
i.e. one ds between each of three pairs of variable regions. [0211]
Tetradstetrabody as employed herein refers to a tetrabody
comprising four intra-variable region disulfide bonds i.e. one ds
between each variable domain. [0212] Tribody (also referred to a
Fab(scFv).sub.2) as employed herein refers to a Fab fragment with a
first scFv appended to the C-terminal of the light chain and a
second scFv appended to the C-terminal of the heavy the chain.
[0213] dstribody as employed herein refers to a tribody comprising
a dsscFv in one of the two positions. [0214] didstribody or TrYbe
as employed herein refers to a tribody comprising two dsscFvs.
[0215] dsFab as employed herein refers to a Fab with an
intra-variable region disulfide bond. [0216] dsFab' as employed
herein refers to a Fab' with an intra-variable region disulfide
bond. [0217] scFab is a single chain Fab fragment. [0218] scFab' is
a single chain Fab' fragment. [0219] dsscFab is a dsFab as a single
chain. [0220] dsscFab' is a dsFab' as a single chain. [0221] Fabdab
as employed herein refers to a Fab fragment with a domain antibody
appended to the heavy or light chain thereof, optionally via a
linker. [0222] Fab' dab as employed herein refers to a Fab'
fragment with a domain antibody appended to the heavy or light
chain thereof, optionally via a linker. [0223] FabFv as employed
herein refers to a Fab fragment with an additional variable region
appended to the C-terminal of each of the following, the CH1 of the
heavy chain and CL of the light chain see for example
WO2009/040562. The format may be provided as a PEGylated version
thereof see for example WO2011/061492, [0224] Fab'Fv as employed
herein is similar to FabFv, wherein the Fab portion is replaced by
a Fab'. The format may be provided as a PEGylated version thereof.
[0225] FabdsFv as employed herein refers to a FabFv wherein an
intra-Fv disulfide bond stabilises the appended C-terminal variable
regions, see for example WO2010/035012. The format may be provided
as a PEGylated version thereof. [0226] Fab single linker Fv and
Fab' single linker as employed herein refers to a Fab or Fab'
fragment linked to a variable domain, for example by a peptide
linker, and said variable domain is linked to a second variable
domain via an intra-variable domain disulfide bond thereby forming
a dsFv, see for example WO2014/096390. [0227] Fab-scFv (also
referred to as a bibody) as employed herein is a Fab molecule with
a scFv appended on the C-terminal of the light or heavy chain,
optionally via a linker. [0228] Fab'-scFv as employed herein is a
Fab' molecule with a scFv appended on the C-terminal of the light
or heavy chain, optionally via a linker. [0229] FabdsscFv or BYbe
as employed herein is a FabscFv with a disulfide bond between the
variable regions of the single chain Fv. [0230] Fab'dsscFv as
employed herein is a Fab'scFv with a disulfide bond between the
variable regions of the single chain Fv. [0231] FabscFv-dab as
employed herein refers to a Fab with a scFv appended to the
C-terminal of one chain and domain antibody appended to the
C-terminal of the other chain. [0232] Fab'scFv-dab as employed
herein refers to a Fab' with a scFv appended to the C-terminal of
one chain and domain antibody appended to the C-terminal of the
other chain. [0233] FabdsscFv-dab as employed herein refers to a
Fab with a dsscFv appended to the C-terminal of one chain and
domain antibody appended to the C-terminal of the other chain.
[0234] Fab'dsscFv-dab as employed herein refers to a Fab' with a
dsscFv appended to the C-terminal of one chain and domain antibody
appended to the C-terminal of the other chain. [0235] FabscFv
single linker Fv as employed herein refers to a Fab single linker
Fv wherein a domain of the Fv is linked to the heavy or light chain
of the Fab and a scFv is linked to the other Fab chain and the
domains of the Fv are connected by an intra-variable region
disulfide. [0236] FabdsscFv single linker Fv as employed herein
refers to a FabscFv single linker Fv wherein the scFv comprises an
intra-variable region disulfide bond. [0237] Fab'scFv single linker
Fv as employed herein refers to a Fab' single linker Fv wherein a
domain of the Fv is linked to the heavy or light chain of the Fab
and a scFv is linked to the other Fab chain and the domains of the
Fv are connected by an intra-variable region disulfide. [0238] Fab'
dsscFv single linker Fv as employed herein refers to a Fab'scFv
single linker Fv wherein the scFv comprises an intra-variable
region disulfide bond. [0239] FvFabFv as employed herein refers to
a Fab with the domains of a first Fv appended to the N-terminus of
the heavy and light chain of the Fab and the domains of a second Fv
appended to the C-terminus of the heavy and light chain. [0240]
FvFab'Fv as employed herein refers to a Fab' with the domains of a
first Fv appended to the N-terminus of the heavy and light chain of
the Fab' and the domains of a second Fv appended to the C-terminus
of the heavy and light chain. [0241] dsFvFabFv as employed herein
refers to a Fab with the domains of a first Fv appended to the
N-terminus of the heavy and light chain of the Fab wherein the
first Fv comprises an intra-variable region disulfide bond and the
domains of a second Fv appended to the C-terminus of the heavy and
light chain. [0242] FvFabdsFv as employed herein refers to a Fab
with the domains of a first Fv appended to the N-terminus of the
heavy and light chain of the Fab and the domains of a second Fv
appended to the C-terminus of the heavy and light chain and wherein
the second Fv comprises an intra-variable region disulfide bond.
[0243] dsFvFab'Fv as employed herein refers to a Fab' with the
domains of a first Fv appended to the N-terminus of the heavy and
light chain of the Fab' wherein the first Fv comprises an
intra-variable region disulfide bond and the domains of a second Fv
appended to the C-terminus of the heavy and light chain. [0244]
FvFab'dsFv as employed herein refers to a Fab' with the domains of
a first Fv appended to the N-terminus of the heavy and light chain
of the Fab' and the domains of a second Fv appended to the
C-terminus of the heavy and light chain and wherein the second Fv
comprises an intra-variable region disulfide bond. [0245]
dsFvFabdsFv as employed herein refers to a Fab with the domains of
a first Fv appended to the N-terminus of the heavy and light chain
of the Fab wherein the first Fv comprises an intra-variable region
disulfide bond and the domains of a second Fv appended to the
C-terminus of the heavy and light chain and wherein the second Fv
also comprises an intra-variable region disulfide bond. [0246]
dsFvFab'dsFv as employed herein refers to a Fab' with the domains
of a first Fv appended to the N-terminus of the heavy and light
chain of the Fab' wherein the first Fv comprises an intra-variable
region disulfide bond and the domains of a second Fv appended to
the C-terminus of the heavy and light chain and wherein the second
Fv also comprises an intra-variable region disulfide bond. [0247]
FabFvFv as employed herein refers to a Fab fragment with two pairs
of Fvs appended in series to the C-terminal of the heavy and light
chain, see for example WO2011/086091. [0248] Fab'FvFv as employed
herein refers to a Fab' fragment with two pairs of Fvs appended in
series to the C-terminal of the heavy and light chain, see for
example WO2011/086091. [0249] FabdsFvFv as employed herein refers
to a Fab fragment with two pairs of Fvs appended in series to the
C-terminal of the heavy and light chain, see for example
WO2011/086091, wherein the first Fv pair attached directly to the
C-terminal comprise an intra-variable region disulfide bond. [0250]
Fab'dsFvFv as employed herein refers to a Fab' fragment with two
pairs of Fvs appended in series to the C-terminal of the heavy and
light chain, see for example WO2011/086091, wherein the first Fv
pair attached directly to the C-terminal comprise an intra-variable
region disulfide bond. [0251] FabFvdsFv as employed herein refers
to a Fab fragment with two pairs of Fvs appended in series to the
C-terminal of the heavy and light chain, wherein the second Fv pair
at the "C"-terminal of the molecule comprise an intra-variable
region disulfide bond. [0252] Fab
'FvdsFv as employed herein refers to a Fab' fragment with two pairs
of Fvs appended in series to the C-terminal of the heavy and light
chain, wherein the second Fv pair at the "C"-terminal of the
molecule comprise an intra-variable region disulfide bond. [0253]
FabdsFvdsFv as employed herein refers to a Fab fragment with two
pairs of Fvs appended in series to the C-terminal of the heavy and
light chain, wherein the first and second Fv pair comprise an
intra-variable region disulfide bond. [0254] Fab'dsFvdsFv as
employed herein refers to a Fab' fragment with two pairs of Fvs
appended in series to the C-terminal of the heavy and light chain,
wherein the first and second Fv comprise an intra-variable region
disulfide bond. [0255] DiFab as employed herein refers to two Fab
molecules linked via their C-terminus of the heavy chains. [0256]
DiFab' as employed herein refers to two Fab' molecules linked via
one or more disulfide bonds in the hinge region thereof. [0257]
DiFab and DiFab' molecules include chemically conjugated forms
thereof [0258] (FabscFv).sub.2 as employed herein refers to a diFab
molecule with two scFvs appended thereto, for example appended to
the C-terminal of the heavy or light chain, such as the heavy
chain. [0259] (Fab'scFv).sub.2 as employed herein refers to a
diFab' molecule with two scFvs appended thereto, for example
appended to the C-terminal of the heavy or light chain, such as the
heavy chain. [0260] (Fab).sub.2scFvdsFv as employed herein refers
to a diFab with a scFv and dsFv appended, for example one from each
of the heavy chain C-terminal. [0261] (Fab').sub.2scFvdsFv as
employed herein refers to a diFab' with a scFv and dsFv appended,
for example one from each of the heavy chain C-terminal. [0262]
(Fab).sub.2dsscFvdsFv, as employed herein refers to a diFab with a
dsscFv and dsFv appended, for example from the heavy chain
C-terminal. [0263] (Fab').sub.2dsscFvdsFv as employed herein refers
to the a diFab' with a dsscFv and dsFv appended, for example from
the heavy chain C-terminal. [0264] Minibody as employed herein
refers to (VL/VH--CH.sub.3).sub.2. [0265] dsminibody as employed
herein refers to (VL/VH--CH.sub.3).sub.2 wherein one VL/VH
comprises an intra-variable region disulfide bond. [0266]
didsminibody as employed herein refers to a (dsFv-CH3).sub.2 [0267]
scFv-Fc as employed herein refers to a scFv appended to the
N-terminus of a CH2 domain, for example via a hinge, of constant
region fragment --(CH2CH3), such that the molecule has 2 binding
domains. [0268] dsscFv-Fc as employed herein refers to a dsscFv
appended to the N-terminus of a CH2 domain and a scFv appended to
the N-terminus of a second CH2 domain, for example via a hinge, of
constant region fragment --(CH2CH3)2, such that the molecule has 2
binding domains. [0269] didsscFv-Fc as employed herein refers to a
scFv appended to the N-terminus of a CH2 domain, for example via a
hinge, of constant region fragment --(CH2CH3)2, such that the
molecule has 2 binding domains [0270] Tandem scFv-Fc as employed
herein refers to two tandem scFvs, wherein each one is appended in
series to the N-terminus of a CH.sub.2 domain, for example via a
hinge, of constant region fragment --(CH.sub.2CH.sub.3), such that
the molecule has 4 binding domains. [0271] Scdiabody-Fc as employed
herein is two scdiabodies, wherein each one is appended to the
N-terminus of a CH2 domain, for example via a hinge, of constant
region fragment --CH.sub.2CH.sub.3. [0272] ScFv-Fc-scFv as employed
herein refers to four scFvs, wherein one of each is appended to the
N-terminus and the C-terminus of both the heavy and light chain of
a --CH2CH3 fragment. [0273] Scdiabody-CH.sub.3 as employed herein
refers to two scdiabody molecules each linked, for example via a
hinge to a CH.sub.3 domain. [0274] kappa/lambda body` or
`.kappa./.lamda.-body is in the format of a normal IgG with two
heavy chains and two light chains, wherein the two light chains are
different to each other, one is a lambda light chain (VL-CL) and
the other is a kappa light chain (VK-CK). The heavy chain is
identical, even at the CDRs as described in WO2012/023053. [0275]
IgG-scFv as employed herein is a full length antibody with a scFv
on the C-terminal of each of the heavy chains or each of the light
chains. [0276] scFv-IgG as employed herein is a full length
antibody with a scFv on the N-terminal of each of the heavy chains
or each of the light chains. [0277] V-IgG as employed herein is a
full length antibody with a variable domain on the N-terminal of
each of the heavy chains or each of the light chains. [0278] IgG-V
as employed herein is a full length antibody with a variable domain
on the C-terminal of each of the heavy chains or each of the light
chains [0279] DVD-Ig (also known as dual V domain IgG) is a full
length antibody with 4 additional variable domains, one on the
N-terminus of each heavy and each light chain. [0280] Duobody or
`Fab-arm exchange` as employed herein is a bispecific IgG format
antibody where matched and complementary engineered amino acid
changes in the constant domains (typically CH3) of two different
monoclonal antibodies lead, upon mixing, to the formation of
heterodimers. A heavy:light chain pair from the first antibody
will, as a result of the residue engineering, prefer to associate
with a heavy:light chain pair of a second antibody. See for example
WO2008/119353, WO2011/131746 and WO2013/060867
[0281] Where one or more pairs of variable regions in the
multispecific molecule of the present invention comprise a
disulphide bond between VH and VL this may be in any suitable
position such as between two of the residues listed below (unless
the context indicates otherwise Kabat numbering is employed in the
list below). Wherever reference is made to Kabat numbering the
relevant reference is Kabat et al., 1987, in Sequences of Proteins
of Immunological Interest, US Department of Health and Human
Services, NIH, USA.
[0282] In one embodiment the disulfide bond is in a position
selected from the group comprising: [0283] V.sub.H37+V.sub.L95C see
for example Protein Science 6, 781-788 Zhu et al (1997); [0284]
V.sub.H44+V.sub.L100 see for example; Biochemistry 33 5451-5459
Reiter et al (1994); or Journal of Biological Chemistry Vol. 269
No. 28 pp. 18327-18331 Reiter et al (1994); or Protein Engineering,
vol. 10 no. 12 pp. 1453-1459 Rajagopal et al (1997); [0285]
V.sub.H44+V.sub.L105 see for example J Biochem. 118, 825-831 Luo et
al (1995); [0286] V.sub.H45+V.sub.L87 see for example Protein
Science 6, 781-788 Zhu et al (1997); [0287] V.sub.H55+V.sub.L101
see for example FEBS Letters 377 135-139 Young et al (1995); [0288]
V.sub.H100+V.sub.L50 see for example Biochemistry 29 1362-1367
Glockshuber et al (1990); [0289] V.sub.H100b+V.sub.L49; [0290]
V.sub.H98+V.sub.L 46 see for example Protein Science 6, 781-788 Zhu
et al (1997); [0291] V.sub.H101+V.sub.L46; [0292]
V.sub.H105+V.sub.L43 see for example; Proc. Natl. Acad. Sci. USA
Vol. 90 pp. 7538-7542 Brinkmann et al (1993); or Proteins 19, 35-47
Jung et al (1994), [0293] V.sub.H106+V.sub.L57 see for example FEBS
Letters 377 135-139 Young et al (1995) [0294] and a position
corresponding thereto in variable region pair located in the
molecule. [0295] In one embodiment, the disulphide bond is formed
between positions V.sub.H44 and V.sub.L100.
[0296] "Monospecific" as employed herein refers to the ability to
bind a target antigen only once. Thus is one embodiment the
multispecific molecules of the present invention are monspecific
for each antigen
[0297] Thus in one embodiment the binding domains of the
multispecific molecules according to the present disclosure are
monospecific. This is advantageous in some therapeutic applications
because the molecules of the disclosure are not able to cross-link
antigen via binding the target antigen more than once. Thus in one
embodiment bispecific or multispecific molecules of the present
disclosure are not able to cross-link by binding the same target
twice in two different locations, for example on the same cell or
on two different cells.
[0298] Cross-linking, in particular in relation to CD79b on the
same cell or different cells can generate signals in vivo, for
example which stimulate the activity of the target antigen
[0299] In another embodiment, for example where the molecules of
the disclosure comprise at least three binding domains then two or
three binding domains (for example antibodies, fragments or a
combination of an antibody and a fragment) may have different
antigen specificities, for example binding to two or three
different target antigens.
[0300] In one example the multispecific molecules of the present
invention contain no more than one binding domain for CD22 and no
more than one binding domain for CD79. Each binding domain is
monospecific.
[0301] In one embodiment, each antibody or antibody fragment
employed in the multi-specific molecules of the present disclosure
is monovalent.
[0302] Thus in one embodiment the binding domains of the
multispecific molecules of the present disclosure are
monovalent.
[0303] Thus in one embodiment the binding domains of the
multispecific molecules of the present disclosure are monovalent
and monospecific.
[0304] In one embodiment the multispecific molecule of the present
disclosure is comprised of two or more monospecific, monovalent
binding domains such as Fab, Fab', scFv, VH, VL, VHH, Fv, dsFv,
combined or linked in any suitable way to construct a multispecific
molecule, for example as described herein above.
Constant Regions
[0305] The antibody constant region domains of a multispecific
molecule of the present disclosure, if present, may be selected
having regard to the proposed function of the multispecific
antibody molecule, and in particular the effector functions which
may be required. For example, the constant region domains may be
human IgA, IgD, IgE, IgG or IgM domains. In particular, human IgG
constant region domains may be used, especially of the IgG1 and
IgG3 isotypes when the antibody molecule is intended for
therapeutic uses and antibody effector functions are required.
Alternatively, IgG2 and IgG4 isotypes may be used when the antibody
molecule is intended for therapeutic purposes and antibody effector
functions are not required. It will be appreciated that sequence
variants of these constant region domains may also be used. For
example IgG4 molecules in which the serine at position 241 has been
changed to proline as described in Angal et al., 1993, Molecular
Immunology, 1993, 30:105-108 may be used. Accordingly, in the
embodiment where the antibody is an IgG4 antibody, the antibody may
include the mutation S241P.
[0306] In one embodiment, the antibody heavy chain comprises a
CH.sub.1 domain and the antibody light chain comprises a CL domain,
either kappa or lambda.
[0307] In one embodiment, the antibody heavy chain comprises a
CH.sub.1 domain, a CH.sub.2 domain and a CH.sub.3 domain and the
antibody light chain comprises a CL domain, either kappa or
lambda.
[0308] The four human IgG isotypes bind the activating Fc.gamma.
receptors (Fc.gamma.RI, Fc.gamma.RIIa, Fc.gamma.RIIIa), the
inhibitory Fc.gamma.RIIb receptor, and the first component of
complement (C1q) with different affinities, yielding very different
effector functions (Bruhns P. et al., 2009. Specificity and
affinity of human Fcgamma receptors and their polymorphic variants
for human IgG subclasses. Blood. 113(16):3716-25), see also Jeffrey
B. Stavenhagen, et al. Cancer Research 2007 Sep. 15;
67(18):8882-90.
[0309] Binding of IgG to the Fc.gamma.Rs or C1q depends on residues
located in the hinge region and the CH.sub.2 domain. Two regions of
the CH.sub.2 domain are critical for Fc.gamma.Rs and C1q binding,
and have unique sequences in IgG2 and IgG4. Substitutions into
human IgG1 of IgG2 residues at positions 233-236 and IgG4 residues
at positions 327, 330 and 331 have been shown to greatly reduce
ADCC and CDC (Armour K L. et al., 1999. Recombinant human IgG
molecules lacking Fcgamma receptor I binding and monocyte
triggering activities. Eur J Immunol. 29(8):2613-24 and Shields R
L. et al., 2001. High resolution mapping of the binding site on
human IgG1 for Fc gamma RI, Fc gamma RII, Fc gamma RIII, and FcRn
and design of IgG1 variants with improved binding to the Fc gamma
R. J Biol Chem. 276(9):6591-604). Furthermore, Idusogie et al.
demonstrated that alanine substitution at different positions,
including K322, significantly reduced complement activation
(Idusogie E E. et al., 2000. Mapping of the C1q binding site on
rituxan, a chimeric antibody with a human IgG1 Fc. J Immunol.
164(8):4178-84). Similarly, mutations in the CH.sub.2 domain of
murine IgG2A were shown to reduce the binding to Fc.gamma.RI, and
C1q (Steurer W. et al., 1995. Ex vivo coating of islet cell
allografts with murine CTLA4/Fc promotes graft tolerance. J
Immunol. 155(3):1165-74).
[0310] In one embodiment the Fc region employed is mutated, in
particular a mutation described herein. In one embodiment the
mutation is to remove binding and/or effector function.
[0311] In one embodiment the Fc mutation is selected from the group
comprising a mutation to remove binding of the Fc region, a
mutation to increase or remove an effector function, a mutation to
increase half-life and a combination of the same.
[0312] Some antibodies that selectively bind FcRn at pH 6.0, but
not pH 7.4, exhibit a higher half-life in a variety of animal
models. Several mutations located at the interface between the
CH.sub.2 and CH.sub.3 domains, such as T250Q/M428L (Hinton P R. et
al., 2004. Engineered human IgG antibodies with longer serum
half-lives in primates. J Biol Chem. 279(8):6213-6) and
M252Y/S254T/T256E+H433K/N434F (Vaccaro C. et al., 2005. Engineering
the Fc region of immunoglobulin G to modulate in vivo antibody
levels. Nat Biotechnol. 23(10):1283-8), have been shown to increase
the binding affinity to FcRn and the half-life of IgG1 in vivo.
[0313] However, there is not always a direct relationship between
increased FcRn binding and improved half-life (Datta-Mannan A. et
al., 2007. Humanized IgG1 Variants with Differential Binding
Properties to the Neonatal Fc Receptor: Relationship to
Pharmacokinetics in Mice and Primates. Drug Metab. Dispos. 35:
86-94).
[0314] IgG4 subclass show reduced Fc receptor (Fc.gamma.RIIIa)
binding, antibodies of other IgG subclasses generally show strong
binding. Reduced receptor binding in these other IgG subtypes can
be effected by altering, for example replacing one or more amino
acids selected from the group comprising Pro238, Aps265, Asp270,
Asn270 (loss of Fc carbohydrate), Pro329, Leu234, Leu235, Gly236,
Gly237, Ile253, Ser254, Lys288, Thr307, Gln311, Asn434 and
His435.
[0315] In one embodiment a molecule according to the present
disclosure has an Fc of IgG subclass, for example IgG1, IgG2 or
IgG3 wherein the Fc is mutated in one, two or all following
positions S228, L234 and/or D265.
[0316] In one embodiment the mutations in the Fc region are
independently selected from S228P, L234A, L235A, L235A, L235E and
combinations thereof.
[0317] It may be desired to either reduce or increase the effector
function of an Fc region. Antibodies that target cell-surface
molecules, especially those on immune cells, abrogating effector
functions is required. In some embodiments, for example for the
treatment of autoimmunity, enhanced Fc binding on immune cells by
increasing negative Fc receptor binding (FcgRIIb or CD32b) may be
desirable see Stavenhagen J B, et al Advances in Enzyme Regulation
2007 Dec. 3 and Veri M C, et al. Arthritis Rheum, 2010 Mar. 30;
62(7):1933-43. Conversely, for antibodies intended for oncology
use, increasing effector functions may improve the therapeutic
activity.
[0318] Numerous mutations have been made in the CH.sub.2 domain of
human IgG1 and their effect on ADCC and CDC tested in vitro
(Idusogie E E. et al., 2001. Engineered antibodies with increased
activity to recruit complement. J Immunol. 166(4):2571-5). Notably,
alanine substitution at position 333 was reported to increase both
ADCC and CDC. Lazar et al. described a triple mutant
(S239D/I332E/A330L) with a higher affinity for Fc.gamma.RIIIa and a
lower affinity for Fc.gamma.RIIb resulting in enhanced ADCC (Lazar
G A. et al., 2006. Engineered antibody Fc variants with enhanced
effector function. PNAS 103(11): 4005-4010). The same mutations
were used to generate an antibody with increased ADCC (Ryan M C. et
al., 2007. Antibody targeting of B-cell maturation antigen on
malignant plasma cells. Mol. Cancer Ther., 6: 3009 3018). Richards
et al. studied a slightly different triple mutant
(S239D/I332E/G236A) with improved Fc.gamma.RIIIa affinity and
Fc.gamma.Rlla/Fc.gamma.RIIb ratio that mediates enhanced
phagocytosis of target cells by macrophages (Richards J O et al
2008. Optimization of antibody binding to Fcgamma RIIa enhances
macrophage phagocytosis of tumor cells. Mol Cancer Ther.
7(8):2517-27).
[0319] Due to their lack of effector functions, IgG4 antibodies
represent a suitable IgG subclass for receptor blocking without
cell depletion. IgG4 molecules can exchange half-molecules in a
dynamic process termed Fab-arm exchange. This phenomenon can occur
between therapeutic antibodies and endogenous IgG4. The S228P
mutation has been shown to prevent this recombination process
allowing the design of less unpredictable therapeutic IgG4
antibodies (Labrijn A F. et al., 2009. Therapeutic IgG4 antibodies
engage in Fab-arm exchange with endogenous human IgG4 in vivo. Nat
Biotechnol. 27(8):767-71). This technology may be employed to
create bispecific antibody molecules.
[0320] It will also be understood by one skilled in the art that
antibodies may undergo a variety of post-translational
modifications. The type and extent of these modifications often
depends on the host cell line used to express the antibody as well
as the culture conditions. Such modifications may include
variations in glycosylation, methionine oxidation, diketopiperazine
formation, aspartate isomerization and asparagine deamidation. A
frequent modification is the loss of a carboxy-terminal basic
residue (such as lysine or arginine) due to the action of
carboxypeptidases (as described in Harris, R J. Journal of
Chromatography 705:129-134, 1995). Accordingly, the C-terminal
lysine of the antibody heavy chain may be absent.
Affinity
[0321] The multispecific molecules of the present invention
comprise a binding domain specific to the antigen CD45 and a
binding domain specific to the antigen CD79a and/or CD79b.
[0322] In one embodiment a binding domain employed in the molecules
of the present disclosure is specific to CD45.
[0323] In one embodiment a binding domain employed in the molecules
of the present disclosure is specific to CD79a.
[0324] In one embodiment a binding domain employed in the molecules
of the present disclosure is specific to CD79b.
[0325] In one embodiment a binding domain employed in the molecules
of the present disclosure is specific to CD79 complex, i.e. it
recognises an epitope present in the complex and specific thereto,
for example an epitope comprising an interaction between CD79a and
CD79b.
[0326] CD45 (also known as PTPRC) is a known protein. CD45 is a
member of the protein tyrosine phosphatase (PTP) family. PTPs are
known to be signaling molecules that regulate a variety of cellular
processes including cell growth, differentiation, mitotic cycle,
and oncogenic transformation. This PTP contains an extracellular
domain, a single transmembrane segment and two tandem
intracytoplasmic catalytic domains, and thus belongs to receptor
type PTP. Various isoforms of CD45 exist: CD45RA, CD45RB, CD45RC,
CD45RAB, CD45RAC, CD45RBC, CD45RO, CD45R (ABC). CD45RA is located
on naive T cells and CD45RO is located on memory T cells. CD45
splice variant isoforms A, B and C are expressed differentially on
human B cells. CD45 is a member of the Protein Tyrosine Phosphatase
(PTP) family: Its intracellular (COOH-terminal) region contains two
PTP catalytic domains, and the extracellular region is highly
variable due to alternative splicing of exons 4, 5, and 6
(designated A, B, and C, respectively), plus differing levels of
glycosylation. The CD45 isoforms detected are cell type-,
maturation, and activation state-specific. In general the long form
of the protein (A, B or C) is expressed on naive or unactivated B
cells and the mature or truncated form of CD45 (RO) is expressed on
activated or mature/memory B cells.
[0327] The human sequence is available in UniProt entry number
P08575 and provided herein in SEQ ID NO:143, or amino acids 24-1304
of SEQ ID NO:143, lacking the signal peptide. The murine version in
UniProt entry P06800. The present disclosure relates to all forms
of CD45, from any species. In one embodiment CD45 refers to the
human form of the protein and natural variants and isoforms
thereof.
[0328] In one embodiment the affinity of the binding domain for
CD45 in a molecule of the present disclosure is about 100 nM or
stronger such as about 50 nM, 20 nM, 10 nM, 1 nM, 500 pM, 250 pM,
200 pM, 100 pM or stronger, in particular a binding affinity of 50
pM or stronger. The binding domain for CD79 may bind to CD79a
and/or CD79b.
[0329] CD79a (also known as immunoglobulin alpha and B-cell antigen
receptor complex-associated protein alpha chain) is a known
protein. Expression of CD79a is restricted to B lymphocytes. The
human sequence is available in UniProt under entry P11912 (SEQ ID
NO: and without signal sequence amino acids 33-226 of SEQ ID NO:
141). The murine version is available in UniProt under entry 11911.
The present disclosure relates to all forms of CD79a from any
species, in particular human and any natural variants thereof. In
one embodiment CD79a refers to the human form of the protein.
[0330] CD79b (also known as immunoglobulin associated beta and
cluster differentiation 79B) is a known protein. Expression of
CD79b is restricted to B lymphocytes. The human sequence is
available in UniProt under entry P40259 (SEQ ID NO:142 and without
signal sequence amino acids 29-229 of SEQ ID NO:142). The murine
version in UniProt under entry P15530. The present disclosure
relates to all forms of CD79b, from any species, in particular
human and any natural variants thereof. In one embodiment CD79b
refers to the human form of the protein.
[0331] In one embodiment the binding domain specific to CD79 binds
CD79a.
[0332] In one embodiment the binding domain specific to CD79 binds
CD79b.
[0333] In one embodiment the binding domain specific to CD79 binds
a complex of CD79a and CD79b
[0334] In one embodiment the affinity of the binding domain for
CD79 in a molecule of the present disclosure is about 100 nM or
stronger such as about 50 nM, 20 nM, 10 nM, 1 nM, 500 pM, 250 pM,
200 pM, 100 pM or stronger, in particular a binding affinity of 50
pM or stronger. In one embodiment the affinity of the binding
domain for CD79a in a molecule of the present disclosure is about
100 nM or stronger such as about 50 nM, 20 nM, 10 nM, 1 nM, 500 pM,
250 pM, 200 pM, 100 pM or stronger, in particular a binding
affinity of 50 pM or stronger.
[0335] In one embodiment the affinity of the binding domain for
CD79b in a molecule of the present disclosure is about 100 nM or
stronger such as about 50 nM, 20 nM, 10 nM, 1 nM, 500 pM, 250 pM,
200 pM, 100 pM or stronger, in particular a binding affinity of 50
pM or stronger.
[0336] It will be appreciated that the affinity of the binding
domain for CD45 may be the same or different from the affinity of
the binding domain for CD79.
[0337] In one embodiment, the multi-specific antibody molecules of
the present disclosure or antibody/fragment components thereof are
processed to provide improved affinity for a target antigen or
antigens. Such variants can be obtained by a number of affinity
maturation protocols including mutating the CDRs (Yang et al., J.
Mol. Biol., 254, 392-403, 1995), chain shuffling (Marks et al.,
Bio/Technology, 10, 779-783, 1992), use of mutator strains of E.
coli (Low et al J. Mol. Biol., 250, 359-368, 1996), DNA shuffling
(Patten et al Curr. Opin. Biotechnol., 8, 724-733, 1997), phage
display (Thompson et al., J. Mol. Biol., 256, 77-88, 1996) and
sexual PCR (Crameri et al Nature, 391, 288-291, 1998). Vaughan et
al (supra) discusses these methods of affinity maturation.
Antibodies & Generation of the Same
[0338] Binding domains for use in the present invention may be
generated by any suitable method known in the art, for example CDRs
may be taken from non-human antibodies including commercially
available antibodies and grafted into human frameworks or
alternatively chimeric antibodies can be prepared with non-human
variable regions and human constant regions etc.
[0339] Typically the binding domains for use in the present
invention are binding domains derived from antibodies which bind
the selected antigen, such as antibodies which bind CD45, CD79a
and/or CD79b.
[0340] Examples of CD45 and CD79 antibodies are known in the art
and these may be employed directly in the molecules of the present
invention or screened for suitability using the methods described
herein, and subsequently modified if necessary, for example
humanised, using the methods described herein. Therapeutic
anti-CD45 and anti-CD79 antibodies have been described in the art,
for example anti-CD45 antibodies disclosed in US2011/0076270,
anti-CD79b antibodies disclosed in WO2014/011521 and
WO2015/021089.
[0341] Examples of CD45 antibodies include rat monoclonal YTH54,
YTH25.4, mouse monoclonal from Miltenyi clone 5B1 and clone 30F11,
rat monoclonal YAML568, from BD Bioscience mouse monoclonal clone
2D1 catalog No. 347460, from Novus mouse monoclonal antibody 5D3A3
catalog No. NBP2-37293, mouse monoclonal HI30 catalog No.
NBP1-79127, mouse monoclonal 4A8A4C7A2 catalog No. NBP1-47428,
mouse monoclonal 2B11 catalog No. NBP2-32934, rat monoclonal
YTH24.5 catalog No. NB100-63828, rabbit monoclonal Y321 catalog No.
NB110-55701, mouse monoclonal PD7/26/16 catalog No. NB120-875, from
Santa Cruz mouse monoclonal from clone B8 catalog No. sc-28369,
mouse monoclonal from clone F10-89-4 catalog No. sc-52490, rabbit
monoclonal from clone H-230 catalog No. sc-25590, goat monoclonal
from clone N-19 catalog No. sc-1123, mouse monoclonal from clone
OX1 catalog No. sc-53045, rat monoclonal (T29/33) catalog No
sc-18901, rat monoclonal (YAML 501.4) catalog No. sc65344, rat
monoclonal (YTH80.103) catalog No sc-59071, mouse monoclonal
(35105) catalog No. sc-53201, mouse monoclonal (35-Z6) catalog No.
sc-1178, mouse monoclonal (158-4D3) catalog No. sc-52386, mouse
monoclonal to CD45RO (UCH-L1) catalog No. sc-1183, mouse monoclonal
to CD45RO (2Q1392) catalog No. sc-70712. CD45 antibodies are also
disclosed in WO2005/026210, WO02/072832 and WO2003/048327
incorporated herein by reference.
[0342] Commercially available anti-CD79a antibodies include mouse
monoclonal LS-B4504 (LSBio) from clone HM57, mouse monoclonal
LS-B8330, mouse monoclonal LS-C44954, rabbit monoclonal LS-B9093,
mouse monoclonal LS-B8513 from clone JCB117, rabbit monoclonal
LS-C210607 from clone SP18, mouse monoclonal LS-C175441 from clone
5E2, mouse monoclonal LS-C338670 from clone 3D3, mouse monoclonal
LS-C88120 from clone HM47/A9, mouse monoclonal LS-C191714, mouse
monoclonal LS-C87592, mouse monoclonal LS-C44955, mouse monoclonal
LS-C95934, mouse monoclonal LS-C121584, mouse monoclonal
LS-C121585, mouse monoclonal LS-C204347, mouse monoclonal
LS-C88122, Abcam mouse monoclonal ab3121 [HM47/A9], rabbit
monoclonal ab79414, and rabbit monoclonal ab133483.
[0343] Commercially available CD79b antibodies include mouse
monoclonal Abcam antibody ab33295, rat monoclonal ab23826, mouse
monoclonal ab103422, rabbit monoclonal ab134103, rabbit monoclonal
ab134147, and rabbit monoclonal ab183343.
[0344] Such commercially available antibodies may be useful tools
in the discovery of therapeutic antibodies.
[0345] The skilled person may generate antibodies for use in the
multi-specific molecules of the invention using any suitable method
known in the art.
[0346] Antigen polypeptides, for use in generating antibodies for
example for use to immunize a host or for use in panning, such as
in phage display, may be prepared by processes well known in the
art from genetically engineered host cells comprising expression
systems or they may be recovered from natural biological sources.
In the present application, the term "polypeptides" includes
peptides, polypeptides and proteins. These are used interchangeably
unless otherwise specified. The antigen polypeptide may in some
instances be part of a larger protein such as a fusion protein for
example fused to an affinity tag or similar. In one embodiment the
host may be immunised with a cell transfected with the relevant
protein or polypeptide, for example co-transfected with CD79a and
CD79b.
[0347] Antibodies generated against an antigen polypeptide may be
obtained, where immunisation of an animal is necessary, by
administering the polypeptides to an animal, preferably a non-human
animal, using well-known and routine protocols, see for example
Handbook of Experimental Immunology, D. M. Weir (ed.), Vol 4,
Blackwell Scientific Publishers, Oxford, England, 1986). Many
warm-blooded animals, such as rabbits, mice, rats, sheep, cows,
camels or pigs may be immunized. However, mice, rabbits, pigs and
rats are generally most suitable. Monoclonal antibodies may be
prepared by any method known in the art such as the hybridoma
technique (Kohler & Milstein, 1975, Nature, 256:495-497), the
trioma technique, the human B-cell hybridoma technique (Kozbor et
al 1983, Immunology Today, 4:72) and the EBV-hybridoma technique
(Cole et al Monoclonal Antibodies and Cancer Therapy, pp 77-96,
Alan R Liss, Inc., 1985).
[0348] Antibodies may also be generated using single lymphocyte
antibody methods by cloning and expressing immunoglobulin variable
region cDNAs generated from single lymphocytes selected for the
production of specific antibodies by, for example, the methods
described by Babcook, J. et al 1996, Proc. Natl. Acad. Sci. USA
93(15):7843-78481; WO92/02551; WO2004/051268 and WO2004/106377.
[0349] The antibodies for use in the present disclosure can also be
generated using various phage display methods known in the art and
include those disclosed by Brinkman et al. (in J. Immunol. Methods,
1995, 182: 41-50), Ames et al. (J. Immunol. Methods, 1995,
184:177-186), Kettleborough et al. (Eur. J. Immunol. 1994,
24:952-958), Persic et al. (Gene, 1997 187 9-18), Burton et al.
(Advances in Immunology, 1994, 57:191-280) and WO90/02809;
WO91/10737; WO92/01047; WO92/18619; WO93/11236; WO95/15982;
WO95/20401; and U.S. Pat. Nos. 5,698,426; 5,223,409; 5,403,484;
5,580,717; 5,427,908; 5,750,753; 5,821,047; 5,571,698; 5,427,908;
5,516,637; 5,780,225; 5,658,727; 5,733,743; 5,969,108, and
WO20011/30305.
[0350] In one example the multi-specific molecules of the present
disclosure are fully human, in particular one or more of the
variable domains are fully human.
[0351] Fully human molecules are those in which the variable
regions and the constant regions (where present) of both the heavy
and the light chains are all of human origin, or substantially
identical to sequences of human origin, not necessarily from the
same antibody. Examples of fully human antibodies may include
antibodies produced, for example by the phage display methods
described above and antibodies produced by mice in which the murine
immunoglobulin variable and optionally the constant region genes
have been replaced by their human counterparts e.g. as described in
general terms in EP0546073, U.S. Pat. Nos. 5,545,806, 5,569,825,
5,625,126, 5,633,425, 5,661,016, 5,770,429, EP 0438474 and
EP0463151.
[0352] In one example the binding domains of the multi-specific
molecules according to the disclosure are humanised.
[0353] Humanised (which include CDR-grafted antibodies) as employed
herein refers to molecules having one or more complementarity
determining regions (CDRs) from a non-human species and a framework
region from a human immunoglobulin molecule (see, e.g. U.S. Pat.
No. 5,585,089; WO91/09967). It will be appreciated that it may only
be necessary to transfer the specificity determining residues of
the CDRs rather than the entire CDR (see for example, Kashmiri et
al., 2005, Methods, 36, 25-34). Humanised antibodies may optionally
further comprise one or more framework residues derived from the
non-human species from which the CDRs were derived.
[0354] As used herein, the term "humanised antibody molecule"
refers to an antibody molecule wherein the heavy and/or light chain
contains one or more CDRs (including, if desired, one or more
modified CDRs) from a donor antibody (e.g. a murine monoclonal
antibody) grafted into a heavy and/or light chain variable region
framework of an acceptor antibody (e.g. a human antibody). For a
review, see Vaughan et al, Nature Biotechnology, 16, 535-539, 1998.
In one embodiment rather than the entire CDR being transferred,
only one or more of the specificity determining residues from any
one of the CDRs described herein above are transferred to the human
antibody framework (see for example, Kashmiri et al., 2005,
Methods, 36, 25-34). In one embodiment only the specificity
determining residues from one or more of the CDRs described herein
above are transferred to the human antibody framework. In another
embodiment only the specificity determining residues from each of
the CDRs described herein above are transferred to the human
antibody framework.
[0355] When the CDRs or specificity determining residues are
grafted, any appropriate acceptor variable region framework
sequence may be used having regard to the class/type of the donor
antibody from which the CDRs are derived, including mouse, primate
and human framework regions. Suitably, the humanised antibody
according to the present invention has a variable domain comprising
human acceptor framework regions as well as one or more of the CDRs
provided herein.
[0356] Examples of human frameworks which can be used in the
present disclosure are KOL, NEWM, REI, EU, TUR, TEI, LAY and POM
(Kabat et al supra). For example, KOL and NEWM can be used for the
heavy chain, REI can be used for the light chain and EU, LAY and
POM can be used for both the heavy chain and the light chain.
Alternatively, human germline sequences may be used; these are
available at: http://www2.mrc-lmb.cam.ac.uk/vbase/list2.php.
[0357] In a humanised antibody molecule of the present disclosure,
the acceptor heavy and light chains do not necessarily need to be
derived from the same antibody and may, if desired, comprise
composite chains having framework regions derived from different
chains.
[0358] The framework regions need not have exactly the same
sequence as those of the acceptor antibody. For instance, unusual
residues may be changed to more frequently-occurring residues for
that acceptor chain class or type. Alternatively, selected residues
in the acceptor framework regions may be changed so that they
correspond to the residue found at the same position in the donor
antibody (see Reichmann et al 1998, Nature, 332, 323-324). Such
changes should be kept to the minimum necessary to recover the
affinity of the donor antibody. A protocol for selecting residues
in the acceptor framework regions which may need to be changed is
set forth in WO91/09967.
[0359] Derivatives of frameworks may have 1, 2, 3 or 4 amino acids
replaced with an alternative amino acid, for example with a donor
residue.
[0360] Donor residues are residues from the donor antibody, i.e.
the antibody from which the CDRs were originally derived, in
particular the residue in a corresponding location from the donor
sequence is adopted. Donor residues may be replaced by a suitable
residue derived from a human receptor framework (acceptor
residues).
[0361] The residues in antibody variable domains are conventionally
numbered according to a system devised by Kabat et al. This system
is set forth in Kabat et al., 1987, in Sequences of Proteins of
Immunological Interest, US Department of Health and Human Services,
NIH, USA (hereafter "Kabat et al. (supra)"). This numbering system
is used in the present specification except where otherwise
indicated.
[0362] The Kabat residue designations do not always correspond
directly with the linear numbering of the amino acid residues. The
actual linear amino acid sequence may contain fewer or additional
amino acids than in the strict Kabat numbering corresponding to a
shortening of, or insertion into, a structural component, whether
framework or complementarity determining region (CDR), of the basic
variable domain structure. The correct Kabat numbering of residues
may be determined for a given antibody by alignment of residues of
homology in the sequence of the antibody with a "standard" Kabat
numbered sequence.
[0363] The CDRs of the heavy chain variable domain are located at
residues 31-35 (CDR-H1), residues 50-65 (CDR-H2) and residues
95-102 (CDR-H3) according to the Kabat numbering system. However,
according to Chothia (Chothia, C. and Lesk, A. M. J. Mol. Biol.,
196, 901-917 (1987)), the loop equivalent to CDR-H1 extends from
residue 26 to residue 32. Thus unless indicated otherwise `CDR-H1`
as employed herein is intended to refer to residues 26 to 35, as
described by a combination of the Kabat numbering system and
Chothia's topological loop definition.
[0364] The CDRs of the light chain variable domain are located at
residues 24-34 (CDR-L1), residues 50-56 (CDR-L2) and residues 89-97
(CDR-L3) according to the Kabat numbering system.
[0365] In one example there is provided a binding domain comprising
a heavy chain variable region (VH), specific for CD79 which
comprises three CDRs, wherein CDR H1 has the sequence given in SEQ
ID NO: 78, CDR H2 has the sequence given in SEQ ID NO: 79, and CDR
H3 has the sequence given in SEQ ID NO: 80.
[0366] In one embodiment there is provided a binding domain
comprising a heavy chain variable region (VH), specific for CD79
comprising 3 heavy chain CDRs SEQ ID NO: 88 for CDRH1, SEQ ID NO:
89 for CDRH2 and SEQ ID NO: 90 for CDRH3.
[0367] In one embodiment there is provided the binding domain
comprising a light chain variable region specific for CD79
comprising 3 light chain CDRs SEQ ID NO: 75 for CDRL1, SEQ ID NO:
76 for CDRL2 and SEQ ID NO: 77 for CDRL3.
[0368] In one embodiment there is provided binding domain
comprising a light chain variable region specific for CD79
comprising 3 light chain CDRs SEQ ID NO: 85 for CDRL1, SEQ ID NO:
86 for CDRL2 and SEQ ID NO: 87 for CDRL3.
[0369] In one example there is provided a binding domain comprising
a heavy chain variable region (VH), specific for CD79 which
comprises three CDRs, wherein CDR H1 has the sequence given in SEQ
ID NO: 78, CDR H2 has the sequence given in SEQ ID NO: 79, and CDR
H3 has the sequence given in SEQ ID NO: 80 and a light chain
variable region (VL) which comprises three CDRs, wherein CDR L1 has
the sequence given in SEQ ID NO: 75, CDR L2 has the sequence given
in SEQ ID NO: 76 and CDR L3 has the sequence given in SEQ ID NO:
77.
[0370] In one example there is provided a binding domain comprising
a heavy chain variable region (VH), specific for CD79 which
comprises three CDRs, wherein CDR H1 has the sequence given in SEQ
ID NO: 88, CDR H2 has the sequence given in SEQ ID NO: 89, and CDR
H3 has the sequence given in SEQ ID NO: 90 and a light chain
variable region (VL) which comprises three CDRs, wherein CDR L1 has
the sequence given in SEQ ID NO: 85, CDR L2 has the sequence given
in SEQ ID NO: 86 and CDR L3 has the sequence given in SEQ ID NO:
87.
[0371] In one embodiment a multispecific molecule according to the
present disclosure comprises a binding domain specific to CD45
which comprises 3 heavy chain CDRS selected from the group
comprising SEQ ID NO: 98, 99, 100, 108, 109, 110, 118, 119, 120,
128, 129 and 130, In one embodiment a multispecific molecule
according to the present disclosure comprises a binding domain
specific to CD45 which comprises 3 light chain CDRS selected from
the group comprising SEQ ID NO: 95, 97, 97, 105, 106, 107, 115,
116, 117, 125, 126 and 127.
[0372] In one embodiment a multispecific molecule according to the
present disclosure comprises a binding domain specific to CD45
which comprises 3 heavy chain CDRS selected from the group
comprising SEQ ID NO: 98, 99, 100, 108, 109, 110, 118, 119, 120,
128, 129, 130, and 3 light chain CDRS selected from the group
comprising SEQ ID NO: 95, 97, 97, 105, 106, 107, 115, 116, 117,
125, 126 and 127.
[0373] In one embodiment there is provided a binding domain
comprising a heavy chain variable region (VH), specific for CD45
comprising 3 heavy chain CDRs SEQ ID NO: 98 for CDRH1, SEQ ID NO:
99 for CDRH2 and SEQ ID NO: 100 for CDRH3.
[0374] In one embodiment there is provided a binding domain
comprising a heavy chain variable region (VH), specific for CD45
comprising 3 heavy chain CDRs SEQ ID NO: 108 for CDRH1, SEQ ID NO:
109 for CDRH2 and SEQ ID NO: 110 for CDRH3.
[0375] In one embodiment there is provided a binding domain
comprising a heavy chain variable region (VH), specific for CD45
comprising 3 heavy chain CDRs SEQ ID NO: 118 for CDRH1, SEQ ID NO:
119 for CDRH2 and SEQ ID NO: 120 for CDRH3.
[0376] In one embodiment there is provided a binding domain
comprising a heavy chain variable region (VH), specific for CD45
comprising 3 heavy chain CDRs SEQ ID NO: 128 for CDRH1, SEQ ID NO:
129 for CDRH2 and SEQ ID NO: 130 for CDRH3.
[0377] In one embodiment there is provided a binding domain
comprising a light chain variable region specific for CD45
comprising 3 light chain CDRs SEQ ID NO: 95 for CDRL1, SEQ ID NO:
96 for CDRL2 and SEQ ID NO: 97 for CDRL3.
[0378] In one embodiment there is provided binding domain
comprising a light chain variable region specific for CD45
comprising 3 light chain CDRs SEQ ID NO: 105 for CDRL1, SEQ ID NO:
106 for CDRL2 and SEQ ID NO: 107 for CDRL3.
[0379] In one embodiment there is provided binding domain
comprising a light chain variable region specific for CD45
comprising 3 light chain CDRs SEQ ID NO: 115 for CDRL1, SEQ ID NO:
116 for CDRL2 and SEQ ID NO: 117 for CDRL3.
[0380] In one embodiment there is provided binding domain
comprising a light chain variable region specific for CD45
comprising 3 light chain CDRs SEQ ID NO: 125 for CDRL1, SEQ ID NO:
126 for CDRL2 and SEQ ID NO: 127 for CDRL3.
[0381] In one example there is provided a binding domain specific
to CD45 comprising a heavy chain variable region (VH), which
comprises three CDRs, wherein CDR H1 has the sequence given in SEQ
ID NO: 98, CDR H2 has the sequence given in SEQ ID NO: 99, and CDR
H3 has the sequence given in SEQ ID NO: 100 and a light chain
variable region (VL) which comprises three CDRs, wherein CDR L1 has
the sequence given in SEQ ID NO: 95, CDR L2 has the sequence given
in SEQ ID NO: 96 and CDR L3 has the sequence given in SEQ ID NO:
97.
[0382] In one example there is provided a binding domain specific
to CD45 comprising a heavy chain variable region (VH), which
comprises three CDRs, wherein CDR H1 has the sequence given in SEQ
ID NO: 108, CDR H2 has the sequence given in SEQ ID NO: 109, and
CDR H3 has the sequence given in SEQ ID NO: 110 and a light chain
variable region (VL) which comprises three CDRs, wherein CDR L1 has
the sequence given in SEQ ID NO: 105, CDR L2 has the sequence given
in SEQ ID NO: 106 and CDR L3 has the sequence given in SEQ ID NO:
107. In one example there is provided a binding domain specific to
CD45 comprising a heavy chain variable region (VH), which comprises
three CDRs, wherein CDR H1 has the sequence given in SEQ ID NO:
118, CDR H2 has the sequence given in SEQ ID NO: 119, and CDR H3
has the sequence given in SEQ ID NO: 120 and a light chain variable
region (VL) which comprises three CDRs, wherein CDR L1 has the
sequence given in SEQ ID NO: 115, CDR L2 has the sequence given in
SEQ ID NO: 116 and CDR L3 has the sequence given in SEQ ID NO: 117.
In one example there is provided a binding domain specific to CD45
comprising a heavy chain variable region (VH), which comprises
three CDRs, wherein CDR H1 has the sequence given in SEQ ID NO:
128, CDR H2 has the sequence given in SEQ ID NO: 129, and CDR H3
has the sequence given in SEQ ID NO: 130 and a light chain variable
region (VL) which comprises three CDRs, wherein CDR L1 has the
sequence given in SEQ ID NO: 125, CDR L2 has the sequence given in
SEQ ID NO: 126 and CDR L3 has the sequence given in SEQ ID NO: 127.
In one example the present invention provides a multispecific
molecule comprising a binding domain specific to the antigen CD79
and a binding domain specific to the antigen CD45 wherein these
pairs of binding domains each comprise 6 CDRs from a pair of CD79
and CD45 antibodies, for example selected from the following pairs
antibodies; 4447 and 4122, 4447 and 4129, 4447 and 4131, 4447 and
4133, 4450 and 4122, 4450 and 4129, 4450 and 4131 and 4447 and
4133.
[0383] The sequences of these CD79 antibodies (antibody 4447 and
antibody 4450), including VH, VL and CDR sequences are provided
herein below. The sequences of these CD45 antibodies (antibodies
4122, 4129, 4131 and 4133) including VH, VL and CDR sequences are
provided herein below, and may be combined as binding domains in
molecules of the present invention. In one embodiment the
disclosure extends to an antibody sequence disclosed herein.
[0384] In one example there is provided a binding domain specific
to albumin comprising a heavy chain variable region (VH), which
comprises three CDRs, wherein CDR H1 has the sequence given in SEQ
ID NO: 131, CDR H2 has the sequence given in SEQ ID NO: 132, and
CDR H3 has the sequence given in SEQ ID NO: 133 and a light chain
variable region (VL) which comprises three CDRs, wherein CDR L1 has
the sequence given in SEQ ID NO: 134, CDR L2 has the sequence given
in SEQ ID NO: 135 and CDR L3 has the sequence given in SEQ ID NO:
136.
[0385] In one example there is provided a binding domain specific
to albumin comprising a heavy chain variable region (VH) having the
sequence given in SEQ ID NO:137 and a light chain variable region
(VL) having the sequence given in SEQ ID NO:139.
[0386] In one example there is provided a binding domain specific
to albumin comprising a heavy chain variable region (VH) having the
sequence given in SEQ ID NO:138 and a light chain variable region
(VL) having the sequence given in SEQ ID NO:140.
[0387] In one example the binding domains are humanised.
[0388] In one example one or more CDRs provided herein may be
modified to remove undesirable residues or sites, such as cysteine
residues or aspartic acid (D) isomerisation sites or asparagine (N)
deamidation sites. In one example an Asparagine deamidation site
may be removed from one or more CDRs by mutating the asparagine
residue (N) and/or a neighbouring residue to any other suitable
amino acid. In one example an asparagine deamidation site such as
NG or NS may be mutated, for example to NA or NT.
[0389] In one example an Aspartic acid isomerisation site may be
removed from one or more CDRs by mutating the aspartic acid residue
(D) and/or a neighbouring residue to any other suitable amino acid.
In one example an aspartic acid isomerisation site such as DG or DS
may be mutated, for example to EG, DA or DT.
[0390] For example one or more cysteine residues in any one of the
CDRs may be substituted with another amino acid, such as
serine.
[0391] In one example an N-glycosylation site such as NLS may be
removed by mutating the asparagine residue (N) to any other
suitable amino acid, for example to SLS or QLS. In one example an
N-glycosylation site such as NLS may be removed by mutating the
serine residue (S) to any other residue with the exception of
threonine (T).
[0392] The skilled person is able to test variants of CDRs or
humanised sequences in any suitable assay such as those described
herein to confirm activity is maintained.
[0393] Specific binding to antigen may be tested using any suitable
assay including for example ELISA or surface plasmon resonance
methods such as BIACORE where binding to antigen (CD45 and/or CD79)
may be measured. Such assays may use isolated natural or
recombinant CD45 or CD79 (a or b) or a suitable fusion
protein/polypeptide. In one example binding is measured using
recombinant CD45 (SEQ ID NO: 143 or amino acids 23-1304 of SEQ ID
NO:143) or CD79 such as the sequence provided in SEQ ID NO:141 and
SEQ ID NO:142 and amino acids 33-226 of SEQ ID NO:141 and amino
acids 29-229 of SEQ ID NO:142) by for example surface plasmon
resonance, such as BIACORE. Alternatively the proteins may be
expressed on a cell, such as a HEK cell and affinity measured
employing a flow cytometry based affinity determination.
[0394] The antibody sequences provided by the present invention may
be used to identify further antibodies and hence binding domains
suitable for use in the multispecific molecules of the present
invention. Antibodies which cross-block the binding of an antibody
molecule according to the present invention to CD79 in particular,
an antibody molecule comprising the heavy chain sequence given in
SEQ ID NO:73 and the light chain sequence given in SEQ ID NO:71 or
an antibody molecule comprising the heavy chain sequence given in
SEQ ID NO:83 and the light chain sequence given in SEQ ID NO:81 may
be similarly useful in binding CD79 and therefore similarly useful
in the multispecific molecules of the present invention.
Accordingly, the present invention also provides a multi-specific
molecule comprising a binding domain specific to the antigen CD45
and a binding domain specific to the antigen CD79b wherein the
binding domain for CD79b cross-blocks the binding of any one of the
antibody molecules described herein above to CD79 and/or is
cross-blocked from binding CD79 by any one of those antibodies. In
one embodiment, such an antibody binds to the same epitope as an
antibody described herein above.
[0395] Similarly antibodies which cross-block the binding of an
antibody molecule according to the present invention to CD45, in
particular, an antibody molecule comprising the heavy chain
sequence given in SEQ ID NO:93 and the light chain sequence given
in SEQ ID NO:91 or an antibody molecule comprising the heavy chain
sequence given in SEQ ID NO:103 and the light chain sequence given
in SEQ ID NO:101, or an antibody molecule comprising the heavy
chain sequence given in SEQ ID NO:113 and the light chain sequence
given in SEQ ID NO:111, or an antibody molecule comprising the
heavy chain sequence given in SEQ ID NO:123 and the light chain
sequence given in SEQ ID NO:121, or an antibody molecule comprising
the heavy chain sequence given in SEQ ID NO:133 and the light chain
sequence given in SEQ ID NO:131 may be similarly useful in binding
CD45 and therefore similarly useful in the multispecific molecules
of the present invention. Accordingly, the present invention also
provides a multi-specific molecule comprising a binding domain
specific to the antigen CD45 and a binding domain specific to the
antigen CD79 wherein the binding domain for CD45 cross-blocks the
binding of any one of the antibody molecules described herein above
to CD45 and/or is cross-blocked from binding CD45 by any one of
those antibodies. In one embodiment, such an antibody binds to the
same epitope as an antibody described herein above. In another
embodiment the cross-blocking antibody binds to an epitope which
borders and/or overlaps with the epitope bound by an antibody
described herein above.
[0396] In another embodiment the cross-blocking neutralising
antibody binds to an epitope which borders and/or overlaps with the
epitope bound by an antibody described herein above. Cross-blocking
antibodies can be identified using any suitable method in the art,
for example by using competition ELISA or BIACORE assays where
binding of the cross blocking antibody to antigen (CD45 and/or
CD79) prevents the binding of an antibody of the present invention
or vice versa. Such cross blocking assays may use isolated natural
or recombinant CD45 or CD79 (a and/or b) or a suitable fusion
protein/polypeptide. In one example binding and cross-blocking is
measured using recombinant CD45 (SEQ ID NO: 143) or CD79.
[0397] Alternatively or in addition, the antibodies according to
this aspect of the invention may be cross-blocked from binding to
antigen (CD45 or CD79) by an a binding domain disclosed herein, for
example comprising CDRs derived from the the heavy chain variable
sequence given in and the light chain sequence given in SEQ ID
NO:71 and 73, 81 and 83, 91 and 93, 101 and 103, 111 and 113, and
121 and 123. Also provided therefore is a multi-specific molecule
comprising a binding domain specific to the antigen CD45 and a
binding domain specific to the antigen CD79b wherein the binding
domain for CD79b cross-blocks the binding of any one of the
antibody molecules described herein above to CD79b and/or is
cross-blocked from binding CD79 by any one of those antibodies by
greater than 80%, for example by greater than 85%, such as by
greater than 90%, in particular by greater than 95% and optionally
wherein the binding domain for CD45 cross-blocks the binding of any
one of the antibody molecules described herein above to CD45 and/or
is cross-blocked from binding CD45 by any one of those antibodies
by greater than 80%, for example by greater than 85%, such as by
greater than 90%, in particular by greater than 95%.
[0398] In one aspect, there is provided a multi-specific antibody
molecule comprising or consisting of: [0399] a) a polypeptide chain
of formula (I):
[0399] V.sub.H--CH.sub.1--X--(V.sub.1).sub.p;
[0400] b) a polypeptide chain of formula (II):
V.sub.L--C.sub.L--Y--(V.sub.2).sub.q; [0401] wherein: [0402]
V.sub.H represents a heavy chain variable domain; [0403] CH.sub.1
represents a domain of a heavy chain constant region, for example
domain 1 thereof; [0404] X represents a bond or linker, for example
an amino acid linker; [0405] Y represents a bond or linker, for
example an amino acid linker; [0406] V.sub.1 represents a dab,
scFv, dsscFv or dsFv; [0407] V.sub.L represents a variable domain,
for example a light chain variable domain; [0408] C.sub.L
represents a domain from a constant region, for example a light
chain constant region domain, such as Ckappa; [0409] V.sub.2
represents a dab, scFv, dsscFv or dsFv; [0410] p is 0 or 1; [0411]
q is 0 or 1; and when p is 1 q is 0 or 1 and when q is 1 p is 0 or
1 i.e. p and q do not both represent 0
[0412] In one embodiment the multispecific antibody molecule
comprises no more than one binding domain for CD45 and no more than
one binding domain for CD79
[0413] In one embodiment q is 0 and p is 1.
[0414] In one embodiment q is 1 and p is 1.
[0415] In one embodiment V.sub.1 is a dab and V.sub.2 is a dab and
together they form a single binding domain of a co-operative pair
of variable regions, such as a cognate VH/VL pair, which are
optionally linked by a disulphide bond.
[0416] In one embodiment V.sub.H and V.sub.L are specific to, CD79,
for example CD79a or CD79b.
[0417] In one embodiment the V.sub.1 is specific to, CD79, for
example CD79a or CD79b.
[0418] In one embodiment the V.sub.2 is specific to, CD79, for
example CD79a or CD79b.
[0419] In one embodiment the V.sub.1 and V.sub.2 together (eg as
binding domain) are specific to, CD79, for example CD79a or CD79b
and V.sub.H and V.sub.L are specific to, CD45.
[0420] In one embodiment the V.sub.1 is specific to, CD45.
[0421] In one embodiment the V.sub.2 is specific to, CD45.
[0422] In one embodiment the V.sub.1 and V.sub.2 together (eg as
one binding domain) are specific to, CD45 and V.sub.H and V.sub.L
are specific to CD79.
[0423] In one embodiment the V.sub.1 is specific to CD45, V.sub.2
is specific to albumin and V.sub.H and V.sub.L are specific to
CD79.
[0424] In one embodiment the V.sub.1 is specific to albumin,
V.sub.2 is specific to CD45 and V.sub.H and V.sub.L are specific to
CD79.
[0425] In one embodiment the V.sub.1 is specific to CD79, V.sub.2
is specific to albumin and V.sub.H and V.sub.L are specific to
CD45.
[0426] In one embodiment the V.sub.1 is specific to albumin,
V.sub.2 is specific to CD79 and V.sub.H and V.sub.L are specific to
CD45.
[0427] In one embodiment the V.sub.1 is a dsscFv specific to CD45,
V.sub.2 is a dsscFv specific to albumin and V.sub.H and V.sub.L are
specific to CD79.
[0428] In one embodiment the V.sub.1 is a dsscFv specific to
albumin, V.sub.2 is a dscFv specific to CD45 and V.sub.H and
V.sub.L are specific to CD79.
[0429] In one embodiment the V.sub.1 is a dsscFv specific to CD79,
V.sub.2 is a dsscFv specific to albumin and V.sub.H and V.sub.L are
specific to CD45.
[0430] In one embodiment the V.sub.1 is a dsscFv specific to
albumin, V.sub.2 is a dsscFv specific to CD79 and V.sub.H and
V.sub.L are specific to CD45.
[0431] V1, V2, VH and VL in the constructs above may each represent
a binding domain and incorporate any of the sequences provided
herein.
[0432] X and Y represent any suitable linker, for example X and Y
may be SGGGGSGGGGS (SEQ ID NO:17).
[0433] In one embodiment, when V.sub.1 and/or V.sub.2 are a dab,
dsFv or a dsscFv, the disulfide bond between the variable domains
V.sub.H and V.sub.L of V.sub.1 and/or V.sub.2 is formed between
positions V.sub.H44 and V.sub.L100.
[0434] The present disclosure also extends to novel polypeptide
sequences disclosed herein and sequences at least 80% similar or
identical thereto, for example 85% or greater, such 90% or greater,
in particular by 95% or greater similarity or identity.
[0435] "Identity", as used herein, indicates that at any particular
position in the aligned sequences, the amino acid residue is
identical between the sequences. "Similarity", as used herein,
indicates that, at any particular position in the aligned
sequences, the amino acid residue is of a similar type between the
sequences. For example, leucine may be substituted for isoleucine
or valine.
[0436] Other amino acids which can often be substituted for one
another include but are not limited to: [0437] phenylalanine,
tyrosine and tryptophan (amino acids having aromatic side chains);
[0438] lysine, arginine and histidine (amino acids having basic
side chains); [0439] aspartate and glutamate (amino acids having
acidic side chains); [0440] asparagine and glutamine (amino acids
having amide side chains); and [0441] cysteine and methionine
(amino acids having sulphur-containing side chains).
[0442] Degrees of identity and similarity can be readily calculated
(Computational Molecular Biology, Lesk, A. M., ed., Oxford
University Press, New York, 1988; Biocomputing. Informatics and
Genome Projects, Smith, D. W., ed., Academic Press, New York, 1993;
Computer Analysis of Sequence Data, Part 1, Griffin, A. M., and
Griffin, H. G., eds., Humana Press, New Jersey, 1994; Sequence
Analysis in Molecular Biology, von Heinje, G., Academic Press,
1987, Sequence Analysis Primer, Gribskov, M. and Devereux, J.,
eds., M Stockton Press, New York, 1991, the BLAST software
available from NCBI (Altschul, S. F. et al., 1990, J. Mol. Biol.
215:403-410; Gish, W. & States, D. J. 1993, Nature Genet.
3:266-272. Madden, T. L. et al., 1996, Meth. Enzymol. 266:131-141;
Altschul, S. F. et al., 1997, Nucleic Acids Res. 25:3389-3402;
Zhang, J. & Madden, T. L. 1997, Genome Res. 7:649-656,).
[0443] In particular in one aspect the present inventon provides
the CD45 and CD79 antibodies described herein in any suitable
antibody format.
[0444] Accordingly in one aspect the present invention provides
anti-CD45 antibodies or fragments thereof containing one or more of
the binding domains described herein above comprising the CDRs
provided herein and in SEQ ID NOS 95, 96, 97, 98, 99 and 100
(antibody 4122) or 105, 106, 107, 108, 109 and 110 (antibody 4129)
or 115, 116, 117, 118, 119 and 120 (antibody 4131) or 125, 126,
127, 128, 129 and 130 (antibody 4133). Also provided are anti-CD79
antibodies or fragments thereof containing one or more of the
binding domains described herein above comprising the CDRs provided
herein and in SEQ ID NOS 75, 76, 77, 78, 79 and 80 (antibody 4447)
or SEQ ID NOs 85, 86, 87, 88, 89 and 90 (antibody 4450).
[0445] Said CDRs may be incorporated into any suitable antibody
framework and into any suitable antibody format. Such antibodies
include whole antibodies and functionally active fragments or
derivatives thereof which may be, but are not limited to,
monoclonal, humanised, fully human or chimeric antibodies.
Accordingly, such antibodies may comprise a complete antibody
molecule having full length heavy and light chains or a fragment
thereof and may be, but are not limited to Fab, modified Fab, Fab',
F(ab').sub.2, Fv, single domain antibodies, scFv, bi, tri or
tetra-valent antibodies, Bis-scFv, diabodies, triabodies,
tetrabodies and epitope-binding fragments of any of the above (see
for example Holliger and Hudson, 2005, Nature Biotech.
23(9):1126-1136; Adair and Lawson, 2005, Drug Design
Reviews--Online 2(3), 209-217). The methods for creating and
manufacturing these antibody fragments are well known in the art
(see for example Verma et al., 1998, Journal of Immunological
Methods, 216, 165-181). Multi-valent antibodies may comprise
multiple specificities or may be monospecific (see for example WO
92/22853 and WO05/113605). It will be appreciated that this aspect
of the invention also extends to variants of these anti-CD45 and
CD79 antibodies including humanised versions and modified versions,
including those in which amino acids have been mutated in the CDRs
to remove one or more isomerisation, deamidation, glycosylation
site or cysteine residue as described herein above.
Linkers
[0446] The teaching herein of linkers in one context can equally be
applied to linkers in different contexts where a linker is
employed, such as in any multispecific molecule of the present
invention.
[0447] In one embodiment, the linker employed in a molecule of the
disclosure is an amino acid linker 50 residues or less in length,
for example selected from a sequence shown in sequence 5 to 70.
TABLE-US-00001 TABLE 1 Hinge linker sequences SEQ ID NO: SEQUENCE 5
DKTHTCAA 6 DKTHTCPPCPA 7 DKTHTCPPCPATCPPCPA 8
DKTHTCPPCPATCPPCPATCPPCPA 9 DKTHTCPPCPAGKPTLYNSLVMSDTAGTCY 10
DKTHTCPPCPAGKPTHVNVSVVMAEVDGTCY 11 DKTHTCCVECPPCPA 12
DKTHTCPRCPEPKSCDTPPPCPRCPA 13 DKTHTCPSCPA
TABLE-US-00002 TABLE 2 Flexible linker sequences SEQ ID NO:
SEQUENCE 14 SGGGGSE 15 DKTHTS 16 (S)GGGGS 17 (S)GGGGSGGGGS 18
(S)GGGGSGGGGSGGGGS 19 (S)GGGGSGGGGSGGGGSGGGGS 20
(S)GGGGSGGGGSGGGGSGGGGSGGGGS 21 AAAGSG-GASAS 22 AAAGSG-XGGGS-GASAS
23 AAAGSG-XGGGSXGGGS-GASAS 24 AAAGSG-XGGGSXGGGSXGGGS-GASAS 25
AAAGSG-XGGGSXGGGSXGGGSXGGGS-GASAS 26 AAAGSG-XS-GASAS 27
PGGNRGTTTTRRPATTTGSSPGPTQSHY 28 ATTTGSSPGPT 29 ATTTGS 30 GS 31
EPSGPISTINSPPSKESHKSP 32 GTVAAPSVFIFPPSD 33 GGGGIAPSMVGGGGS 34
GGGGKVEGAGGGGGS 35 GGGGSMKSHDGGGGS 36 GGGGNLITIVGGGGS 37
GGGGVVPSLPGGGGS 38 GGEKSIPGGGGS 39 RPLSYRPPFPFGFPSVRP 40
YPRSIYIRRRHPSPSLTT 41 TPSHLSHILPSFGLPTFN 42 RPVSPFTFPRLSNSWLPA 43
SPAAHFPRSIPRPGPIRT 44 APGPSAPSHRSLPSRAFG 45 PRNSIHFLHPLLVAPLGA 46
MPSLSGVLQVRYLSPPDL 47 SPQYPSPLTLTLPPHPSL 48 NPSLNPPSYLHRAPSRIS 49
LPWRTSLLPSLPLRRRP 50 PPLFAKGPVGLLSRSFPP 51 VPPAPVVSLRSAHARPPY 52
LRPTPPRVRSYTCCPTP- 53 PNVAHVLPLLTVPWDNLR 54 CNPLLPLCARSPAVRTFP
[0448] (S) is optional in sequences 17 to 20.
[0449] Examples of rigid linkers include the peptide sequences
GAPAPAAPAPA (SEQ ID NO:69), PPPP (SEQ ID NO:70) and PPP.
[0450] Other linkers are shown in Table 3:
TABLE-US-00003 SEQ ID NO: SEQUENCE 55 DLCLRDWGCLW 56 DICLPRWGCLW 57
MEDICLPRWGCLWGD 58 QRLMEDICLPRWGCLWEDDE 59 QGLIGDICLPRWGCLWGRSV 60
QGLIGDICLPRWGCLWGRSVK 61 EDICLPRWGCLWEDD 62 RLMEDICLPRWGCLWEDD 63
MEDICLPRWGCLWEDD 64 MEDICLPRWGCLWED 65 RLMEDICLARWGCLWEDD 66
EVRSFCTRWPAEKSCKPLRG 67 RAPESFVCYWETICFERSEQ 68 EMCYFPGICWM
Effector Molecules
[0451] If desired a multispecific molecule for use in the present
invention may be conjugated to one or more effector molecule(s). It
will be appreciated that the effector molecule may comprise a
single effector molecule or two or more such molecules so linked as
to form a single moiety that can be attached to the multispecific
molecules of the present invention. Where it is desired to obtain
an antibody or multispecific molecule according to the present
disclosure linked to an effector molecule, this may be prepared by
standard chemical or recombinant DNA procedures in which the
antibody fragment is linked either directly or via a coupling agent
to the effector molecule. Techniques for conjugating such effector
molecules to antibodies are well known in the art (see, Hellstrom
et al., Controlled Drug Delivery, 2nd Ed., Robinson et al., eds.,
1987, pp. 623-53; Thorpe et al., 1982, Immunol. Rev., 62:119-58 and
Dubowchik et al., 1999, Pharmacology and Therapeutics, 83, 67-123).
Particular chemical procedures include, for example, those
described in WO 93/06231, WO 92/22583, WO 89/00195, WO 89/01476 and
WO 03/031581. Alternatively, where the effector molecule is a
protein or polypeptide the linkage may be achieved using
recombinant DNA procedures, for example as described in WO 86/01533
and EP0392745.
[0452] In one embodiment the multispecific molecules of the present
disclosure may comprise an effector molecule.
[0453] The term effector molecule as used herein includes, for
example, antineoplastic agents, drugs, toxins, biologically active
proteins, for example enzymes, other antibody or antibody
fragments, synthetic or naturally occurring polymers, nucleic acids
and fragments thereof e.g. DNA, RNA and fragments thereof,
radionuclides, particularly radioiodide, radioisotopes, chelated
metals, nanoparticles and reporter groups such as fluorescent
compounds or compounds which may be detected by NMR or ESR
spectroscopy.
[0454] Examples of effector molecules may include cytotoxins or
cytotoxic agents including any agent that is detrimental to (e.g.
kills) cells. Examples include combrestatins, dolastatins,
epothilones, staurosporin, maytansinoids, spongistatins, rhizoxin,
halichondrins, roridins, hemiasterlins, taxol, cytochalasin B,
gramicidin D, ethidium bromide, emetine, mitomycin, etoposide,
tenoposide, vincristine, vinblastine, colchicin, doxorubicin,
daunorubicin, dihydroxy anthracin dione, mitoxantrone, mithramycin,
actinomycin D, 1-dehydrotestosterone, glucocorticoids, procaine,
tetracaine, lidocaine, propranolol, and puromycin and analogs or
homologs thereof.
[0455] Effector molecules also include, but are not limited to,
antimetabolites (e.g. methotrexate, 6-mercaptopurine,
6-thioguanine, cytarabine, 5-fluorouracil decarbazine), alkylating
agents (e.g. mechlorethamine, thioepa chlorambucil, melphalan,
carmustine (BSNU) and lomustine (CCNU), cyclothosphamide, busulfan,
dibromomannitol, streptozotocin, mitomycin C, and
cis-dichlorodiamine platinum (II) (DDP) cisplatin), anthracyclines
(e.g. daunorubicin (formerly daunomycin) and doxorubicin),
antibiotics (e.g. dactinomycin (formerly actinomycin), bleomycin,
mithramycin, anthramycin (AMC), calicheamicins or duocarmycins),
and anti-mitotic agents (e.g. vincristine and vinblastine).
[0456] Other effector molecules may include chelated radionuclides
such as .sup.111In and .sup.90Y, Lu.sup.177, Bismuth.sup.213,
Californium.sup.252, Iridium.sup.192 and
Tungsten.sup.188/Rhenium.sup.188; or drugs such as but not limited
to, alkylphosphocholines, topoisomerase I inhibitors, taxoids and
suramin.
[0457] Other effector molecules include proteins, peptides and
enzymes. Enzymes of interest include, but are not limited to,
proteolytic enzymes, hydrolases, lyases, isomerases, transferases.
Proteins, polypeptides and peptides of interest include, but are
not limited to, immunoglobulins, toxins such as abrin, ricin A,
pseudomonas exotoxin, or diphtheria toxin, a protein such as
insulin, tumour necrosis factor, .alpha.-interferon,
.beta.-interferon, nerve growth factor, platelet derived growth
factor or tissue plasminogen activator, a thrombotic agent or an
anti-angiogenic agent, e.g. angiostatin or endostatin, or, a
biological response modifier such as a lymphokine, interleukin-1
(IL-1), interleukin-2 (IL-2), granulocyte macrophage colony
stimulating factor (GM-CSF), granulocyte colony stimulating factor
(G-CSF), nerve growth factor (NGF) or other growth factor and
immunoglobulins.
[0458] Other effector molecules may include detectable substances
useful for example in diagnosis. Examples of detectable substances
include various enzymes, prosthetic groups, fluorescent materials,
luminescent materials, bioluminescent materials, radioactive
nuclides, positron emitting metals (for use in positron emission
tomography), and nonradioactive paramagnetic metal ions. See
generally U.S. Pat. No. 4,741,900 for metal ions which can be
conjugated to antibodies for use as diagnostics. Suitable enzymes
include horseradish peroxidase, alkaline phosphatase,
beta-galactosidase, or acetylcholinesterase; suitable prosthetic
groups include streptavidin, avidin and biotin; suitable
fluorescent materials include umbelliferone, fluorescein,
fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine
fluorescein, dansyl chloride and phycoerythrin; suitable
luminescent materials include luminol; suitable bioluminescent
materials include luciferase, luciferin, and aequorin; and suitable
radioactive nuclides include .sup.125I, .sup.131I, .sup.111In and
.sup.99Tc.
[0459] In another example the effector molecule may increase the
half-life of the antibody in vivo, and/or reduce immunogenicity of
the antibody and/or enhance the delivery of an antibody across an
epithelial barrier to the immune system. Examples of suitable
effector molecules of this type include polymers, albumin, albumin
binding proteins or albumin binding compounds such as those
described in WO05/117984.
[0460] Where the effector molecule is a polymer it may, in general,
be a synthetic or a naturally occurring polymer, for example an
optionally substituted straight or branched chain polyalkylene,
polyalkenylene or polyoxyalkylene polymer or a branched or
unbranched polysaccharide, e.g. a homo- or
hetero-polysaccharide.
[0461] Specific optional substituents which may be present on the
above-mentioned synthetic polymers include one or more hydroxy,
methyl or methoxy groups.
[0462] Specific examples of synthetic polymers include optionally
substituted straight or branched chain poly(ethyleneglycol),
poly(propyleneglycol) poly(vinylalcohol) or derivatives thereof,
especially optionally substituted poly(ethyleneglycol) such as
methoxypoly(ethyleneglycol) or derivatives thereof.
Functional Assays and Screening Formats
[0463] Typically suitable binding domains for use in the present
invention can be identified by testing one or more binding domain
pairs in a functional assay. For example a multi specific molecule
comprising a binding domain specific to the antigen CD45 and a
binding domain specific to the antigen CD79a and/or CD79b may be
tested in one or more functional assays.
[0464] A "functional assay," as used herein, is an assay that can
be used to determine one or more desired properties or activities
of the protein complexes, antibody complexes or the mixture of
antibodies subject to the assay conditions. Suitable functional
assays may be binding assays, apoptosis assays, antibody-dependent
cellular cytotoxicity (ADCC) assays, complement-dependent
cytotoxicity (CDC) assays, inhibition of cell growth or
proliferation (cytostatic effect) assays, cell-killing (cytotoxic
effect) assays, cell-signaling assays, cytokine production assays,
antibody production and isotype switching, and cellular
differentiation assays.
[0465] The efficacy of multispecific antibodies according to the
present disclosure can be compared to individual antibodies or
mixtures of antibodies (or fragments) in such models by methods
generally known to one of ordinary skill in the art.
[0466] The functional assays may be repeated a number of times as
necessary to enhance the reliability of the results. Various
statistical tests known to the skilled person can be employed to
identify statistically significant results and thus identify
multispecific molecules with biological functions.
[0467] Examples of suitable functional assays are described in the
Examples herein and include measuring the ability of a
multispecific molecule of the present invention to inhibit B cell
activation following stimulation with anti-IgM, as measured by
detecting the inhibition of markers of B cell activation such as
phosphorylated Akt expression, phosphorylated P38 expression,
PLC.gamma. signalling, CD40 expression, CD71 expression and/or CD86
expression.
[0468] When establishing a functional assay for screening the
skilled person can set a suitable threshold over which an
identified activity is deemed a `hit`. Where more than one
functional assay is used the threshold for each assay may be set at
a suitable level to establish a manageable hit rate, in one example
the hit rate may be 3-5%, in one example the criteria set when
searching for pairs of binding domains that inhibit B cell function
may be at least 30% inhibition of at least two phospho-readouts in
a B cell activation assay.
[0469] In one example a multispecific molecule of the present
invention has an IC50 of less than 5 nM for inhibition of CD86
expression in anti-IgM stimulated B cells.
[0470] In one embodiment in vivo assays, such as animal models,
including mouse tumor models, models of auto-immune disease,
virus-infected or bacteria-infected rodent or primate models, and
the like, may be employed to test molecules of the present
disclosure.
[0471] An example of a suitable format for screening and discovery
of binding domains is described herein below.
[0472] Screening to identify binding domains for use in the present
invention may employ a bispecific protein complex.
[0473] "Bispecific protein complex" as used herein refers to a
molecule comprising two proteins (A and B referred to herein as
bispecific components) which are retained together by a
heterodimeric-tether. In one embodiment one or both of the proteins
have a binding domain, for example one or both of the proteins are
antibodies or fragments thereof.
[0474] Typically the bispecific protein complex has the formula
A-X:Y-B wherein: [0475] A-X is a first fusion protein; [0476] Y-B
is a second fusion protein; [0477] X:Y is a heterodimeric-tether;
[0478] A comprises a first binding domain; [0479] B comprises a
second binding domain; [0480] X is a first binding partner of a
binding pair; [0481] Y is a second binding partner of the binding
pair; and [0482] : is an interaction (such as a binding
interaction) between X and Y.
[0483] "Fusion proteins" as employed herein comprise a protein
component, for example A or B fused to another entity, for example
a binding partner X or Y (as appropriate). In embodiment the fusion
protein is a translational protein expressed by a recombinant
techniques from a genetic construct, for example expressed in a
host from a DNA construct.
[0484] The function of the tether X:Y is to retain the proteins A
and B in proximity to each other so that synergistic function of A
and B can be realised.
[0485] "heterodimeric-tether" as used herein refers to a tether
comprising two different binding partners X and Y which form a
interaction (such as a binding) between each other which has an
overall affinity that is sufficient to retain the two binding
partners together. In one embodiment X and/or Y are unsuitable for
forming homodimers.
[0486] Heterodimerically-tethered and heterodimeric-tether are used
interchangeably herein.
[0487] In one embodiment "unsuitable for forming homodimers" as
employed herein refers to formation of the heterodimers of X-Y are
more preferable, for example stable, such as thermodynamically
stable and/or physically stable (for example evidenced by lack of
aggregation), once formed.
[0488] In one embodiment the X-Y interaction is more favourable
than the X-X or Y-Y interaction. This reduces the formation of
homodimers X-X or Y-Y when the fusion proteins A-X and B-Y are
mixed. This also renders removal of homodimers relatively simple,
for example, one purification step, such as column chromatography
provides substantially pure fusion proteins and/or bispecific
protein complexes according to the present disclosure.
[0489] In one embodiment a purification step is provided after
expression of the fusion protein. Thus in one embodiment prior to
in vitro mixing the fusion protein(s) is/are provided in
substantially pure form. Substantially pure form as employed herein
refers to wherein the fusion protein is in the range 85 to 100%,
for example 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% monomer.
In one embodiment no purification is required after the bispecific
protein complex formation. In one embodiment the ratio of fusion
proteins employed in the in vitro mixing step of the present method
is A-X to B-Y 0.8:1 to 3:1, such as 1.5:1 or 2:1.
[0490] In one embodiment the ratio of fusion proteins employed in
the in vitro mixing step of the present method is B-Y to A-X 0.8:1
to 3:1, such as 1.5:1 or 2:1.
[0491] In one embodiment the ratio is 1:1.
[0492] In one embodiment one (or at least one) of the binding
partners is incapable of forming a homodimer, for example an amino
acid sequence of the binding partner is mutated to eliminate or
minimise the formation of homodimers.
[0493] In one embodiment both of the binding partners are incapable
of forming a homodimer, for example one of the binding partners is
a peptide and the other binding partner is a V.sub.HH specific to
said peptide.
[0494] In one embodiment an scFv employed in the molecules of the
present disclosure is incapable of forming a homodimer.
[0495] Incapable of forming homodimers as employed herein, refers
to a low or zero propensity to form homodimers. Low as employed
herein refers to 5% or less, such as 4, 3, 2, 1, 0.5% or less
aggregate.
[0496] Small amounts of aggregate in the fusion proteins or
residual in the heterodimerically-tethered bispecific protein
complex generally has minimal effect on the method of the present
disclosure.
[0497] In one embodiment: is a binding interaction, for example
based on attractive forces such as Van der Waals forces, such as
hydrogen bonding and electrostatic interactions, for example, based
on antibody specificity for an antigen, such as a peptide.
[0498] In one embodiment: is a covalent bond formed from a specific
chemical interaction, such as click chemistry.
[0499] In one embodiment: is not a covalent bond.
[0500] "Form the complex" as employed herein refers to an
interaction, including a binding interactions or a chemical
reaction, which is sufficiently specific and strong when the fusion
protein components A-X and B-Y are brought into contact under
appropriate conditions that the complex is assembled and the fusion
proteins are retained together.
[0501] "Retained together" as employed herein refers to the holding
of the components (the fusion proteins) in the proximity of each
other, such that after binding the complex can be handled as if it
were one molecule, and in many instances behaves and acts like a
single molecule. In one embodiment the retention renders the
complex suitable for use in the method disclosed herein, i.e.
suitable for use in at least one functional screen.
[0502] In one embodiment the binding interaction is reversible.
[0503] Specificity when in relation to X and Y as employed herein
refers where the binding partners X and Y in the interaction only
recognise each other or have significantly higher affinity for each
other in comparison to non-partners, for example at least 2, 3, 4,
5, 6, 7, 8, 9, 10 times higher affinity.
[0504] In one embodiment, the binding interaction between X and Y
has a low dissociation constant. Examples of a low dissociation
constant include 1-9.times.10.sup.-2s.sup.-1 or less, for example
1-9.times.10.sup.-3s.sup.-1, 1-9.times.10.sup.-4s.sup.-1,
1-9.times.10.sup.-5s.sup.-1, 1-9.times.10.sup.-6s.sup.-1 or
1-9.times.10.sup.-7s.sup.-1. Particularly suitable dissociation
constants include 1.times.10.sup.-4s.sup.-1 or less, for example
1.times.10.sup.-5s.sup.-1, 1.times.10.sup.-6s.sup.-1 or
1.times.10.sup.-7s.sup.-1.
[0505] Whilst not wishing to be bound by theory it is thought that
the low dissociation constant (also referred to as off rate) allows
the molecules to be sufficiently stable to render the bispecific
protein complex useful, in particular in functional screening
assays.
[0506] In one embodiment, the affinity of X and Y for each other is
5 nM or stronger, for example 4 nM, 3 nM, 2 nM, 1 nM or
stronger.
[0507] In one embodiment, the affinity of X and Y for each other is
900 pM or stronger, such as 800, 700, 600, 500, 400, 300, 200, 100
or 50 pM or stronger.
[0508] In another embodiment, the affinity of X and Y for each
other is 10 pM or stronger, for example 9, 8, 7, 6 or 5 pM.
[0509] Affinity is a value calculated from the on and off rate of
an interaction. The term "affinity" as used herein refers to the
strength of the sum total of non-covalent interactions between a
single binding site of a molecule (e.g. an antibody) and its
binding partner (e.g. a peptide). The affinity of a molecule for
its binding partner can generally be represented by the equilibrium
dissociation constant (K.sub.D). Affinity can be measured by common
methods known in the art, including those described herein, such as
surface plasmon resonance methods, in particular BIACORE.
[0510] In one embodiment, multiple bispecific protein complexes
according to the present disclosure are tested in parallel or
essentially simultaneously.
[0511] Simultaneously as employed herein refers to the where the
samples/molecules/complexes are analysed in the same analysis, for
example in the same "run".
[0512] In one embodiment simultaneously refers to concomitant
analysis where the signal output is analysed by the instrument at
essentially the same time. This signal may require deconvolution to
interpret the results obtained.
[0513] Advantageously, testing multiple bispecific protein
complexes allows for more efficient screening of a large number of
bispecific protein complexes and the identification of new and
interesting relationships. Clearly different variable regions to
the target antigens of interesting CD45 and CD79 can give access to
subtle nuances in biological function.
[0514] In one embodiment, the multiple bispecific protein complexes
are tested by using a multiplex as defined above and subjecting the
same to one or more functional assays.
[0515] The term "biological function" as used herein refers to an
activity that is natural to or the purpose of the biological entity
being tested, for example a natural activity of a cell, protein or
similar. Ideally the presence of the function can be tested using
an in vitro functional assay, including assays utilizing living
mammalian cells. Natural function as employed herein includes
aberrant function, such as functions associated with cancers.
[0516] A relevant "biological comparator" as employed herein refers
to a suitable entity for assessing activity, in the same assay as
that employed for the bispecific protein complex, to establish if
there is any change or novel activity or function. Suitable
comparators for A-X:Y-B may include purified protein (including
recombinant proteins) in a natural form or presented in the same
format as the bispecific i.e. where A and B are the same entity,
such as A-X:Y-A or B-X:Y-B. Alternatively the fusion protein A-X or
B-Y in an uncomplexed form may be employed as a comparator.
Alternatively, multiple comparators of different formats (in
particular as described herein) may be employed. The person skilled
in the art is able to identify and include a suitable
control/comparator based on common general knowledge or information
that is found in the literature.
[0517] The term "synergistic function" as used herein refers to
biological activity that is not observed or higher than observed
when the first and second proteins of a bispecific protein complex
of the present disclosure are not employed together, for example
activity which is only observed in a bispecific form. Therefore,
"synergistic" includes novel biological function.
[0518] The present disclosure provides a molecule with at least
specificity to CD45 and CD79 with a novel biological function.
[0519] Novel biological function as employed herein refers to
function which is not apparent or absent until the two or more
synergistic entities [protein A and protein B] are brought together
(as a bispecific or otherwise) or a previously unidentified
function.
[0520] Higher as employed herein refers to an increase in activity
including an increase from zero i.e. some activity in the
bispecific where the individual uncomplexed bispecific component or
components has/have no activity in the relevant functional assay,
also referred to herein as new activity or novel biological
function. Higher as employed herein also includes a greater than
additive function in the bispecific in a relevant functional assay
in comparison to the individual uncomplexed bispecific components
or bivalent binding domains, for example 10, 20, 30, 40, 50, 60,
70, 80, 90, 100, 200, 300% or more increase in a relevant
activity.
[0521] In one embodiment the novel synergistic function is a higher
inhibitory activity.
[0522] In one embodiment the multispecific antibody molecule of the
present invention has a higher inhibitory activity than the sum of
the activity of a bivalent binding domain to CD45 and a bivalent
binding domain to CD79a provided alone or in admixture
[0523] In one embodiment, at least one of the first binding
partner, X, and the second binding partner, Y, of the binding pair
are independently selected from a peptide and a protein; for
example the first binding partner or second binding partner is a
peptide.
[0524] Suitable peptides include the group comprising GCN4, Fos/Jun
(human and murine Fos have a Uniprot number P01100 and P01101
respectively and human and murine jun have a Uniprot number P05412
and P05627 respectively), human influenza hemagglutinin (HA),
polyhistidine (His), green fluorescent protein (GFP) and FLAG.
Other peptides are also contemplated as suitable for use in the
present disclosure and particularly suitable peptides are affinity
tags for protein purification because such peptides have a tendency
to bind with high affinity to their respective binding
partners.
[0525] The term "peptide" as used herein refers to a short polymer
of amino acids linked by peptide bonds, wherein the peptide
contains in the range of 2 to 100 amino acids, for example 5 to 99,
such as 6 to 98, 7 to 97 or 8 to 96. In one embodiment a peptide
employed in the present disclosure is an amino acid sequence of 50
amino acid residues or less, for example 40, 30, 10 or less. The
peptides used in the present disclosure are of a sufficient length
to be fit for purpose, for example if the peptide is a linker, it
needs to be suitably long to allow the fragment which it links to
perform its biological function; alternatively if the peptide is a
binding partner, it must be capable of binding specifically to
another entity such as an antibody. In one embodiment, the other
binding partner of the binding pair (the alternative first or
second binding partner) is a protein.
[0526] Protein as employed herein refers to an amino acid sequence
of 100 amino acids or more. In one embodiment a "protein" as
employed herein refers to an amino acid sequence with a secondary
or tertiary structure.
[0527] In one embodiment, the first protein, A, and/or second
protein, B, of the bispecific protein complex is an antibody or
antibody fragment. Such a bispecific protein complex may be
referred to as a bispecific antibody complex.
[0528] In one embodiment each antibody or fragment employed in the
bispecific antibody complex of the disclosure comprises one binding
site.
[0529] The full length antibody or antibody fragment employed in
the fusion proteins (A-X or B-Y) may be monospecific, multivalent
or bispecific.
[0530] Advantageously, the use of two bispecific antibody or
antibody fragments allows the molecules of the present disclosure,
such as the bispecific antibody complex described herein to
potentially be specific for up to 4 different antigens (i.e. the
complex may be tetraspecific). This allows avidity type effects to
be investigated.
[0531] In one embodiment, the antibody or antibody fragment
employed in the molecules of the present disclosure or components
thereof, such as the first fusion protein A-X is a monospecific
antibody or antibody fragment, for example a Fab, Fab', scFv or
similar, and in particular is specific to CD45.
[0532] In one embodiment, the antibody or antibody fragment
employed in the molecules of the present disclosure or components
thereof, such as the second fusion protein B-Y is a monospecific
antibody or antibody fragment, for example a Fab, Fab', scFv or
similar, and in particular is specific to CD79a and/or CD79b.
[0533] In one embodiment, the antibody or antibody fragment
employed in the molecules of the present disclosure or components
thereof, such as the second fusion protein B-Y is multivalent, that
is has two or more binding domains.
[0534] In one embodiment, the antibody or antibody fragment
employed in the molecules of the present disclosure or components
thereof, such as the first fusion protein A-X is monovalent and the
antibody or antibody fragment employed in the molecules of the
present disclosure or components thereof, such as the second fusion
protein B-X is monovalent.
[0535] Thus in one embodiment the binding domains of the
multispecific molecules of the present disclosure are
monovalent.
[0536] Thus in one embodiment the binding domains of the
multispecific molecules of the present disclosure are monovalent
and monospecific.
[0537] In one embodiment, the antibody or antibody fragment
employed in the molecules of the present disclosure or components
thereof, such as the first fusion protein A-X is monovalent and the
antibody or antibody fragment employed in the molecules of the
present disclosure or components thereof, such as the second fusion
protein B-Y is multivalent.
[0538] In one embodiment, the antibody or antibody fragment
employed in the molecules of the present disclosure or components
thereof, such as the first fusion protein A-X is multivalent and
the antibody or antibody fragment employed in the molecules of the
present disclosure or components thereof, such as the second fusion
protein B-Y is monovalent.
[0539] In one embodiment, the antibody or antibody fragment
employed in the molecules of the present disclosure or components
thereof, such as the first fusion protein A-X is multivalent and
the antibody or antibody fragment employed in the molecules of the
present disclosure or components thereof, such as the second fusion
protein B-Y is multivalent.
[0540] In one embodiment, a first antibody, a second antibody or
both the first and second antibody of a the molecules of the
present disclosure or components thereof, such as a bispecific
antibody complex may be an IgG format, for example an anti-CD45
and/or anti-CD79 antibody may be provided in an IgG format.
[0541] In one embodiment, an antibody fragment is selected from the
group consisting of: a fragment antigen (Fab) fragment, a single
chain variable fragment (scFv) and a single domain antibody (sdAb),
such as a scFv, is employed in the first (A-X) or second fusion
protein (B-Y). Advantageously, the small size of a scFv may
facilitate the correct folding of the bispecific antibody
complexes.
[0542] In one embodiment, the first (A), second antibody/fragment
(B) or both the first and second antibody/fragment of the
bispecific antibody complex of the present disclosure may be a Fab.
In one embodiment, the first, second antibody/fragment or both the
first and second antibody/fragment of the bispecific antibody
complex of the present disclosure is/are a Vim. "Fusion protein" as
employed in the context of a bispecific complex of the present
disclosure refers to a protein, for example an antibody or antibody
fragment attached to a binding partner. For convenience bispecific
protein complexes of the present disclosure are referred to herein
as A-X:Y-B. However, this nomenclature is not intended to limit how
the fusion protein A-X and B-Y are designed because our experiments
indicate that binding partners X and Y can be reversed i.e. A-Y and
B-X without adversely impacting on the method. Thus A and B and X
and Y are nominal labels referred to for assisting the explanation
of the present technology. "Attached" as employed herein refers to
connected or joined directly or indirectly via a linker, such as a
peptide linker examples of which are discussed below. Directly
connected includes fused together (for example a peptide bond) or
conjugated chemically.
[0543] "Binding partner" as employed herein refers to one component
part of a binding pair.
[0544] In one embodiment, the affinity of the binding partners is
high, 5 nM or stronger, such as 900, 800, 700, 600, 500, 400, 300
pM or stronger.
[0545] "Binding pair" as employed herein refers to two binding
partners which specifically bind to each other. Examples of a
binding pair include a peptide and an antibody or binding fragment
specific thereto, or an enzyme and ligand, or an enzyme and an
inhibitor of that enzyme.
[0546] In one embodiment, the first binding partner (X) is selected
from the group comprising: a full length antibody, a Fab, a Fab', a
scFv, a peptide and a sdAb, wherein examples of a sdAb include VH
or VL or V.sub.HH.
[0547] In one embodiment, the second partner (Y) is selected from
the group comprising: a full length antibody, a Fab, a Fab', a
scFv, a peptide and a sdAb, wherein examples of a sdAb include VH
or VL or V.sub.HH.
[0548] In one embodiment, where A is an antibody or fragment
thereof the first binding partner (X) is attached to the C-terminal
of the heavy or light chain of the first antibody or antibody
fragment, for example, the first binding partner is attached to the
C-terminal of the heavy chain of the first antibody or antibody
fragment.
[0549] In another embodiment, where B is an antibody or fragment
thereof the second binding partner (Y) is attached to the
C-terminal of the heavy or light chain of the second antibody or
antibody fragment, for example the second binding partner is
attached to the C-terminal of the heavy chain of the second
antibody or antibody fragment.
[0550] In one embodiment X is attached to the C-terminal of the
heavy chain of the antibody or fragment (protein A) and Y is
attached to the C-terminal of the antibody or fragment (protein
B).
[0551] In one embodiment X is attached via a linker (such as ASGGGG
or ASGGGGSG) to the C-terminal of the heavy chain of the antibody
or fragment (protein A) and Y is attached via a linker (such as
ASGGGG or ASGGGGSG) to the C-terminal of the antibody or fragment
(protein B).
[0552] In one embodiment, the first or second binding partner (X or
Y) is a peptide.
[0553] Examples of a suitable binding pair may include GCN4 (SEQ ID
NO: 1) or a variant thereof and 52SR4 (SEQ ID NO:3) or a variant
thereof, which is a scFv specific for GCN4.
[0554] In a one embodiment, the first binding partner (nominally X)
is GCN4 (for example as shown in SEQ ID NO:1) or a variant thereof
(for example without the His tag) and the second binding partner
(nominally Y) is a scFv specific for GCN4 (for example as shown in
SEQ ID NO:3) or a variant thereof.
[0555] In a one embodiment, the first binding partner (nominally X)
is a sFv specific for GCN4 (for example as shown in SEQ ID NO:3) or
a variant thereof and the second binding partner (nominally Y) is
GCN4 (for example as shown in SEQ ID NO:1) or a variant thereof.
GCN4 variants include an amino acid sequence with at least 80%,
85%, 90%, 91%, 92%, 93%, 94% 95%, 96%, 97% or 98%, or 99% identity
to SEQ ID NO:1. GCN4 variants also include an amino acid having at
least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%
to a sequence encoded by a nucleotide sequence SEQ ID NO:2, or a
nucleotide sequence which hybridises to SEQ ID NO: 2 under
stringent conditions.
[0556] A suitable scFv specific to GCN4 is 52SR4 (SEQ ID NO: 3) or
a variant thereof. Variants of 52SR4 include an amino acid sequence
with at least 80%, or 85%, or 90%, or 95%, or 98%, or 99% identity
to SEQ ID NO: 3. 52SR4 variants also include an amino acid sequence
having at least at least 80%, or 85%, or 90%, or 95%, or 98%, or
99% to a sequence encoded by a nucleotide sequence SEQ ID NO:4, or
a nucleotide sequence which hybridises to SEQ ID NO: 2 under
stringent conditions.
[0557] The present inventors have found that the single chain
antibody 52SR4 and peptide GCN4, are a binding pair suitable for
use in the bispecific protein complexes of the present disclosure.
Alternatively, any suitable antibody/fragment and antigen (such as
a peptide) may be employed as X and Y.
[0558] In one embodiment, the first binding partner (X) and the
second binding partner (Y) are a protein.
[0559] In one embodiment, the first binding partner (X) is an
enzyme or an active fragment thereof and the second binding partner
(Y) is a ligand or vice versa.
[0560] In one embodiment, the first binding partner (X) is an
enzyme or an active fragment thereof and the second binding partner
(Y) is an inhibitor of that enzyme or vice versa.
[0561] "Active fragment" as employed herein refers to an amino acid
fragment, which is less than the whole amino acid sequence for the
entity and retains essentially the same biological activity or a
relevant biological activity, for example greater than 50% activity
such as 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,
98%, 99% or 100%.
[0562] In another embodiment, the first binding partner X is
glutathione (GSH) and the second binding partner Y is
glutathione-S-transferase (GST) or vice versa.
[0563] In another embodiment, X is Fos and Y is Jun or vice
versa.
[0564] In another embodiment, X is His and Y is anti-His or vice
versa.
[0565] In another embodiment, the binding pair is clamodulin
binding peptide and Y is calmodulin or vice versa.
[0566] In another embodiment, X is maltose-binding protein and Y is
an anti-maltose binding protein or fragment thereof or vice
versa.
[0567] Other enzyme-ligand combinations are also contemplated for
use in binding partners. Also suitable are affinity tags known in
the art for protein purification because these have a tendency to
bind with high affinity to their respective binding partners.
[0568] Degrees of identity and similarity can be readily calculated
(Computational Molecular Biology, Lesk, A. M., ed., Oxford
University Press, New York, 1988; Biocomputing. Informatics and
Genome Projects, Smith, D. W., ed., Academic Press, New York, 1993;
Computer Analysis of Sequence Data, Part 1, Griffin, A. M., and
Griffin, H. G., eds., Humana Press, New Jersey, 1994; Sequence
Analysis in Molecular Biology, von Heinje, G., Academic Press,
1987, Sequence Analysis Primer, Gribskov, M. and Devereux, J.,
eds., M Stockton Press, New York, 1991, the BLAST software
available from NCBI (Altschul, S. F. et al., 1990, J. Mol. Biol.
215:403-410; Gish, W. & States, D. J. 1993, Nature Genet.
3:266-272. Madden, T. L. et al., 1996, Meth. Enzymol. 266:131-141;
Altschul, S. F. et al., 1997, Nucleic Acids Res. 25:3389-3402;
Zhang, J. & Madden, T. L. 1997, Genome Res. 7:649-656,).
[0569] In one embodiment, the first or second binding partner (X or
Y) is a protein or peptide.
[0570] In one embodiment, the first and second fusion proteins
comprise one or more peptide linkers. The linkers may be
incorporated at various locations in the fusion proteins. For
example, a linker may be introduced between a binding partner and
the protein attached thereto.
[0571] In one embodiment, the linker is a peptide linker.
[0572] The term "peptide linker" as used herein refers to a peptide
with amino acid sequences. A range of suitable peptide linkers will
be known to the person of skill in the art.
[0573] In one embodiment, the peptide linker may be of synthetic
origin, i.e. prepared by synthetic chemistry techniques.
[0574] In one embodiment, the binding partners of the bispecific
protein complexes are joined to their respective proteins via
peptide linkers.
[0575] In one embodiment the fusion proteins is a translational
fusion, that is a fusion protein expressed in a host cells
comprising a genetic construct from which the fusion protein is
expressed.
[0576] In one embodiment the fusion protein is prepared by
conjugating the A to X or B to Y optionally via a peptide
linker.
[0577] In one embodiment, the peptide linker is 50 amino acids in
length or less, for example 20 amino acids of less.
[0578] Generally it will be more efficient to express the fusion
protein recombinantly and therefore a direct peptide bond or a
peptide linker that can be expressed by a host cell may be
advantageous.
[0579] In one aspect, there is provided a method of producing a
bispecific protein complex of the present disclosure, comprising
the steps of: [0580] (a) producing a first fusion protein (A-X),
comprising a binding domain specific to CD45 or CD79 a and/or CD79b
(A), attached to a first binding partner (X) of a binding pair;
[0581] (b) producing a second fusion protein (B-Y), comprising a
binding domain specific to CD45 or CD79a and/or CD79b (B), attached
to a second binding partner (Y) of a binding pair; [0582] wherein
at least the first fusion protein or the second fusion protein
comprises a binding domain specific to CD45 and the remaining
fusion protein comprises a binding domain specific to CD79a and/or
CD79b, and [0583] (c) mixing the first (A-X) and second fusion
proteins (B-Y) together prepared in step a) and b).
[0584] In particular, the heterodimerically-tethered bispecific
protein complex is prepared by mixing A-X and B-Y in vitro. Thus in
one embodiment the method comprises an in vitro mixing step
bringing A-X and B-Y into contact.
[0585] Thus generally the fusion proteins A-X and B-Y are not
co-expressed in the same cell. This is advantageous because it
allows, for example 100 A-X fusion proteins and 100 A-Y fusion
proteins to be expressed separately and optionally purified, and
through subsequent mixing of the 200 fusion proteins in the various
permutations can provide 10,000 heterodimerically-tethered
bispecific protein complexes.
[0586] In contrast prior art methods require co-expression of
bispecifics and thus for 10,000 complexes, 10,000 transfections,
expressions and purifications are required.
[0587] The binding partners X and Y have affinity for each other
and act as biological equivalent of VELCRO or a bar and magnet and
hold the complex together. Advantageously, this means that the
fusion proteins A-X and Y-B can be readily assembled into a
bispecific protein complex simply by mixing the fusion proteins
together. Thus the bispecific protein complex of the present
disclosure has a modular structure which allows for two different
proteins to be easily assembled in order to produce large panels of
permutations of bispecific protein complexes with different
combinations of antigen specificities in, for example a grid-like
fashion. This allows for the efficient and systematic screening of
a large number of bispecific protein complexes in order to detect
additive, synergistic or novel biological function.
[0588] Given X and Y are specific for each other this significantly
reduces the ability to form homodimers. X and Y are collectively
referred to herein as a binding pair or binding partners. In one
embodiment X does not have high affinity for other Xs. In one
embodiment Y does not have high affinity for other Ys.
Advantageously, when X and Y do not form homodimers, this prevents
the formation of undesired monospecific protein complexes,
increases yield of the desired bispecific protein complexes, and
removes the need for onerous purification steps to remove the
monospecific protein complexes.
[0589] This rapid assembly of bispecific protein complexes, the
level of yield and/or purity cannot be obtained efficiently by
prior art methods, in particular prior art methods generally
require extensive purification steps.
[0590] Advantageously, the X and Y components allow a multiplex
comprising bispecific protein complexes made up of different
permutations of fusion proteins to be assembled rapidly and
easily.
[0591] In one embodiment the proteins A and B are antibodies or
antibody fragments. When the antibody or antibody fragments are
held together as a complex via X and Y, this forms a bispecific
antibody complex.
[0592] The mixing is generally effected in conditions where the X
and Y can interact. In one embodiment, the fusion proteins are
incubated in cell culture media under cell culturing conditions,
for example the fusion proteins are incubated for 90 minutes in a
37.degree. C./5% CO.sub.2 environment.
[0593] In one embodiment the fusions proteins of the present
disclosure are mixed in an aqueous environment, for example one
fusion protein may be bound to a solid surface such as a bead or a
plate and the other fusion protein can be introduced thereto in an
aqueous solution/suspension. The solid phase allows excess
components and reagents to be washed away readily. In one
embodiment neither fusion is attached a solid phase and are simply
admixed in a liquid/solution/medium.
[0594] Advantageously, the method of the present disclosure can be
employed to prepare complexes formed between heterogenous pairs
(i.e. between the first fusion protein [A-X] and second fusion
protein [B-Y]) wherein interactions between homogenous pairs (i.e.
between two first fusion proteins [A-X] or two second fusion
proteins [B-Y]) are minimised. Thus the present method allows large
numbers of bispecific protein complexes to be prepared, with
minimal or no contamination with homodimeric complexes. This level
of purity and yield is not possible using the prior art
methods.
[0595] In one embodiment the complexes formed require no further
purification steps.
[0596] In one embodiment the complexes formed require one
purification step, for example column chromatography.
[0597] In one embodiment the method further comprises at least one
purification step, for example after expression of a fusion protein
according to the present disclosure.
[0598] A "functional assay," as used herein, is an assay that can
be used to determine one or more desired properties or activities
of the protein complexes, antibody complexes or the mixture of
antibodies subject to the assay conditions. Suitable functional
assays may be binding assays, apoptosis assays, antibody-dependent
cellular cytotoxicity (ADCC) assays, complement-dependent
cytotoxicity (CDC) assays, inhibition of cell growth or
proliferation (cytostatic effect) assays, cell-killing (cytotoxic
effect) assays, cell-signaling assays, cytokine production assays,
antibody production and isotype switching, and cellular
differentiation assays, In one embodiment in vivo assays, such as
animal models, including mouse tumor models, models of auto-immune
disease, virus-infected or bacteria-infected rodent or primate
models, and the like, may be employed to test molecules of the
present disclosure.
[0599] In the context of bispecific antibody complexes, the
efficacy of bispecific antibody complexes according to the present
disclosure can be compared to individual antibodies or mixtures of
antibodies (or fragments) in such models by methods generally known
to one of ordinary skill in the art.
[0600] The functional assays may be repeated a number of times as
necessary with or without different samples of a particular
bispecific antibody complex to enhance the reliability of the
results. Various statistical tests known to the skilled person can
be employed to identify statistically significant results and thus
identify bispecific antibody complexes with biological functions,
and in particular to identify optimal variable region pairs for use
in multispecific molecule of the present invention.
Compositions and Medical Uses
[0601] In one aspect there is provided a molecule according to the
present disclosure or a component, such as a fusion protein, a
heterodimerically-tethered bispecific protein complex, a
composition comprising a molecule of the invention, including a
fusion protein or said bispecific protein complex, a multiplex,
array, library as defined herein.
[0602] In one embodiment the molecules of the present disclosure,
for example an antibody described herein, a multispecific molecule
and/or a bispecific protein complexes are suitable for therapeutic
applications and may provide novel therapies for treating diseases.
Thus in a further aspect, there is provided a molecule of the
present disclosure, for example a bispecific protein complex as
described above, for use in therapy. The molecules of the present
disclosure including the multispecific molecules and bispecific
protein complexes described herein are suitable for treating a
range of diseases, such as cancer.
[0603] The molecules of the present disclosure, including the
multispecific molecules and bispecific protein complexes described
herein are also particularly suited for inhibiting B cell function
in order to control immune and autoimmune reactions in various
autoimmune diseases.
[0604] Thus, the present disclosure extends to a method of treating
a disease in a patient, comprising the administration of a
therapeutically effect amount of a molecule of the present
disclosure, for example a multispecific molecule or bispecific
protein complex of the present disclosure. In one aspect, there is
provided a pharmaceutical composition comprising one or more
molecules of the present disclosure, for example a multispecific
molecule of the present disclosure.
[0605] Various different components can be included in the
composition, including pharmaceutically acceptable carriers,
excipients and/or diluents. The composition may, optionally,
comprise further molecules capable of altering the characteristics
of the population of multispecific molecules of the invention
thereby, for example, reducing, stabilizing, delaying, modulating
and/or activating the function of the antibodies. The composition
may be in solid, or liquid form and may be, inter alia, be in the
form of a powder, a tablet, a solution or an aerosol.
[0606] The present invention also provides a pharmaceutical or
diagnostic composition comprising an antibody molecule or a
multispecific molecule of the present invention in combination with
one or more of a pharmaceutically acceptable excipient, diluent or
carrier. Accordingly, provided is the use of a multispecific
molecule of the invention for use in the treatment and for the
manufacture of a medicament for the treatment of a pathological
condition or disorder.
Pathological Conditions
[0607] The pathological condition or disorder, may, for example be
selected from the group consisting of infections (viral, bacterial,
fungal and parasitic), endotoxic shock associated with infection,
arthritis such as rheumatoid arthritis, asthma such as severe
asthma, chronic obstructive pulmonary disease (COPD), pelvic
inflammatory disease, Alzheimer's Disease, inflammatory bowel
disease, Crohn's disease, ulcerative colitis, Peyronie's Disease,
coeliac disease, gallbladder disease, Pilonidal disease,
peritonitis, psoriasis, vasculitis, surgical adhesions, stroke,
Type I Diabetes, lyme disease, meningoencephalitis, autoimmune
uveitis, immune mediated inflammatory disorders of the central and
peripheral nervous system such as multiple sclerosis, lupus (such
as systemic lupus erythematosus) and Guillain-Barr syndrome, Atopic
dermatitis, autoimmune hepatitis, fibrosing alveolitis, Grave's
disease, IgA nephropathy, idiopathic thrombocytopenic purpura,
Meniere's disease, pemphigus, primary biliary cirrhosis,
sarcoidosis, scleroderma, Wegener's granulomatosis, other
autoimmune disorders, pancreatitis, trauma (surgery),
graft-versus-host disease, transplant rejection, heart disease
including ischaemic diseases such as myocardial infarction as well
as atherosclerosis, intravascular coagulation, bone resorption,
osteoporosis, osteoarthritis, periodontitis, hypochlorhydia and
cancer, including breast cancer, lung cancer, gastric cancer,
ovarian cancer, hepatocellular cancer, colon cancer, pancreatic
cancer, esophageal cancer, head & neck cancer, kidney, and
cancer, in particular renal cell carcinoma, prostate cancer, liver
cancer, melanoma, sarcoma, myeloma, neuroblastoma, placental
choriocarcinoma, cervical cancer, and thyroid cancer, and the
metastatic forms thereof.
[0608] In one embodiment the disorder is cancer, for example
Leukemia, including lyphocytic leukemia, such as acute
lymphoblastic leukemia or chronic lymphocytic leukemia; or
myelogenus leukemia, such as acture myelogenous leukemia or chronic
myelogenous leukemia.
[0609] In one embodiment autoimmune disease includes: --Acute
disseminated encephalomyelitis (adem), acute necrotizing
hemorrhagic leukoencephalitis, Addison's disease, adrenal
insufficiency, hypocortisolism, alopecia areata, amyloidosis,
ankylosing spondylitis, spondyloarthritis, Strumpell-marie disease,
anti-GBM/anti-TBM nephritis, antiphospholipid syndrome (aps),
autoimmune angioedema, autoimmune aplastic anemia, autoimmune
dysautonomia, autoimmune hepatitis, autoimmune hyperlipidemia,
autoimmune immunodeficiency, autoimmune inner ear disease (AIED),
autoimmune lymphoproliferative syndrome (ALPS), Canale-Smith
syndrome, autoimmune myocarditis, autoimmune oophoritis, autoimmune
pancreatitis (AIP), autoimmune polyglandular syndromes (types I, II
& III), autoimmune retinopathy (AR), autoimmune
thrombocytopenic purpura (ATP), autoimmune thyroid disease,
autoimmune urticaria, axonal/neuronal neuropathies, balo disease,
Behcet's disease, bullous pemphigoid, cardiomyopathy, Castleman
disease, coeliac disease, chagas disease, chronic inflammatory
demyelinating polyneuropathy (CIDP), chronic recurrent multifocal
ostomyelitis (CRMO), Churg-Strauss syndrome, cicatricial
pemphigoid/benign mucosal pemphigoid (CP), Crohn's disease,
inflammatory bowel disease, colitis, enteritis, ileitis, Cogans
syndrome, cold agglutinin disease, congenital heart block,
Coxsackie myocarditis, crest disease, cryoglobulinemia,
demyelinating neuropathies, dermatitis herpetiformis, Duhring's
disease, dermatomyositis, diabetes, type I, discoid lupus
erythematosus (DLE), Dressler's syndrome, endometriosis,
epidermolysis bullosa (EB) and eb acquisita (EBA), eosinophilic
gastroenteritis, esophagitis, eosinophilic fasciitis, schulman's
syndrome, erythema nodosum, experimental allergic
encephalomyelitis, Evans syndrome, fibrosing alveolitis, giant cell
arteritis (temporal arteritis), giant cell myocarditis,
glomerulonephritis (non-proliferative: focal segmental
glomerulosclerosis and membranous glomerulonephritis.
proliferative: IgA nephropathy), goodpasture's syndrome,
granulomatosis with polyangiitis (GPA) (formerly called Wegener's
granulomatosis), Graves' disease, Guillain-Barre syndrome, Miller
Fisher syndrome, acute motor axonal neuropathy, acute motor sensory
axonal neuropathy, acute panautonomic neuropathy, Bickerstaff's
brainstem encephalitis, Hashimoto's encephalitis, Hashimoto's
thyroiditis, hemolytic anemia, Henoch-Schonlein purpura, herpes
gestationis, hypogammaglobulinemia, idiopathic pulmonary fibrosis,
idiopathic thrombocytopenic purpura (ITP), IgA nephropathy (IGAN),
berger's syndrome, synpharyngitic glomerulonephritis, IgA
pemphigus, IgG4-related sclerosing disease, immune-regulated
infertility, inclusion body myositis, insulin-dependent diabetes
mellitus, interstitial cystitis, Isaac's syndrome, neuromyotonia,
juvenile arthritis, juvenile myositis, Kawasaki syndrome,
Lambert-Eaton syndrome, leukocytoclastic vasculitis, lichen planus,
lichen sclerosus, ligneous conjunctivitis, linear IgA dermatosis
(LAD), pemphigoid, lupus (SLE), lyme disease, Meniere's disease,
microscopic polyangiitis (MPA), mixed connective tissue disease
(MCTD), monoclonal gammaopathy, Mooren's ulcer, Mucha-Habermann
disease, multiple sclerosis, myasthenia gravis, myositis,
narcolepsy, neuromyelitis optica (devic's), neuromyotonia, Isaac's
syndrome (acquired, paraneoplastic, hereditary), neutropenia,
ocular cicatricial pemphigoid, optic neuritis, oophoritis,
opsoclonus-myoclonus syndrome, orchitis, palindromic rheumatism,
pandas (pediatric autoimmune neuropsychiatric disorders associated
with streptococcus), paraneoplastic autoimmune multiorgan syndrome
(PAMS), paraneoplastic cerebellar degeneration, paraneoplastic
pemphigus (PNP), paroxysmal nocturnal hemoglobinuria (PNH), Parry
Romberg syndrome, Parsonnage-Turner syndrome, pars planitis
(peripheral uveitis), pempgigoid gestationis (PG), pemphigus
vulgaris (PV), pemphigus folliaceus (PF), peripheral neuropathy,
perivenous encephalomyelitis, pernicious anemia, Poems syndrome,
polyarteritis nodosa (PAN), polymyalgia rheumatic, polymyositis,
postmyocardial infarction syndrome, postpericardiotomy syndrome,
progesterone dermatitis primary biliary cirrhosis, Hanot syndrome,
primary sclerosing cholangitis (PSC), sclerosong cholangitis,
psoriasis, psoriatic arthritis, pyoderma gangrenosum, pure red cell
aplasia, Rasmussen's encephalitis, chronic focal encephalitis
(CFE), Raynauds phenomenon, reactive arthritis, Reiter's syndrome,
recoverin-associated retinopathy (RAR), reflex sympathetic
dystrophy, Reiter's syndrome, relapsing polychondritis, restless
legs syndrome, retroperitoneal fibrosis, rheumatic fever,
rheumatoid arthritis, sarcoidosis, Schmidt syndrome, scleritis,
scleroderma, systemic sclerosis, sjogren's syndrome, sperm &
testicular autoimmunity, stiff person/man syndrome, subacute
bacterial endocarditis (SBE), Susac's syndrome, sympathetic
ophthalmia, Takayasu's arteritis, temporal arteritis/giant cell
arteritis, thromboangiitis obliterans, Buerger's disease,
thrombocytopenic purpura (TTP), Tolosa-Hunt syndrome, transverse
myelitis, ulcerative colitis, undifferentiated connective tissue
disease (UCTD), uveitis, polymyalgia rheumatica, Takayasu's
arteritis, temporal arteritis, Buerger's disease, cutaneous
vasculitis, Kawasaki disease, polyarteritis nodosa, Behcet's
syndrome, Churg-Strauss syndrome, cutaneous vasculitis,
Henoch-Schonlein purpura, microscopic polyangiitis, Wegener's
granulomatosis, golfer's vasculitis, vesiculobullous dermatosis,
Vitiligowegener's granulomatosis (now termed granulomatosis with
polyangiitis (GPA).
[0610] In one embodiment the autoimmune disease is selected from
the group comprising or consisting of: --ANCA vasculitis, IgA
nephropathy (Berger's), pemphigus vulgaris/bullous pemphigoid, ITP,
primary biliary cirrhosis, autoimmune thyroiditis (Grave's
disease), hashimoto's disease, lupus nephritis, membranous
glomerulonephritis (or membranous nephropathy), APS, myasthenia
gravis, neuromyelitis optica, primary Sjogren's, autoimmune
neutropaenia, autoimmune pancreatitis, dermatosmyositis, autoimmune
uveitis, autoimmune retinopathy, Behcet's disease, IPF, systemic
sclerosis, liver fibrosis, autoimmune hepatitis, primary sclerosing
cholangitis, vitiligo, goodpasture's syndrome, pulmonary alveolar
proteinosis, chronic autoimmune urticarial, psoriasis, rheumatoid
arthritis, psoriatic arthritis, axial spodyloarthritis,
transplantation (including GvHD), asthma, COPD, giant cell
arteritis, refractory autoimmune cytopaenias, Evans syndrome
(autoimmune haemolytic anaemia), type I diabetes, sarcoidosis,
polymyositis, ulcerative colitis, Crohn's disease, coeliac disease,
Waldenstrom's macroglobulinaemia, focal segmental
glomerulosclerosis, chronic Lyme disease (Lyme borreliosis), lichen
planus, Stiff person syndrome, dilated cardiomyopathy, autoimmune
(lymphocytic) oophoritis, epidermolysis bullosa acquisita,
autoimmune atrophic gastritis, pernicious anaemia, atopic
dermatitis, atherosclerosis, multiple sclerosis, Rasmussen's
encephalitis, Guillain-Barre syndrome and acquired neuromyotonia,
stroke. In one embodiment the disorder is cancer, for example
leukemia, for example lyphocytic leukemia, such as acute
lymphoblastic leukemia or chronic lymphocytic leukemia; or
myelogenus leukemia, such as acture myelogenous leukemia or chronic
myelogenous leukemia; or lymphoma, such as diffuse large B cell
lymphoma or Hodgkin's or non-Hodkin's lymphoma.
[0611] The present invention also provides a pharmaceutical or
diagnostic composition comprising a molecule of the present
disclosure, such as a multispecific molecule described herein in
combination with one or more of a pharmaceutically acceptable
excipient, diluent or carrier. Accordingly, provided is the use of
a molecule of the present disclosure, such as a multispecific
molecule as described herein for use in treatment and in the
manufacture of a medicament.
[0612] The composition will usually be supplied as part of a
sterile, pharmaceutical composition that will normally include a
pharmaceutically acceptable carrier. A pharmaceutical composition
of the present invention may additionally comprise a
pharmaceutically-acceptable adjuvant.
[0613] The present invention also provides a process for
preparation of a pharmaceutical or diagnostic composition
comprising adding and mixing the multispecific molecule of the
present invention together with one or more of a pharmaceutically
acceptable excipient, diluent or carrier.
[0614] The term "pharmaceutically acceptable excipient" as used
herein refers to a pharmaceutically acceptable formulation carrier,
solution or additive to enhance the desired characteristics of the
compositions of the present disclosure. Excipients are well known
in the art and include buffers (e.g., citrate buffer, phosphate
buffer, acetate buffer and bicarbonate buffer), amino acids, urea,
alcohols, ascorbic acid, phospholipids, proteins (e.g., serum
albumin), EDTA, sodium chloride, liposomes, mannitol, sorbitol, and
glycerol. Solutions or suspensions can be encapsulated in liposomes
or biodegradable microspheres. The formulation will generally be
provided in a substantially sterile form employing sterile
manufacture processes.
[0615] This may include production and sterilization by filtration
of the buffered solvent solution used for the formulation, aseptic
suspension of the antibody in the sterile buffered solvent
solution, and dispensing of the formulation into sterile
receptacles by methods familiar to those of ordinary skill in the
art.
[0616] The pharmaceutically acceptable carrier should not itself
induce the production of antibodies harmful to the individual
receiving the composition and should not be toxic. Suitable
carriers may be large, slowly metabolised macromolecules such as
proteins, polypeptides, liposomes, polysaccharides, polylactic
acids, polyglycolic acids, polymeric amino acids, amino acid
copolymers and inactive virus particles.
[0617] Pharmaceutically acceptable salts can be used, for example
mineral acid salts, such as hydrochlorides, hydrobromides,
phosphates and sulphates, or salts of organic acids, such as
acetates, propionates, malonates and benzoates.
[0618] Pharmaceutically acceptable carriers in therapeutic
compositions may additionally contain liquids such as water,
saline, glycerol and ethanol. Such carriers enable the
pharmaceutical compositions to be formulated as tablets, pills,
dragees, capsules, liquids, gels, syrups, slurries and suspensions,
for ingestion by the patient.
[0619] The molecules of the disclosure such as a multispecific
molecule described herein can be delivered dispersed in a solvent,
e.g., in the form of a solution or a suspension. It can be
suspended in an appropriate physiological solution, e.g.,
physiological saline, a pharmacologically acceptable solvent or a
buffered solution. Buffered solutions known in the art may contain
0.05 mg to 0.15 mg disodium edetate, 8.0 mg to 9.0 mg NaCl, 0.15 mg
to 0.25 mg polysorbate, 0.25 mg to 0.30 mg anhydrous citric acid,
and 0.45 mg to 0.55 mg sodium citrate per 1 ml of water so as to
achieve a pH of about 4.0 to 5.0. As mentioned supra a suspension
can made, for example, from lyophilised antibody.
[0620] A thorough discussion of pharmaceutically acceptable
carriers is available in Remington's Pharmaceutical Sciences (Mack
Publishing Company, N.J. 1991).
[0621] The term "therapeutically effective amount" as used herein
refers to an amount of a therapeutic agent needed to treat,
ameliorate or prevent a targeted disease or condition, or to
exhibit a detectable therapeutic or preventative effect. For any
antibody, the therapeutically effective amount can be estimated
initially either in cell culture assays or in animal models,
usually in rodents, rabbits, dogs, pigs or primates. The animal
model may also be used to determine the appropriate concentration
range and route of administration. Such information can then be
used to determine useful doses and routes for administration in
humans.
[0622] The precise therapeutically effective amount for a human
subject will depend upon the severity of the disease state, the
general health of the subject, the age, weight and gender of the
subject, diet, time and frequency of administration, drug
combination(s), reaction sensitivities and tolerance/response to
therapy. This amount can be determined by routine experimentation
and is within the judgement of the clinician. Generally, a
therapeutically effective amount will be from 0.01 mg/kg to 50
mg/kg, for example 0.1 mg/kg to 20 mg/kg. Alternatively, the dose
may be 1 to 500 mg per day such as 10 to 100, 200, 300 or 400 mg
per day. Pharmaceutical compositions may be conveniently presented
in unit dose forms containing a predetermined amount of an active
agent of the invention.
[0623] Compositions may be administered individually to a patient
or may be administered in combination (e.g. simultaneously,
sequentially or separately) with other agents, drugs or
hormones.
[0624] The dose at which the multispecific molecule of the present
disclosure is administered depends on the nature of the condition
to be treated, the extent of the inflammation present and on
whether the antibody molecule is being used prophylactically or to
treat an existing condition. The frequency of dose will depend on
the half-life of the multispecific molecule and the duration of its
effect. If the multispecific molecule has a short half-life (e.g. 2
to 10 hours) it may be necessary to give one or more doses per day.
Alternatively, if the multispecific molecule has a long half-life
(e.g. 2 to 15 days) it may only be necessary to give a dosage once
per day, once per week or even once every 1 or 2 months.
[0625] In the present disclosure, the pH of the final formulation
is not similar to the value of the isoelectric point of the
multispecific molecule, for if the pH of the formulation is 7 then
a pI of from 8-9 or above may be appropriate. Whilst not wishing to
be bound by theory it is thought that this may ultimately provide a
final formulation with improved stability, for example the antibody
or fragment remains in solution.
[0626] The pharmaceutical compositions of this invention may be
administered by any number of routes including, but not limited to,
oral, intravenous, intramuscular, intra-arterial, intramedullary,
intrathecal, intraventricular, transdermal, transcutaneous (for
example, see WO98/20734), subcutaneous, intraperitoneal,
intranasal, enteral, topical, sublingual, intravaginal or rectal
routes. Hyposprays may also be used to administer the
pharmaceutical compositions of the invention.
[0627] Direct delivery of the compositions will generally be
accomplished by injection, subcutaneously, intraperitoneally,
intravenously or intramuscularly, or delivered to the interstitial
space of a tissue. The compositions can also be administered into a
specific tissue of interest. Dosage treatment may be a single dose
schedule or a multiple dose schedule.
[0628] Where the product is for injection or infusion, it may take
the form of a suspension, solution or emulsion in an oily or
aqueous vehicle and it may contain formulatory agents, such as
suspending, preservative, stabilising and/or dispersing agents.
Alternatively, the multispecific molecule may be in dry form, for
reconstitution before use with an appropriate sterile liquid. If
the composition is to be administered by a route using the
gastrointestinal tract, the composition will need to contain agents
which protect the antibody from degradation but which release the
bispecific protein complex once it has been absorbed from the
gastrointestinal tract. A nebulisable formulation according to the
present disclosure may be provided, for example, as single dose
units (e.g., sealed plastic containers or vials) packed in foil
envelopes. Each vial contains a unit dose in a volume, e.g., 2 ml,
of solvent/solution buffer.
[0629] The term "variant" as used herein refers to peptide or
protein that contains at least one amino acid sequence or
nucleotide sequence alteration as compared to the amino acid or
nucleotide sequence of the corresponding wild-type peptide or
protein. A variant may comprise at least 80%, or 85%, or 90%, or
95%, or 98% or 99% sequence identity to the corresponding wild-type
peptide or protein. However, it is possible for a variant to
comprise less than 80% sequence identity, provided that the variant
exhibits substantially similar function to its corresponding
wild-type peptide or protein.
[0630] In one embodiment the construct of the present disclosure is
at least trispecific. In this situation the further specificity may
be directed to any antigen of interest, for example antigens to
extend half-life such as albumin or Fc neonatal receptor (FcRn);
antigens for effector function such as activating or inhibiting Fc
receptors or costimulatory molecules; tissue or cell targeting
antigens; or antigens to aid blood/brain barrier (BBB) transfer
such as transferrin receptor or LRP1.
[0631] The disclosure also extends to compositions, such as
pharmaceutical compositions comprising said novel formats with the
particular antigen specificity.
[0632] In a further aspect the disclosure includes use of the
formats and the compositions in treatment. The present invention
also provides a process for preparation of a pharmaceutical or
diagnostic composition comprising adding and mixing the antibody
molecule of the present invention together with one or more of a
pharmaceutically acceptable excipient, diluent or carrier.
[0633] The antibody molecule may be the sole active ingredient in
the pharmaceutical or diagnostic composition or may be accompanied
by other active ingredients including other antibody ingredients or
non-antibody ingredients such as steroids or other drug
molecules.
[0634] The pharmaceutical compositions suitably comprise a
therapeutically effective amount of the antibody of the invention.
The term "therapeutically effective amount" as used herein refers
to an amount of a therapeutic agent needed to treat, ameliorate or
prevent a targeted disease or condition, or to exhibit a detectable
therapeutic or preventative effect. For any antibody, the
therapeutically effective amount can be estimated initially either
in cell culture assays or in animal models, usually in rodents,
rabbits, dogs, pigs or primates. The animal model may also be used
to determine the appropriate concentration range and route of
administration. Such information can then be used to determine
useful doses and routes for administration in humans.
[0635] The precise therapeutically effective amount for a human
subject will depend upon the severity of the disease state, the
general health of the subject, the age, weight and gender of the
subject, diet, time and frequency of administration, drug
combination(s), reaction sensitivities and tolerance/response to
therapy. This amount can be determined by routine experimentation
and is within the judgement of the clinician. Generally, a
therapeutically effective amount will be from 0.01 mg/kg to 500
mg/kg, for example 0.1 mg/kg to 200 mg/kg, such as 100 mg/Kg.
Pharmaceutical compositions may be conveniently presented in unit
dose forms containing a predetermined amount of an active agent of
the invention per dose.
[0636] Compositions may be administered individually to a patient
or may be administered in combination (e.g. simultaneously,
sequentially or separately) with other agents, drugs or
hormones.
[0637] Agents as employed herein refers to an entity which when
administered has a physiological affect.
[0638] Drug as employed herein refers to a chemical entity which at
a therapeutic dose has an appropriate physiological affect.
[0639] In one embodiment the antibodies or fragments according to
the present disclosure are employed with an immunosuppressant
therapy, such as a steroid, in particular prednisone.
[0640] In one embodiment the antibodies or fragments according to
the present disclosure are employed with Rituximab or other B cell
therapies.
[0641] In one embodiment the antibodies or fragments according to
the present disclosure are employed with any B cell or T cell
modulating agent or immunomodulator. Examples include methotrexate,
microphenyolate and azathioprine.
[0642] The dose at which the antibody molecule of the present
invention is administered depends on the nature of the condition to
be treated, the extent of the inflammation present and on whether
the antibody molecule is being used prophylactically or to treat an
existing condition.
[0643] The frequency of dose will depend on the half-life of the
antibody molecule and the duration of its effect. If the antibody
molecule has a short half-life (e.g. 2 to 10 hours) it may be
necessary to give one or more doses per day. Alternatively, if the
antibody molecule has a long half life (e.g. 2 to 15 days) and/or
long lasting pharmacodynamics (PD) profile it may only be necessary
to give a dosage once per day, once per week or even once every 1
or 2 months.
[0644] In one embodiment the dose is delivered bi-weekly, i.e.
twice a month.
[0645] In one embodiment doses are spaced to allow anti-drug (in
this case anti-antibody) responses to waine before administration
of further dose.
[0646] Half life as employed herein is intended to refer to the
duration of the molecule in circulation, for example in
serum/plasma.
[0647] Pharmacodynamics as employed herein refers to the profile
and in particular duration of the biological action of the molecule
according the present disclosure.
[0648] The pharmaceutically acceptable carrier should not itself
induce the production of antibodies harmful to the individual
receiving the composition and should not be toxic. Suitable
carriers may be large, slowly metabolised macromolecules such as
proteins, polypeptides, liposomes, polysaccharides, polylactic
acids, polyglycolic acids, polymeric amino acids, amino acid
copolymers and inactive virus particles.
[0649] Pharmaceutically acceptable salts can be used, for example
mineral acid salts, such as hydrochlorides, hydrobromides,
phosphates and sulphates, or salts of organic acids, such as
acetates, propionates, malonates and benzoates.
[0650] Pharmaceutically acceptable carriers in therapeutic
compositions may additionally contain liquids such as water,
saline, glycerol and ethanol. Additionally, auxiliary substances,
such as wetting or emulsifying agents or pH buffering substances,
may be present in such compositions. Such carriers enable the
pharmaceutical compositions to be formulated as tablets, pills,
dragees, capsules, liquids, gels, syrups, slurries and suspensions,
for ingestion by the patient.
[0651] Suitable forms for administration include forms suitable for
parenteral administration, e.g. by injection or infusion, for
example by bolus injection or continuous infusion. Where the
product is for injection or infusion, it may take the form of a
suspension, solution or emulsion in an oily or aqueous vehicle and
it may contain formulatory agents, such as suspending,
preservative, stabilising and/or dispersing agents. Alternatively,
the antibody molecule may be in dry form, for reconstitution before
use with an appropriate sterile liquid.
[0652] Once formulated, the compositions of the invention can be
administered directly to the subject. The subjects to be treated
can be animals. However, in one or more embodiments the
compositions are adapted for administration to human subjects.
[0653] Suitably in formulations according to the present
disclosure, the pH of the final formulation is not similar to the
value of the isoelectric point of the antibody or fragment, for
example if the pI of the protein is in the range 8-9 or above then
a formulation pH of 7 may be appropriate. Whilst not wishing to be
bound by theory it is thought that this may ultimately provide a
final formulation with improved stability, for example the antibody
or fragment remains in solution. In one example the pharmaceutical
formulation at a pH in the range of 4.0 to 7.0 comprises: 1 to 200
mg/mL of an antibody molecule according to the present disclosure,
1 to 100 mM of a buffer, 0.001 to 1% of a surfactant, a) 10 to 500
mM of a stabiliser, b) 10 to 500 mM of a stabiliser and 5 to 500 mM
of a tonicity agent, or c) 5 to 500 mM of a tonicity agent.
[0654] The pharmaceutical compositions of this invention may be
administered by any number of routes including, but not limited to,
oral, intravenous, intramuscular, intra-arterial, intramedullary,
intrathecal, intraventricular, transdermal, transcutaneous (for
example, see WO98/20734), subcutaneous, intraperitoneal,
intranasal, enteral, topical, sublingual, intravaginal or rectal
routes. Hyposprays may also be used to administer the
pharmaceutical compositions of the invention. Typically, the
therapeutic compositions may be prepared as injectables, either as
liquid solutions or suspensions. Solid forms suitable for solution
in, or suspension in, liquid vehicles prior to injection may also
be prepared.
[0655] Direct delivery of the compositions will generally be
accomplished by injection, subcutaneously, intraperitoneally,
intravenously or intramuscularly, or delivered to the interstitial
space of a tissue. The compositions can also be administered into a
lesion. Dosage treatment may be a single dose schedule or a
multiple dose schedule.
[0656] It will be appreciated that the active ingredient in the
composition will be an antibody molecule. As such, it will be
susceptible to degradation in the gastrointestinal tract. Thus, if
the composition is to be administered by a route using the
gastrointestinal tract, the composition will need to contain agents
which protect the antibody from degradation but which release the
antibody once it has been absorbed from the gastrointestinal
tract.
[0657] A thorough discussion of pharmaceutically acceptable
carriers is available in Remington's Pharmaceutical Sciences (Mack
Publishing Company, N.J. 1991).
[0658] In one embodiment the formulation is provided as a
formulation for topical administrations including inhalation.
[0659] Suitable inhalable preparations include inhalable powders,
metering aerosols containing propellant gases or inhalable
solutions free from propellant gases. Inhalable powders according
to the disclosure containing the active substance may consist
solely of the abovementioned active substances or of a mixture of
the abovementioned active substances with physiologically
acceptable excipient.
[0660] These inhalable powders may include monosaccharides (e.g.
glucose or arabinose), disaccharides (e.g. lactose, saccharose,
maltose), oligo- and polysaccharides (e.g. dextranes), polyalcohols
(e.g. sorbitol, mannitol, xylitol), salts (e.g. sodium chloride,
calcium carbonate) or mixtures of these with one another. Mono- or
disaccharides are suitably used, the use of lactose or glucose,
particularly but not exclusively in the form of their hydrates.
[0661] Particles for deposition in the lung require a particle size
less than 10 microns, such as 1-9 microns for example from 1 to 5
.mu.m. The particle size of the active ingredient (such as the
antibody or fragment) is of primary importance.
[0662] The propellent gases which can be used to prepare the
inhalable aerosols are known in the art. Suitable propellent gases
are selected from among hydrocarbons such as n-propane, n-butane or
isobutane and halohydrocarbons such as chlorinated and/or
fluorinated derivatives of methane, ethane, propane, butane,
cyclopropane or cyclobutane. The abovementioned propellent gases
may be used on their own or in mixtures thereof.
[0663] Particularly suitable propellent gases are halogenated
alkane derivatives selected from among TG 11, TG 12, TG 134a and
TG227. Of the abovementioned halogenated hydrocarbons, TG134a
(1,1,1,2-tetrafluoroethane) and TG227
(1,1,1,2,3,3,3-heptafluoropropane) and mixtures thereof are
particularly suitable.
[0664] The propellent-gas-containing inhalable aerosols may also
contain other ingredients such as cosolvents, stabilisers,
surface-active agents (surfactants), antioxidants, lubricants and
means for adjusting the pH. All these ingredients are known in the
art.
[0665] The propellant-gas-containing inhalable aerosols according
to the invention may contain up to 5% by weight of active
substance. Aerosols according to the invention contain, for
example, 0.002 to 5% by weight, 0.01 to 3% by weight, 0.015 to 2%
by weight, 0.1 to 2% by weight, 0.5 to 2% by weight or 0.5 to 1% by
weight of active ingredient.
[0666] Alternatively topical administrations to the lung may also
be by administration of a liquid solution or suspension
formulation, for example employing a device such as a nebulizer,
for example, a nebulizer connected to a compressor (e.g., the Pari
LC-Jet Plus.RTM. nebulizer connected to a Pari Master.RTM.
compressor manufactured by Pari Respiratory Equipment, Inc.,
Richmond, Va.).
[0667] The antibody or multispecific molecule of the invention can
be delivered dispersed in a solvent, e.g., in the form of a
solution or a suspension. It can be suspended in an appropriate
physiological solution, e.g., saline or other pharmacologically
acceptable solvent or a buffered solution. Buffered solutions known
in the art may contain 0.05 mg to 0.15 mg disodium edetate, 8.0 mg
to 9.0 mg NaCl, 0.15 mg to 0.25 mg polysorbate, 0.25 mg to 0.30 mg
anhydrous citric acid, and 0.45 mg to 0.55 mg sodium citrate per 1
ml of water so as to achieve a pH of about 4.0 to 5.0. A suspension
can employ, for example, lyophilised antibody.
[0668] The therapeutic suspensions or solution formulations can
also contain one or more excipients. Excipients are well known in
the art and include buffers (e.g., citrate buffer, phosphate
buffer, acetate buffer and bicarbonate buffer), amino acids, urea,
alcohols, ascorbic acid, phospholipids, proteins (e.g., serum
albumin), EDTA, sodium chloride, liposomes, mannitol, sorbitol, and
glycerol. Solutions or suspensions can be encapsulated in liposomes
or biodegradable microspheres. The formulation will generally be
provided in a substantially sterile form employing sterile
manufacture processes.
[0669] This may include production and sterilization by filtration
of the buffered solvent/solution used for the formulation, aseptic
suspension of the antibody in the sterile buffered solvent
solution, and dispensing of the formulation into sterile
receptacles by methods familiar to those of ordinary skill in the
art.
[0670] Nebulizable formulation according to the present disclosure
may be provided, for example, as single dose units (e.g., sealed
plastic containers or vials) packed in foil envelopes. Each vial
contains a unit dose in a volume, e.g., 2 mL, of solvent/solution
buffer.
[0671] The antibodies disclosed herein may be suitable for delivery
via nebulisation.
[0672] It is also envisaged that the antibody of the present
invention may be administered by use of gene therapy. In order to
achieve this, DNA sequences encoding the heavy and light chains of
the antibody molecule under the control of appropriate DNA
components are introduced into a patient such that the antibody
chains are expressed from the DNA sequences and assembled in
situ.
[0673] In one embodiment, the molecule of the present disclosure,
such as a bispecific protein complex described herein may be used
to functionally alter the activity of the antigen or antigens of
interest. For example, the bispecific protein complex may
neutralize, antagonize or agonise the activity of said antigen or
antigens, directly or indirectly.
[0674] The present disclosure also extends to a kit, comprising a
molecule of the present disclosure or a component thereof. In one
embodiment the kit comprises: [0675] a) one or more fusion proteins
(A-X) comprising a first antibody or antibody fragment (A) specific
to CD45 or CD79a and/or CD79b attached to a first binding partner
of a binding pair (X); and [0676] b) one or more fusion proteins
(B-Y) comprising a second antibody or antibody fragment
[0677] (B) specific to CD45 or CD79a and/or CD79b attached to a
second binding partner of the binding pair (Y), wherein the latter
is specific for the first binding partner; for example wherein the
first binding partner (X) is a peptide or polypeptide and the
second binding (Y) partner is an antibody or antibody fragment
specific thereto;
wherein Y the second binding partner is specific to the first
binding partner X and the second binding partner is, for example an
antibody or antibody fragment specific thereto; and the specific
interaction (such as a binding interaction) of the two binding
partners forms a heterodimer-tether which physically brings the two
fusion proteins from a) and b) together to form a bispecific
protein complex; and wherein at least one of A or B is specific to
CD45 and the other is specific to CD79a and/or CD79b, and the
fusion protein(s) is/are in a complexed or a non-complexed
form.
[0678] Advantageously, the kit may comprise bispecific protein
complexes of the present disclosure, or may comprise fusion
proteins which are in a complexed or non-complexed form. In the
former case, the bispecific protein complexes are ready for use
"out of the box" which provides convenience and ease of use,
whereas in the latter case, the bispecific protein complexes can be
assembled according to the user's requirements by using combining
different fusion proteins.
[0679] In another embodiment, the kit further comprises
instructions for use.
[0680] In yet another embodiment, the kit further comprises one or
more reagents for performing one or more functional assays.
[0681] In one embodiment, molecules of the present disclosure
including fusion proteins, bispecific proteins complexes or
compositions comprising same are provided for use as a laboratory
reagent.
Further Aspects
[0682] In a further aspect, there is provided a nucleotide
sequence, for example a DNA sequence encoding a construct as
described herein including a multispecific molecule or a fusion
protein as defined above.
[0683] In one embodiment, there is provided a nucleotide sequence,
for example a DNA sequence encoding a construct as described herein
including a multispecific molecule or a bispecific protein complex
or an antibody according to the present disclosure.
[0684] The disclosure herein also extends to a vector comprising a
nucleotide sequence as defined above.
[0685] The term "vector" as used herein refers to a nucleic acid
molecule capable of transporting another nucleic acid to which it
has been linked. An example of a vector is a "plasmid," which is a
circular double stranded DNA loop into which additional DNA
segments may be ligated. Another type of vector is a viral vector,
wherein additional DNA segments may be ligated into the viral
genome. Certain vectors are capable of autonomous replication in a
host cell into which they are introduced (e.g., bacterial vectors
having a bacterial origin of replication and episomal mammalian
vectors). Other vectors (e.g., non-episomal mammalian vectors) can
be integrated into the genome of a host cell, where they are
subsequently replicated along with the host genome. In the present
specification, the terms "plasmid" and "vector" may be used
interchangeably as a plasmid is the most commonly used form of
vector.
[0686] General methods by which the vectors may be constructed,
transfection methods and culture methods are well known to those
skilled in the art. In this respect, reference is made to "Current
Protocols in Molecular Biology", 1999, F. M. Ausubel (ed), Wiley
Interscience, New York and the Maniatis Manual produced by Cold
Spring Harbor Publishing.
[0687] The term "selectable marker" as used herein refers to a
protein whose expression allows one to identify cells that have
been transformed or transfected with a vector containing the marker
gene. A wide range of selection markers are known in the art. For
example, typically the selectable marker gene confers resistance to
drugs, such as G418, hygromycin or methotrexate, on a host cell
into which the vector has been introduced. The selectable marker
can also be a visually identifiable marker such as a fluorescent
marker for example. Examples of fluorescent markers include
rhodamine, FITC, TRITC, Alexa Fluors and various conjugates
thereof.
[0688] Also provided is a host cell comprising one or more cloning
or expression vectors comprising one or more DNA sequences encoding
an antibody of the present disclosure. Any suitable host
cell/vector system may be used for expression of the DNA sequences
encoding the antibody molecule of the present disclosure.
Bacterial, for example E. coli, and other microbial systems may be
used or eukaryotic, for example mammalian, host cell expression
systems may also be used. Suitable mammalian host cells include
CHO, myeloma or hybridoma cells.
[0689] The present disclosure also provides a process for the
production of a molecule according to the present disclosure or a
component thereof comprising culturing a host cell containing a
vector of the present disclosure under conditions suitable for
leading to expression of protein from DNA encoding the molecule of
the present disclosure, and isolating the molecule.
[0690] The molecules of the present disclosure including the
bispecific protein complexes described herein may be used in
diagnosis/detection kits. The kits may, for example comprise
bispecific antibody complexes that are specific for two antigens,
both of which are present on the same cell type, and wherein a
positive diagnosis can only be made if both antigens are
successfully detected. By using a molecule of the present
disclosure such as a bispecific antibody complexes described herein
rather than two separate antibodies or antibody fragments in a
non-complexed form, the specificity of the detection can be greatly
enhanced.
[0691] In one embodiment, the molecules of the present disclosure
such as the bispecific antibody complexes are fixed on a solid
surface. The solid surface may for example be a chip, or an ELISA
plate.
[0692] Further provided is the use of a molecule according to the
present disclosure, for example a bispecific protein complex
described herein for detecting in a sample the presence of a first
and a second peptide, whereby the said molecules are used as
detection agents.
[0693] The molecules of the present disclosure such as the
bispecific antibody complexes described herein may for example be
conjugated to a fluorescent marker which facilitates the detection
of bound antibody-antigen complexes. Such bispecific antibody
complexes can be used for immunofluorescence microscopy.
Alternatively, the bispecific antibody complexes may also be used
for western blotting or ELISA.
[0694] In one embodiment, there is provided a process for purifying
a molecule according to the present disclosure or a component
thereof
[0695] In one embodiment, there is provided a process for purifying
a molecule according the present disclosure or a component thereof
comprising the steps: performing anion exchange chromatography in
non-binding mode such that the impurities are retained on the
column and the antibody is maintained in the unbound fraction. The
step may, for example be performed at a pH about 6-8.
[0696] The process may further comprise an initial capture step
employing cation exchange chromatography, performed for example at
a pH of about 4 to 5.
[0697] The process may further comprise of additional
chromatography step(s) to ensure product and process related
impurities are appropriately resolved from the product stream.
[0698] The purification process may also comprise of one or more
ultra-filtration steps, such as a concentration and diafiltration
step.
[0699] "Purified form" as used supra is intended to refer to at
least 90% purity, such as 91, 92, 93, 94, 95, 96, 97, 98, 99% w/w
or more pure.
[0700] In the context of this specification "comprising" is to be
interpreted as "including".
[0701] Aspects of the disclosure comprising certain elements are
also intended to extend to alternative embodiments "consisting" or
"consisting essentially" of the relevant elements.
[0702] Positively recited embodiments may be employed herein as a
basis for a disclaimer.
[0703] All references referred to herein are specifically
incorporated by reference.
[0704] The sub-headings herein are employed to assist in
structuring the specification and are not intended to be used to
construct the meaning of technical terms herein.
[0705] Sequences of the disclosure are provided herein below.
TABLE-US-00004 GCN4(7P14P) sequences SEQ ID NO: 1
ASGGGRMKQLEPKVEELLPKNYHLENEVARLKKLVGERHHHHHH wherein the amino
acids in bold are optional SEQ ID NO: 2
GCTAGCGGAGGCGGAAGAATGAAACAACTTGAACCCAAGGTTGAAGAATT
GCTTCCGAAAAATTATCACTTGGAAAATGAGGTTGCCAGATTAAAGAAAT
TAGTTGGCGAACGCCATCACCATCACCATCAC 52SR4 ds scFv- sequence SEQ ID NO:
3 DAVVTQESALTSSPGETVTLTCRSSTGAVTTSNYASWVQEKPDHLFTGLI
GGTNNRAPGVPARFSGSLIGDKAALTITGAQTEDEAIYFCVLWYSDHWVF
GCGTKLTVLGGGGGSGGGGSGGGGSGGGGSDVQLQQSGPGLVAPSQSLSI
TCTVSGFLLTDYGVNWVRQSPGKCLEWLGVIWGDGITDYNSALKSRLSVT
KDNSKSQVFLKMNSLQSGDSARYYCVTGLFDYWGQGTTLTVSSAAAHHHH HHEQKLISEEDL-
SEQ ID NO: 4 GATGCGGTGGTGACCCAGGAAAGCGCGCTGACCAGCAGCCCGGGCGAAAC
CGTGACCCTGACCTGCCGCAGCAGCACCGGCGCGGTGACCACCAGCAACT
ATGCGAGCTGGGTGCAGGAAAAACCGGATCATCTGTTTACCGGCCTGATT
GGCGGCACCAACAACCGCGCGCCGGGCGTGCCGGCGCGCTTTAGCGGCAG
CCTGATTGGCGATAAAGCGGCGCTGACCATTACCGGCGCGCAGACCGAAG
ATGAAGCGATTTATTTTTGCGTGCTGTGGTATAGCGACCATTGGGTGTTT
GGCTGCGGCACCAAACTGACCGTGCTGGGTGGAGGCGGTGGCTCAGGCGG
AGGTGGCTCAGGCGGTGGCGGGTCTGGCGGCGGCGGCAGCGATGTGCAGC
TGCAGCAGAGCGGCCCGGGCCTGGTGGCGCCGAGCCAGAGCCTGAGCATT
ACCTGCACCGTGAGCGGCTTTCTCCTGACCGATTATGGCGTGAACTGGGT
GCGCCAGAGCCCGGGCAAATGCCTGGAATGGCTGGGCGTGATTTGGGGCG
ATGGCATTACCGATTATAACAGCGCGCTGAAAAGCCGCCTGAGCGTGACC
AAAGATAACAGCAAAAGCCAGGTGTTTCTGAAAATGAACAGCCTGCAGAG
CGGCGATAGCGCGCGCTATTATTGCGTGACCGGCCTGTTTGATTATTGGG
GCCAGGGCACCACCCTGACCGTGAGCAGCGCGGCCGCCCATCACCATCAC
CATCACGAACAGAAACTGATTAGCGAAGAAGATCTGTAATAG
CD79b Antibodies
TABLE-US-00005 [0706] Ab 4447 Rabbit Ab 4447 VL region SEQ ID NO:
71 AQVLTQTPSP VSAPVGGTVT INCQASQSVV SGNYLAWLQQ KPGQPPKQLI
HSASTLASGV SSRFSGSGS G TQFTLTISGV QCEDAATYYC LGEFSCSSHD CNAFGGGTEV
VVK Rabbit Ab 4447 VL region SEQ ID NO: 72 gcccaagtgc tgacccagac
tccgtcccct gtgtctgcac ctgtgggagg cacagtcacc atcaattgcc aggccagtca
gagtgttgtt agtggcaatt acctagcctg gcttcagcag aaaccagggc agcctcccaa
gcaactgatc cattctgcat ccactctggc atctggggtc tcatcgcggt tcagcggcag
tggatctggg acacaattca ctctcaccat cagcggcgtg cagtgtgaag atgctgccac
ttactactgt ctaggcgaat ttagttgtag tagtcatgat tgtaatgctt tcggcggagg
gaccgaggtg gtggtcaaa Rabbit Ab 4447 VH region SEQ ID NO: 73
QSLEESGGRL VTPGTPLTLT CTVSGFSLSN YAVSWVRQAP GEGLEWIGII YIETGTTWYA
NWAKGRFTIS KTSTTVDLTI TSPSTEDTAT YFCAREPYEP YDDSNIYYGM DPWGPGTLVT
VSS Rabbit Ab 4447 VH region SEQ ID NO: 74 cagtcgctgg aggagtccgg
gggtcgcctg gtcacgcctg ggacacccct gacactcacc tgcaccgtct ctggattctc
cctcagtaac tatgcagtaa gctgggtccg ccaggctcca ggggagggac tggaatggat
cgggatcatt tatattgaaa ctggtaccac atggtacgcg aactgggcga aaggccgatt
caccatctcc aaaacctcga ccacggtgga tctgacaatc accagtccgt caaccgagga
cacggccacc tatttctgtg ccagagaacc ttatgaacct tatgatgata gtaatattta
ctacggcatg gacccctggg gcccaggcac cctcgtcacc gtctcgagt CDRL1 SEQ ID
NO: 75 QASQSVVSGNYLA CDRL2 SEQ ID NO: 76 SASTLAS CDRL3 SEQ ID NO:
77 LGEFSCSSHDCNA CDRH1 SEQ ID NO: 78 GFSLSNYAVS CDRH2 SEQ ID NO: 79
IIYIETGTTWYANWAKG CDRH3 SEQ ID NO: 80 EPYEPYDDSNIYYGMDP
[0707] The disclosure also extends to a derivative of SEQ ID NO: 77
wherein one or both cysteine are replaced with another amino acid
for example serine, in particular where the first cys is replaced
by serine and the second cys remains unchanged, or the first
cysteine remains unchanged and the second cysteine is replaced by
serine, or where both cysteines are replaced by serine.
TABLE-US-00006 Ab 4450 Rabbit Ab 4450 VL region SEQ ID NO: 81
AIDMTQTPSP VSAAVGGTVT INCQSSQSIY NNNDLAWYQQ KPGQPPKLLI YEASKLASGV
PSRFKGSGSG TQFTLTISGV QCDDAATYYC QGGGSGGDGI AFGGGTKVVV E Rabbit Ab
4450 VL region SEQ ID NO: 82 gccattgata tgacccagac tccatccccc
gtgtctgcag ctgtgggagg cacagtcacc atcaattgcc agtccagtca gagtatttat
aataataatg acttagcctg gtatcagcag aaaccagggc agcctcccaa gctcctgatc
tacgaagcat ccaaactggc atctggggtc ccatcgcggt tcaaaggcag tggatctggg
acacagttca ctctcaccat cagtggcgtg cagtgtgatg atgctgccac ttactactgt
cagggcggtg gtagtggtgg tgatggcatt gctttcggcg gagggaccaa ggtggtcgtc
gaa Rabbit Ab 4450 VH region SEQ ID NO: 83 QSVEESGGRL VTPGAPLTLT
CTVSGFSLNN YVMVWVRQAP GKGLEWIGII YVSGNAYYAS WAKGRFTISR TSTTVDLKVT
SLTTEDTATY FCARDAGHSD VDVLDIWGPG TLVTVSS Rabbit Ab 4450 VH region
SEQ ID NO: 84 cagtcggtgg aggagtccgg gggtcgcctg gtcacgcctg
gggcacccct gacactcacc tgcacagtct ctggattctc cctcaataac tatgtaatgg
tctgggtccg ccaggctcca gggaaggggc tggaatggat cggaatcatt tatgttagtg
gtaatgcata ctacgcgagc tgggcaaaag gccgattcac catctccaga acctcgacca
cggtggatct gaaagtgacc agtctgacaa ccgaggacac ggccacctat ttctgtgcca
gagatgctgg tcatagtgat gtcgatgttt tggatatttg gggcccgggc accctcgtca
ccgtctcgag t CDRL1 SEQ ID NO: 85 QSSQSIYNNNDLA CDRL2 SEQ ID NO: 86
EASKLAS CDRL3 SEQ ID NO: 87 QGGGSGGDGIA CDRH1 SEQ ID NO: 88
GFSLNNYVMV CDRH2 SEQ ID NO: 89 IIYVSGNAYYASWAKG CDRH3 SEQ ID NO: 90
DAGHSDVDVLDI
[0708] The disclosure also extends to a derivative of SEQ ID NO: 87
wherein at least one of the amino acids in the motif DG is replaced
by another amino acid, for example the motif is mutated to EG, DA
or DS.
CD45 Antibodies
TABLE-US-00007 [0709] Ab 4122 Rabbit Ab 4122 VL region SEQ ID NO:
91 DIVMTQTPAS VSEPVGGTVT IMCQASQSIS NWLAWYQQKP GQPPKLLIYQ
ASKLASGVPS RFKGSGSGTE YTLTISDLEC ADAATYYCQS YYDSGSNVFF AFGGGTKVVV E
Rabbit Ab 4122 VL region SEQ ID NO: 92 gacattgtga tgacccagac
tccagcctcc gtgtctgaac ctgtgggagg cacagtcacc atcatgtgcc aggccagtca
gagcattagc aattggttag cctggtatca acagaaacca gggcagcctc ccaagctcct
gatctaccag gcatccaaac tggcatctgg ggtcccatcg cggttcaaag gcagtggatc
tgggacagag tacactctca ccatcagcga cctggagtgt gccgatgctg ccacttacta
ctgtcaaagc tattatgata gtggtagtaa tgtttttttt gctttcggcg gagggaccaa
ggtggtggtc gaa Rabbit Ab 4122 VH region SEQ ID NO: 93 LSLEESGGDL
VKPGASLTLT CTASGFSFSA GYWICWVRQA PGKGLEWIAC TYAGRSGSTY YANWVNGRFT
IPKTSSTTVT LQMTSLSGAD TASYFCARGN AGVAVGALWG PGTLVTVSS Rabbit Ab
4122 VH region SEQ ID NO: 94 ctgtcgttgg aggagtccgg gggagacctg
gtcaagcctg gggcatccct gacactcacc tgcacagcct ctggattctc cttcagtgcc
ggctattgga tatgttgggt ccgccaggct ccagggaagg ggctggagtg gatcgcatgc
acttatgctg gtcgtagtgg tagcacttac tacgcgaact gggtgaatgg ccgattcacc
atccccaaaa cctcgtcgac cacggtgact ctgcaaatga ccagtctgtc aggcgcggac
acggccagct atttctgtgc gagaggtaat gctggtgttg ctgttggtgc cttgtggggc
ccaggcaccc tggtcaccgt ctcgagt CDRL1 SEQ ID NO: 95 QASQSISNWLA CDRL2
SEQ ID NO: 96 QASKLAS CDRL3 SEQ ID NO: 97 QSYYDSGSNVFFA CDRH1 SEQ
ID NO: 98 GFSFSAGYWIC CDRH2 SEQ ID NO: 99 CTYAGRSGSTYYANWVNG CDRH3
SEQ ID NO: 100 GNAGVAVGAL
[0710] The disclosure also extends to a derivative of SEQ ID NO: 97
wherein at least one of the amino acids in the motif DS is replaced
by another amino acid, for example the motif is mutated to DA or
DT
[0711] The disclosure also extends to a derivative of SEQ ID NO: 98
wherein cysteine is replaced by another amino acid, for example
serine.
[0712] The disclosure also extends to a derivative of SEQ ID NO: 99
wherein cysteine is replaced by another amino acid, for example
serine.
[0713] The disclosure also extends to a derivative of SEQ ID NO: 99
wherein at least one of the amino acids in the motif NG is replaced
by another amino acid, for example the motif is mutated to NA, NS
or NT.
TABLE-US-00008 Ab 4129 Rabbit Ab 4129 VL region SEQ ID NO: 101
DIVMTQTPAS VEAAVGGTVT INCQASQSIS SWLSWYQQKP GQPPKLLIYG ASNLASGVPS
RFSGSGSGTQ FSLTISDLEC ADAATYYCQS YYDSGSSVFF NFGGGTKVVV K Rabbit Ab
4129 VL region SEQ ID NO: 102 gacattgtga tgacccagac tccagcctcc
gtggaggcag ctgtgggagg cacagtcacc atcaattgcc aagccagtca gagcattagc
agttggttat cctggtatca gcagaaacca gggcagcctc ccaagctcct gatctatggt
gcatccaatc tggcatctgg ggtcccatca cggttcagcg gcagtggatc tgggacacag
ttcagtctca ccatcagcga cctggagtgt gccgatgctg ccacttacta ctgtcaaagc
tattatgata gtggtagtag tgtttttttt aatttcggcg gagggaccaa ggtggtcgtc
aaa Rabbit Ab 4129 VH region SEQ ID NO: 103 QSLEESGGDL VKPGASLTLT
CTASGFSFSA GYWICWVRQA PGKGLEWIAC IYAGSSGSTY YASWAKGRFT IPKTSSTTVT
LQMTSLTGAD TATYFCARGN AGVAVGALWG PGTLVTVSS Rabbit Ab 4129 VH region
SEQ ID NO: 104 cagtcgttgg aggagtccgg gggagacctg gttaagcctg
gggcatccct gacactcacc tgcacagcct ctggattctc cttcagtgcc ggctattgga
tatgttgggt ccgccaggct ccagggaagg ggctggagtg gatcgcatgc atttatgctg
gtagtagtgg tagcacttac tacgcgagct gggcgaaagg ccgattcacc atccccaaaa
cctcgtcgac cacggtgact ctgcaaatga ccagtctgac aggcgcggac acggccacct
atttctgtgc gagaggtaat gctggtgttg ctgttggtgc cttgtggggc ccaggcaccc
tcgtcaccgt ctcgagt CDRL1 SEQ ID NO: 105 QASQSISSWLS CDRL2 SEQ ID
NO: 106 GASNLAS CDRL3 SEQ ID NO: 107 QSYYDSGSSVFFN CDRH1 SEQ ID NO:
108 GFSFSAGYWIC CDRH2 SEQ ID NO: 109 CIYAGSSGSTYYASWAKG CDRH3 SEQ
ID NO: 110 GNAGVAVGAL
[0714] The disclosure also extends to a derivative of SEQ ID NO:
107 wherein at least one of the amino acids in the motif DS is
replaced by another amino acid, for example the motif is mutated to
DA or DT.
[0715] The disclosure also extends to a derivative of SEQ ID NO:
108 wherein cysteine is replaced by another amino acid, for example
serine.
[0716] The disclosure also extends to a derivative of SEQ ID NO:
109 wherein cysteine is replaced by another amino acid, for example
serine.
TABLE-US-00009 Ab 4131 Rabbit Ab 4131 VL region SEQ ID NO: 111
DIVMTQTPAS VSEPVGGSVT IKCQASQSFY NLLAWYQQKP GQPPKLLIYD ASDLASGVPS
RFKGSGSGTD FTLTISDLEC ADAAAYYCQS ADGSSYAFGG GTEVVVK Rabbit Ab 4131
VL region SEQ ID NO: 112 gacattgtga tgacccagac tccagcctcc
gtgtctgaac ctgtgggagg ctcagtcacc atcaagtgcc aggccagtca gagcttttac
aacctcttag cctggtatca gcagaaacca gggcagcctc ccaaactcct gatctatgat
gcatccgatc tggcatctgg ggtcccatcg cggttcaaag gcagtggatc tgggactgat
ttcactctca ccatcagcga cctggagtgt gccgatgctg ccgcttacta ctgtcaaagt
gctgatggta gtagttacgc tttcggcgga gggaccgagg tggtcgtcaa a Rabbit Ab
4131 VH region SEQ ID NO: 113 QEQLEESGGG LVKPEGSLTL TCTASGVSFS
SSYWIYWVRQ APGKGLEWIA CIYTGSSGST YYASWAKGRF TVSETSSTTV TLQMTSLTAA
DTATYFCARA SAWTYGMDLW GPGTLVTVSS Rabbit Ab 4131 VH region SEQ ID
NO: 114 caggagcaat tggaggagtc cgggggaggc ctggtcaagc ctgagggatc
cctgacactc acctgcacag cctctggagt ctccttcagt agcagctatt ggatatactg
ggtccgccag gctccaggga aggggctgga gtggatcgca tgcatttata ctggtagtag
tggtagcact tactacgcga gctgggcgaa aggccgattc accgtctccg aaacctcgtc
gaccacggtg actctgcaaa tgaccagtct gacagccgcg gacacggcca cctatttctg
tgcgagagca agcgcttgga cctacggcat ggacctctgg ggcccgggca ccctcgtcac
cgtctcgagt CDRL1 SEQ ID NO: 115 QASQSFYNLLA CDRL2 SEQ ID NO: 116
DASDLAS CDRL3 SEQ ID NO: 117 QSADGSSYA CDRH1 SEQ ID NO: 118
GVSFSSSYWIY CDRH2 SEQ ID NO: 119 CIYTGSSGSTYYASWAKG CDRH3 SEQ ID
NO: 120 ASAWTYGMDL
[0717] The disclosure also extends to a derivative of SEQ ID NO:
117 wherein at least one of the amino acids in the motif DS is
replaced by another amino acid, for example the motif is mutated to
DA or DT.
[0718] The disclosure also extends to a derivative of SEQ ID NO:
119 wherein cysteine is replaced by another amino acid, for example
serine.
TABLE-US-00010 Ab 4133 Rabbit Ab 4133 VL region SEQ ID NO: 121
AQVLTQTPSP VSAVVGGTVS ISCQASQSVY NNNNLSWYQQ KPGQPPKLLI YDASKLASGV
PSRFKGSGSG TQFTLTISGV QCDDAATYYC LGGYYSSGWY FAFGGGTKVV VK Rabbit Ab
4133 VL region SEQ ID NO: 122 gcgcaagtgc tgacccagac tccatctccc
gtgtctgcag ttgtgggagg cacagtcagc atcagttgcc aggccagtca gagtgtttat
aataacaaca acttatcctg gtatcagcag aaaccagggc agcctcccaa gctcttgatc
tacgatgcat ccaaattggc atctggggtc ccatcccggt tcaaaggcag tggatctggg
acacagttca ctctcaccat cagcggcgtg cagtgtgacg atgctgccac ttactactgt
ctaggcggtt attatagtag tggttggtat tttgctttcg gcggagggac caaggtggtg
gtcaaa Rabbit Ab 4133 VH region SEQ ID NO: 123 QEQLVESGGG
LVQPEGSLTL TCTASGFSFS GNYYMCWVRQ APGKGLEWIG CLYTGSSGST YYASWAKGRF
TISKTSSTTV TLQMTSLTAA DTATYFCARD LGYEIDGYGG LWGQGTLVTV SS Rabbit Ab
4133 VH region SEQ ID NO: 124 caggagcagc tggtggagtc cgggggaggc
ctggtccagc ctgagggatc cctgacacta acctgcacag cttctggatt ctccttcagt
ggcaactact acatgtgctg ggtccgccag gctccaggga aggggctgga gtggatcgga
tgcctttata ctggtagtag tggtagcaca tattacgcga gctgggcgaa aggccgattc
accatctcca aaacctcgtc gaccacggtg actctgcaaa tgaccagtct gacagccgcg
gacacggcca cctatttctg tgcgagagat ctaggttatg aaattgatgg ttatgggggc
ttgtggggcc agggcaccct cgtcaccgtc tcgagt CDRL1 SEQ ID NO: 125
QASQSVYNNNNLS CDRL2 SEQ ID NO: 126 DASKLAS CDRL3 SEQ ID NO: 127
LGGYYSSGWYFA CDRH1 SEQ ID NO: 128 GFSFSGNYYMC CDRH2 SEQ ID NO: 129
CLYTGSSGSTYYASWAKG CDRH3 SEQ ID NO: 130 DLGYEIDGYGGL
[0719] The disclosure also extends to a derivative of SEQ ID NO:
125 wherein the glycosylation site NLS is removed, for example is
mutated to SLS or QLS.
[0720] The disclosure also extends to a derivative of SEQ ID NO:
128 wherein cysteine is replaced by another amino acid, for example
serine.
[0721] The disclosure also extends to a derivative of SEQ ID NO:
130 wherein at least one of the amino acids in the motif DG is
replaced by another amino acid, for example the motif is mutated to
EG, DA or DS.
Serum Albumin Binding Antibodies
TABLE-US-00011 [0722] CDRH1 dAbH1 SEQ ID NO: 131 Gly Ile Asp Leu
Ser Asn Tyr Ala Ile Asn CDRH2 dAbH1 SEQ ID NO: 132 Ile Ile Trp Ala
Ser Gly Thr Thr Phe Tyr Ala Thr Trp Ala Lys Gly CDRH3 dAbH1 SEQ ID
NO: 133 Thr Val Pro Gly Tyr Ser Thr Ala Pro Tyr Phe Asp Leu CDRL1
dAbL1 SEQ ID NO: 134 Gln Ser Ser Pro Ser Val Trp Ser Asn Phe Leu
Ser CDRL2 dAbL1 SEQ ID NO: 135 Glu Ala Ser Lys Leu Thr Ser CDRL3
dAbL1 SEQ ID NO: 136 Gly Gly Gly Tyr Ser Ser Ile Ser Asp Thr Thr
Heavy chain variable domain of anti-albumin antibody (no ds) SEQ ID
NO: 137 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly
Gly Ser Leu Arg Leu Ser Cys Ala Val Ser Gly Ile Asp Leu Ser Asn Tyr
Ala Ile Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Ile Gly
Ile Ile Trp Ala Ser Gly Thr Thr Phe Tyr Ala Thr Trp Ala Lys Gly Arg
Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Val Tyr Leu Gln Met Asn
Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg Thr Val Pro
Gly Tyr Ser Thr Ala Pro Tyr Phe Asp Leu Trp Gly Gln Gly Thr Leu Val
Thr Val Ser Ser Heavy chain variable domain of anti-albumin
antibody (ds) SEQ ID NO: 138 Glu Val Gln Leu Leu Glu Ser Gly Gly
Gly Leu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Val Ser Gly
Ile Asp Leu Ser Asn Tyr Ala Ile Asn Trp Val Arg Gln Ala Pro Gly Lys
Cys Leu Glu Trp Ile Gly Ile Ile Trp Ala Ser Gly Thr Thr Phe Tyr Ala
Thr Trp Ala Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr
Val Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr
Cys Ala Arg Thr Val Pro Gly Tyr Ser Thr Ala Pro Tyr Phe Asp Leu Trp
Gly Gln Gly Thr Leu Val Thr Val Ser Ser Light chain variable domain
of anti-albumin antibody (no ds) SEQ ID NO: 139 Asp Ile Gln Met Thr
Gln Ser Pro Ser Ser Val Ser Ala Ser Val Gly Asp Arg Val Thr Ile Thr
Cys Gln Ser Ser Pro Ser Val Trp Ser Asn Phe Leu Ser Trp Tyr Gln Gln
Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr Glu Ala Ser Lys Leu Thr
Ser Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr
Leu Thr Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gly
Gly Gly Tyr Ser Ser Ile Ser Asp Thr Thr Phe Gly Gly Gly Thr Lys Val
Glu Ile Lys Arg Thr Light chain variable domain of anti-albumin
antibody (ds) SEQ ID NO: 140 Asp Ile Gln Met Thr Gln Ser Pro Ser
Ser Val Ser Ala Ser Val Gly Asp Arg Val Thr Ile Thr Cys Gln Ser Ser
Pro Ser Val Trp Ser Asn Phe Leu Ser Trp Tyr Gln Gln Lys Pro Gly Lys
Ala Pro Lys Leu Leu Ile Tyr Glu Ala Ser Lys Leu Thr Ser Gly Val Pro
Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser
Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gly Gly Gly Tyr Ser
Ser Ile Ser Asp Thr Thr Phe Gly Cys Gly Thr Lys Val Glu Ile Lys Arg
Thr Human CD79a SEQ ID NO: 141 MPGGPGVLQA LPATIFLLFL LSAVYLGPGC
QALWMHKVPA SLMVSLGEDA HFQCPHNSSN NANVTWWRVL HGNYTWPPEF LGPGEDPNGT
LIIQNVNKSH GGIYVCRVQE GNESYQQSCG TYLRVRQPPP RPFLDMGEGT KNRIITAEGI
ILLFCAVVPG TLLLFRKRWQ NEKLGLDAGD EYEDENLYEG LNLDDCSMYE DISRGLQGTY
QDVGSLNIGD VQLEKP Human CD79b SEQ ID NO: 142 MARLALSPVP SHWMVALLLL
LSAEPVPAAR SEDRYRNPKG SACSRIWQSP RFIARKRGFT VKMHCYMNSA SGNVSWLWKQ
EMDENPQQLK LEKGRMEESQ NESLATLTIQ GIRFEDNGIY FCQQKCNNTS EVYQGCGTEL
RVMGFSTLAQ LKQRNTLKDG IIMIQTLLII LFIIVPIFLL LDKDDSKAGM EEDHTYEGLD
IDQTATYEDI VTLRTGEVKW SVGEHPGQE Human CD45 SEQ ID NO: 143
MYLWLKLLAF GFAFLDTEVF VTGQSPTPSP TGLTTAKMPS VPLSSDPLPT HTTAFSPAST
FERENDFSET TTSLSPDNTS TQVSPDSLDN ASAFNTTGVS SVQTPHLPTH ADSQTPSAGT
DTQTFSGSAA NAKLNPTPGS NAISDVPGER STASTFPTDP VSPLTTTLSL AHHSSAALPA
RTSNTTITAN TSDAYLNASE TTTLSPSGSA VISTTTIATT PSKPTCDEKY ANITVDYLYN
KETKLFTAKL NVNENVECGN NTCTNNEVHN LTECKNASVS ISHNSCTAPD KTLILDVPPG
VEKFQLHDCT QVEKADTTIC LKWKNIETFT CDTQNITYRF QCGNMIFDNK EIKLENLEPE
HEYKCDSEIL YNNHKFTNAS KIIKTDFGSP GEPQIIFCRS EAAHQGVITW NPPQRSFHNF
TLCYIKETEK DCLNLDKNLI KYDLQNLKPY TKYVLSLHAY IIAKVQRNGS AAMCHFTTKS
APPSQVWNMT VSMTSDNSMH VKCRPPRDRN GPHERYHLEV EAGNTLVRNE SHKNCDFRVK
DLQYSTDYTF KAYFHNGDYP GEPFILHHST SYNSKALIAF LAFLIIVTSI ALLVVLYKIY
DLHKKRSCNL DEQQELVERD DEKQLMNVEP IHADILLETY KRKIADEGRL FLAEFQSIPR
VFSKFPIKEA RKPFNQNKNR YVDILPYDYN RVELSEINGD AGSNYINASY IDGFKEPRKY
IAAQGPRDET VDDFWRMIWE QKATVIVMVT RCEEGNRNKC AEYWPSMEEG TRAFGDVVVK
INQHKRCPDY IIQKLNIVNK KEKATGREVT HIQFTSWPDH GVPEDPHLLL KLRRRVNAFS
NFFSGPIVVH CSAGVGRTGT YIGIDAMLEG LEAENKVDVY GYVVKLRRQR CLMVQVEAQY
ILIHQALVEY NQFGETEVNL SELHPYLHNM KKRDPPSEPS PLEAEFQRLP SYRSWRTQHI
GNQEENKSKN RNSNVIPYDY NRVPLKHELE MSKESEHDSD ESSDDDSDSE EPSKYINASF
IMSYWKPEVM IAAQGPLKET IGDFWQMIFQ RKVKVIVMLT ELKHGDQEIC AQYWGEGKQT
YGDIEVDLKD TDKSSTYTLR VFELRHSKRK DSRTVYQYQY TNWSVEQLPA EPKELISMIQ
VVKQKLPQKN SSEGNKHHKS TPLLIHCRDG SQQTGIFCAL LNLLESAETE EVVDIFQVVK
ALRKARPGMV STFEQYQFLY DVIASTYPAQ NGQVKKNNHQ EDKIEFDNEV DKVKQDANCV
NPLGAPEKLP EAKEQAEGSE PTSGTEGPEH SVNGPASPAL NQGS
REFERENCES
[0723] 1. Ribosome display efficiently selects and evolves
high-affinity antibodies in vitro from immune libraries. Hanes J,
Jermutus L, Weber-Bornhauser S, Bosshard H R, Pluckthun A. (1998)
Proc. Natl. Acad. Sci. U.S.A. 95, 14130-14135 [0724] 2. Directed in
Vitro Evolution and Crystallographic Analysis of a Peptide-binding
Single Chain Antibody Fragment (scFv) with Low Picomolar Affinity.
Zhand C, Spinelli S, Luginbuhl B, Amstutz P, Cambillau C, Pluckthun
A. (2004) J. Biol. Chem. 279, 18870-18877 [0725] 3. Antigen
recognition by conformational selection. Berger C, Weber-Bornhauser
S, Eggenberger Y, Hanes J, Pluckthun A, Bosshard H. R. (1999)
F.E.B.S. Letters 450, 149-153
EXAMPLES
[0726] The term Fab-Kd-Fab as used in the Examples describes the
bispecific protein complex having the formula A-X:Y-B wherein:
[0727] A-X is a first fusion protein; [0728] Y-B is a second fusion
protein; [0729] X:Y is a heterodimeric-tether; [0730] A comprises a
Fab fragment specific to an antigen such as CD45 or CD79; [0731] B
comprises a Fab fragment specific to an antigen such as CD45 or
CD79; [0732] X is a first binding partner of a binding pair such as
a scFv; [0733] Y is a second binding partner of the binding pair
such as a peptide; and [0734] : is an interaction (such as a
binding interaction) between X and Y.
Example 1--Production of Fab-A (Fab-scFv [A-X]) and Fab-B
(Fab-Peptide [B-Y) for Functional Assays
Cloning Strategy
[0735] Antibody variable region DNA was generated by PCR or gene
synthesis flanking restriction enzyme sites DNA sequence. These
sites were HindIII and XhoI for variable heavy chains and HindIII
and BsiWI for variable light chains. This makes the heavy variable
region amenable to ligating into the two heavy chain vectors (pNAFH
with FabB-Y and pNAFH with FabA-Xds [disulphide stabilised]) as
they have complementary restriction sites. This ligates the
variable region upstream (or 5') to the murine constant regions and
peptide Y (GCN4) or scFv X (52SR4) creating a whole reading frame.
The light chains were cloned into standard in house murine constant
kappa vectors (pMmCK or pMmCK S171C) which again use the same
complimentary restriction sites. The pMmCK S171C vector is used if
the variable region is isolated from a rabbit. The cloning events
were confirmed by sequencing using primers which flank the whole
open reading frame.
Cultivating CHOS
[0736] Suspension CHOS cells were pre-adapted to CDCHO media
(Invitrogen) supplemented with 2 mM (100.times.) glutamx. Cells
were maintained in logarithmic growth phase agitated at 140 rpm on
a shaker incubator (Kuner AG, Birsfelden, Switzerland) and cultured
at 37.degree. C. supplemented with 8% CO.sub.2.
Electroporation Transfection
[0737] Prior to transfection, the cell numbers and viability were
determined using CEDEX cell counter (Innovatis AG. Bielefeld,
Germany) and required amount of cells (2.times.10.sup.8 cells/ml)
were transferred into centrifuge conical tubes and were spun at
1400 rpm for 10 minutes. The Pelleted cells were re-suspended in
sterile Earls Balanced Salts Solution and spun at 1400 rpm for
further 10 minutes. Supernatant was discarded and pellets were
re-suspended to desired cell density.
[0738] Vector DNA at a final concentration of 400 ug for
2.times.10.sup.8 cells/ml mix and 800 .mu.l was pipetted into
Cuvettes (Biorad) and electroporated using in-house electroporation
system.
[0739] Transfected cells were transferred directly into 1.times.3 L
Erlenmeyer Flasks contained ProCHO 5 media enriched with 2 mM
glutmax and antibiotic antimitotic (100.times.) solution (1 in 500)
and Cells were cultured in Kuhner shaker incubator set at
37.degree. C., 5% CO.sub.2 and 140 rpm shaking. Feed supplement 2
g/L ASF (AJINOMOTO) was added at 24 hr post transfection and
temperature dropped to 32.degree. C. for further 13 days culture.
At day four 3 mM Sodium buryrate (n-BUTRIC ACID Sodium Salt, Sigma
B-5887) was added to the culture.
[0740] On day 14, cultures were transferred to tubes and
supernatant separated from the cells after centrifugation for 30
minutes at 4000 rpm. Retained supernatants were further filtered
through 0.22 um SARTO BRAN P Millipore followed by 0.22 .mu.m Gamma
gold filters. Final expression levels were determined by Protein
G-HPLC.
Large Scale (1.0 L) Purification
[0741] The Fab-A and Fab-B were purified by affinity capture using
the AKTA Xpress systems and HisTrap Excel pre-packed nickel columns
(GE Healthcare). The culture supernatants were 0.22 .mu.m sterile
filtered and pH adjusted to neutral, if necessary, with weak acid
or base before loading onto the columns. A secondary wash step,
containing 15-25 mM Imidazole, was used to displace any weakly
bound host cell proteins/non-specific His binders from the nickel
resin. Elution was performed with 10 mM sodium phosphate, pH7.4+1M
NaCl+250 mM Imidazole and 2 ml fractions collected. One column
volume into the elution the system was paused for 10 minutes to
tighten the elution peak, and consequently decrease the total
elution volume. The cleanest fractions were pooled and buffer
exchanged into PBS (Sigma), pH7.4 and 0.22 .mu.m filtered. Final
pools were assayed by A280 Scan, SE-HPLC (G3000 method), SDS-PAGE
(reduced & non-reduced) and for endotoxin using the PTS
Endosafe system.
Example 2--CD45 Fab/CD79Fab Bispecific Complex but not a Mixture of
CD45 and CD79 Fab or Bivalent CD79 Fab Complex Inhibits Akt
Signalling
[0742] Human PBMC derived from platelet apheresis cones were banked
as frozen aliquots. Prior to an assay being performed, cells were
thawed, washed in DMEM (Life Technologies) and allowed to
acclimatise to a 37.degree. C./5% CO.sub.2 environment. During this
period grids of bispecific or bivalent antibodies were created by
diluting equimolar (200 nM) quantities of Fab'-A (Fab-scFv) and
Fab-B (Fab-peptide) or Fab-A (Fab-peptide) and Fab-B (Fab-peptide)
with antigen specificity for the cell surface proteins CD45 and
CD79b in DMEM containing 10% calf serum and 2 mM glutamine. This
grid is shown in Table 4.
TABLE-US-00012 TABLE 4 Grid of bispecific and bivalent combinations
of antibodies with specificity for CD45 and CD79b. (A-X or Y) (B-Y)
Fab B Fab A CD45-Y CD79b-Y CD45-X CD45-X:Y-CD45 CD45-X:Y-CD79b
CD79b-X CD79b-X:Y-CD45 CD79b-X:Y-CD79b CD45-Y CD45-Y:CD79b-Y where
X is a scFv (52SR4) and Y is a peptide (GCN4)
[0743] FabA-X and FabB-Y or Fab-A-Y and Fab-B-Y were incubated
together for 90 minutes (in a 37.degree. C./5% CO.sub.2
environment) before mixing with 2.5.times.10.sup.5 PBMC in V
bottomed 96 well plates. PBMC plus bispecific or bivalent
combinations were then incubated together for a further 90 minutes.
After this time B cells were activated by the addition of 200 nM of
goat F(ab').sub.2 anti-human IgM (Southern Biotechnology) for 8
minutes at 37.degree. C. The signalling reaction was then halted by
adding an equal volume of Cytofix buffer (BD Biosciences). Plates
were then left at room temperature for 15 minutes before
centrifugation at 500 g for 5 minutes. Excess supernatant was
discarded from the cell pellet which was resuspended in flow buffer
(PBS+1% BSA+0.01% NaN.sub.3) and washed once more. Cells were then
resuspended in ice cold Perm Buffer III (BD Biosciences) for 30
minutes before being washed twice in flow buffer. Cells were then
stained with a fluorescently labelled anti-CD20 antibody (BD
Biosciences) and a fluorescently labelled anti-phospho Akt antibody
that recognises a modified serine residue at position 473 on the
protein. Plates were then resuspended and incubated for 1 hour at
room temperature in the dark. After this time plates were washed a
further two times and resuspended in 25 .mu.l of flow buffer.
Cellular expression of CD20 and Akt was measured using an
Intellicyt HTFC flow cytometer.
[0744] Using the data analysis software package FORECYT
(Intellicyt) B cells were identified as distinct from other cell
populations and the geometric mean of Akt levels was calculated for
each well. All data was then expressed as the percentage inhibition
of the maximal response (anti-IgM only) minus the background (cells
only). The relative effect of the combinations of CD45 and CD79b is
shown in Table 5 (.dwnarw.=inhibition, .uparw.=stimulation and =no
overall effect).
TABLE-US-00013 TABLE 5 Table of the relative potency of inhibition
of phosphorylated Akt for bispecific & bivalent combinations of
antibodies with specificity for CD45 & CD79b. (A-X) (B-Y) Fab B
Fab A CD45-Y CD79b-Y CD45-X Not Tested Not Tested CD79b-X
.dwnarw..dwnarw. CD45-Y Not tested
where X is a scFv (52SR4) and Y is a peptide (GCN4).
[0745] This data is also shown in the form of a bar chart (FIG. 1):
the data represents mean values and the error bars are 95%
confidence intervals. The data shows that the bispecific
combination of CD45 with CD79b can inhibit phospho-Akt expression
in B cells stimulated with anti-IgM, whereas combining CD79b-Y with
CD79b-Y, which is a mixture which cannot form a bispecific, does
not.
Example 3--CD45 Fab/CD79Fab Bispecific Complex but not a Mixture of
CD45 and CD79 Fab or Bivalent CD79 Fab' Complex Inhibits
PLC.gamma.2 Signalling
[0746] Human PBMC derived from platelet apheresis cones were banked
as frozen aliquots. Prior to an assay being performed cells were
thawed, washed in DMEM (Life Technologies) and allowed to
acclimatise to a 37.degree. C. /5% CO.sub.2 environment. During
this period grids of bispecific or bivalent antibodies were created
by diluting equimolar (200 nM) quantities of Fab-a (Fab-scFv [A-X])
and Fab'-B (Fab-peptide [B-Y]) or Fab-A (Fab-peptide) and Fab-B
(Fab-peptide with antigen specificity for the cell surface proteins
CD45 and CD79b in DMEM containing 10% calf serum and 2 mM
glutamine. This grid is shown in Table 4.
[0747] Fab'A-X and Fab'B-Y or Fab-A-Y and Fab-B-Y were incubated
together for 90 minutes (in a 37.degree. C./5% CO.sub.2
environment) before mixing with 2.5.times.10.sup.5 PBMC in V
bottomed 96 well plates. PBMC plus bispecific or bivalent
combinations were then incubated together for a further 90 minutes.
After this time B cells were activated by the addition of 200 nM of
goat F(ab')2 anti-human IgM (Southern Biotechnology) for 8 minutes
at 37.degree. C. The signalling reaction was then halted by adding
an equal volume of Cytofix buffer (BD Biosciences). Plates were
then left at room temperature for 15 minutes before centrifugation
at 500 g for 5 minutes. Excess supernatant was discarded from the
cell pellet which was resuspended in flow buffer and washed once
more. Cells were then resuspended in ice cold Perm Buffer III (BD
Biosciences) for 30 minutes before being washed twice in flow
buffer.
[0748] Cells were then stained with a fluorescently labelled
anti-CD20 antibody (BD Biosciences) and a fluorescently labelled
anti-phospho PLC.gamma.2 antibody that recognises a modified
tyrosine residue at position 759 on the protein. Plates were then
resuspended and incubated for 1 hour at room temperature in the
dark. After this time plates were washed a further two times and
resuspended in 25 .mu.l of flow buffer. Cellular expression of CD20
and PLCg2 was measured using an Intellicyt HTFC flow cytometer.
[0749] Using the data analysis software package FORECYT
(Intellicyt) B cells were identified as distinct from other cell
populations and the geometric mean of PLC.gamma.2 levels was
calculated for each well. All data was then expressed as the
percentage inhibition of the maximal response (anti-IgM only) minus
the background (cells only). The relative effect of the combination
of CD45 and CD79b is shown in Table 6 (.dwnarw.=inhibition,
.uparw.=stimulation and =no overall effect).
TABLE-US-00014 TABLE 6 Table of the relative potency of inhibition
of phosphorylated PLCg2 for bispecific and bivalent combinations of
antibodies with specificity for CD45 and CD79b. (A-X or Y) (B-Y)
Fab B Fab A CD45-Y CD79b-Y CD45-X Not Tested Not Tested CD79b-X
.dwnarw..dwnarw..dwnarw. CD45-Y Not tested where X is a scFv and Y
is a peptide
[0750] This data can also be expressed as a bar chart (FIG. 2), the
data represents mean values and the error bars are 95% confidence
intervals. The data shows that the bispecific combination of CD45
with CD79b, inhibit phospho-PLC.gamma.2 expression in B cells
stimulated with anti-IgM, whereas combining CD79b-Y with CD79b-Y,
which is a mixture which cannot form a bispecific, does not.
Example 4 the Bispecific CD45 and CD79b Complex can Potently
Inhibit the Expression of CD86 on B Cells
[0751] Human PBMC derived from platelet apheresis cones were banked
as frozen aliquots. Prior to an assay being performed cells were
thawed, washed in DMEM (Life Technologies) and allowed to
acclimatise to a 37 degree C./5% CO.sub.2 environment. During this
period bispecific combinations were created by diluting equimolar
(500 nM) quantities of Fab-X (Fab-scFv) and Fab-Y (Fab-peptide)
with antigen specificity for the cell surface proteins CD45 and
CD79b in DMEM containing 10% calf serum and 2 mM glutamine. These
combinations were then diluted in 8 stepwise 1 in 2.5 dilutions to
create a dose titration for this combination. Fab-X and Fab-Y were
incubated together for 90 minutes (in a 37 degree C./5% CO.sub.2
environment) before adding 2.5.times.105 PBMC to V bottomed 96 well
plates. PBMC were then added to Fab'-X and Fab'-Y combinations and
incubated together for a further 90 minutes. After this time B
cells were activated by the addition of 200 nM of goat F(ab')2
anti-human IgM (Southern Biotechnology) for 24 hours at 37 degrees
C. To enable detection of cell surface activation markers plates
were placed on ice and washed once in ice cold flow buffer (PBS+1%
BSA+0.01% NaN3). Cells were then stained with a fluorescently
labelled anti-CD19 antibody (BD Biosciences) and a fluorescently
labelled anti-CD86 antibody and incubated on ice for 1 hour in the
dark. After this time plates were washed a further two times and
resuspended in 25 ul of flow buffer. Cellular expression of CD19
and CD86 was measured using an Intellicyt HTFC flow cytometer.
Using the data analysis software package FORECYT (Intellicyt) B
cells were identified as distinct from other cell populations and
the geometric mean of CD86 levels was calculated for each well. All
data was then expressed as the percentage inhibition of the maximal
response (anti-IgM only) minus the background (cells only). As can
be seen in FIG. 3 a titration of the combination of CD45-X/CD79b-Y
was able to inhibit anti-IgM induced CD86 expression on B cells
after 24 hours. The IC50, as extrapolated using a 4 parameter
logistic curve fit using Graphpad Prism 6, was 4.7 nM (the data
represents mean values and the error bars are standard
deviations).
Example 5--the Inhibitory Effect of CD45 and CD79b Bispecific
Protein can be Reproduced with Different Antibody V Regions
[0752] Immunisation:
[0753] DNA encoding antigens CD79a and CD79b and CD45 was obtained
by gene synthesis or commercial sources & cloned into an
expression vector with a strong constitutive promoter. Plasmid DNA
was then transfected into Rab-9 rabbit fibroblast cells (ATCC
CRL-1414) using an in-house electroporation system. For CD79
immunisations, both CD79a and CD79b were co-transfected. Twenty
four hours later cells were checked for antigen expression by flow
cytometry & frozen in aliquots in liquid nitrogen until use. Up
to 6 antigens were immunised per rabbit by either co-expression on
the same cell or making mixtures of singly or multiple transfected
cells. Rabbits were immunised with 3 doses of cells.
[0754] Antibody Discovery:
[0755] B cell cultures were prepared using a method similar to that
described by Zubler et al. (1985). Briefly, spleen or PBMC-derived
B cells from immunized rabbits were cultured at a density of
approximately 2000-5000 cells per well in bar-coded 96-well tissue
culture plates with 200 .mu.l/well RPMI 1640 medium (Gibco BRL)
supplemented with 10% FCS (PAA laboratories ltd), 2% HEPES (Sigma
Aldrich), 1% L-Glutamine (Gibco BRL), 1% penicillin/streptomycin
solution (Gibco BRL), 0.1% .beta.-mercaptoethanol (Gibco BRL), 3%
activated splenocyte culture supernatant and gamma-irradiated
mutant EL4 murine thymoma cells (5.times.10.sup.4/well) for seven
days at 37.degree. C. in an atmosphere of 5% CO.sub.2.
[0756] The presence of antigen-specific antibodies in B cell
culture supernatants was determined using a homogeneous
fluorescence-based binding assay using HEK293 cells co-transfected
with CD79a and CD79b or CD45. Screening involved the transfer of 10
ul of supernatant from barcoded 96-well tissue culture plates into
barcoded 384-well black-walled assay plates containing HEK293 cells
transfected with target antigen (approximately 3000 cells/well)
using a Matrix Platemate liquid handler. Binding was revealed with
a goat anti-rabbit IgG Fc.gamma.-specific Cy-5 conjugate (Jackson).
Plates were read on an Applied Biosystems 8200 cellular detection
system.
[0757] Following primary screening, positive supernatants were
consolidated on 96-well bar-coded master plates using an Aviso Onyx
hit-picking robot and B cells in cell culture plates frozen at
-80.degree. C. Master plates were then screened in a homogeneous
fluorescence-based binding assay on HEK293 cells transfected with
CD79a and CD79b or CD45 antigens or SUPERAVIDIN beads (Bangs
Laboratories) coated with recombinant CD45 protein as a source of
antigen. This was done in order to determine the antigen
specificity for each well.
[0758] To allow recovery of antibody variable region genes from a
selection of wells of interest, a deconvolution step was performed
to enable identification of the antigen-specific B cells in a given
well that contained a heterogeneous population of B cells. This was
achieved using the Fluorescent foci method (Clargo et al., 2014.
Mabs 2014 Jan. 1: 6(1) 143-159; EP1570267B1). Briefly,
Immunoglobulin-secreting B cells from a positive well were mixed
with either HEK293 cells transfected with target antigen or
streptavidin beads (New England Biolabs) coated with biotinylated
target antigen and a 1:1200 final dilution of a goat anti-rabbit
Fc.gamma. fragment-specific FITC conjugate (Jackson). After static
incubation at 37.degree. C. for 1 hour, antigen-specific B cells
could be identified due to the presence of a fluorescent halo
surrounding that B cell. A number of these individual B cell
clones, identified using an Olympus microscope, were then picked
with an Eppendorf micromanipulator and deposited into a PCR tube.
The fluorescent foci method was also used to identify
antigen-specific B cells from a heterogeneous population of B cells
directly from the bone marrow of immunized rabbits.
[0759] Antibody variable region genes were recovered from single
cells by reverse transcription (RT)-PCR using heavy and light chain
variable region-specific primers. Two rounds of PCR were performed,
with the nested secondary PCR incorporating restriction sites at
the 3' and 5' ends allowing cloning of the variable region into
mouse Fab-X and Fab-Y (VH) or mouse kappa (VL) mammalian expression
vectors. Heavy and light chain constructs for the Fab-X and Fab-Y
expression vectors were co-transfected into HEK-293 cells using
Fectin 293 (Life Technologies) or Expi293 cells using Expifectamine
(Life Technologies) and recombinant antibody expressed in 6-well
tissue culture plates in a volume of 5 ml. After 5-7 days
expression, supernatants were harvested. Supernatants were tested
in a homogeneous fluorescence-based binding assay on HEK293 cells
transfected with antigen and SUPERAVIDIN beads (Bangs Laboratories)
coated with recombinant protein or antigen transfected HEK cells.
This was done to confirm the specificity of the cloned antibodies.
Production of small scale Fab A-X and Fab B-Y (Small Scale (50 mL)
Expi293 Transfection)
[0760] The Expi293 cells were routinely sub-cultured in EXPI293
Expression Medium to a final concentration of 0.5.times.10.sup.6
viable cells/mL and were incubated in an orbital shaking incubator
(Multitron, Infors HT) at 120 rpm 8% CO.sub.2 and 37.degree. C.
[0761] On the day of transfection cell viability and concentration
were measured using an automated Cell Counter (Vi-CELL, Beckman
Coulter). To achieve a final cell concentration of
2.5.times.10.sup.6 viable cells/mL the appropriate volume of cell
suspension was added to a sterile 250 mL Erlenmeyer shake flask and
brought up to the volume of 42.5 mL by adding fresh, pre-warmed
EXPI293 Expression Medium for each 50 mL transfection.
[0762] To prepare the lipid-DNA complexes for each transfection a
total of 50 .mu.g of heavy chain and light chain plasmid DNAs were
diluted in OPTI MEM I medium (LifeTechnologies) to a total volume
of 2.5 mL and 135 .mu.L of EXPIFECTAMINE 293 Reagent
(LifeTechnologies) was diluted in OPTI MEM I medium to a total
volume of 2.5 mL. All dilutions were mixed gently and incubate for
no longer than 5 minutes at room temperature before each DNA
solution was added to the respective diluted EXPIFECTAMINE 293
Reagent to obtain a total volume of 5 mL. The DNA-EXPIFECTAMINE 293
Reagent mixtures were mixed gently and incubated for 20-30 minutes
at room temperature to allow the DNA-EXPIFECTAMINE 293 Reagent
complexes to form.
[0763] After the DNA-EXPIFECTAMINE 293 reagent complex incubation
was completed, the 5 mL of DNA-EXPIFECTAMINE 293 Reagent complex
was added to each shake flask. The shake flasks were incubated in
an orbital shaking incubator (Multitron, Infors HT) at 120 rpm, 8%
CO.sub.2 and 37.degree. C.
[0764] Approximately 16-18 hours post-transfection, 250 .mu.L of
EXPIFECTAMINE 293 Transfection Enhancer 1 (LifeTechnologies) and
2.5 mL of EXPIFECTAMINE 293 Transfection Enhancer 2
(LifeTechnologies) were added to each shake flask.
[0765] The cell cultures were harvested 7 days post transfection.
The cells were transferred into 50 mL spin tubes (Falcon) and spun
down for 30 min at 4000 rpm followed by sterile filtration through
a 0.22 um Stericup (Merck Millipore). The clarified and sterile
filtered supernatants were stored at 4.degree. C. Final expression
levels were determined by Protein G-HPLC. Small Scale (50 ml)
Purification:
[0766] Both Fab-X and Fab-Y were purified separately by affinity
capture using a small scale vacuum based purification system.
Briefly, the 50 ml of culture supernatants were 0.22 .mu.m sterile
filtered before 500 .mu.L, of Ni Sepharose beads (GE Healthcare)
were added. The supernatant beads mixture was then tumbled for
about an hour before supernatant was removed by applying vacuum.
Beads were then washed with Wash 1 (50 mM Sodium Phosphate 1 M NaCl
pH 6.2) and Wash 2 (0.5 M NaCl). Elution was performed with 50 mM
sodium acetate, pH4.0+1M NaCl. The eluted fractions buffer
exchanged into PBS (Sigma), pH7.4 and 0.22 .mu.m filtered. Final
pools were assayed by A280 scan, SE-UPLC (BEH200 method), SDS-PAGE
(reduced & non-reduced) and for endotoxin using the PTS
Endosafe system.
[0767] Human PBMC derived from platelet apheresis cones were banked
as frozen aliquots. Prior to an assay being performed cells were
thawed, washed in RPMI 1640 (Life Technologies) and allowed to
acclimatise to a 37.degree. C./5% CO.sub.2 environment. During this
period combinations of bispecific, bivalent or mixtures of
antibodies were created by diluting equimolar (200 nM) quantities
of Fab'-X (Fab-scFv) and Fab'-Y (Fab-peptide) with antigen
specificity for the cell surface proteins CD45 and CD79b in RPMI
1640 containing 10% fetal bovine serum, 50 units/mL Penicillin, 50
.mu.g/mL Streptomycin and 2 mM L-glutamine. These combinations of 3
different CD79b Fab-Ys and 2 different CD45 Fab-Xs are shown in
Table 7.
TABLE-US-00015 TABLE 7 Grid of bispecific proteins with specificity
for CD45 and CD79b. (A-X) (B-Y) Fab B Fab A CD79-Y VR4447 CD79-Y
VR4450 CD79b-y VR4246 CD45-X CD45-X:Y-CD79b CD45-X:Y-CD79b
CD45-X:Y-CD79b VR4131 CD45X CD45-X:Y-CD79b CD45-X:Y-CD79b
CD45-X:Y-CD79b VR4248 where X is a scFv (52SR4) and Y is a peptide
(GCN4)
[0768] FabA-X and FabB-Y were incubated together for 60 minutes (in
a 37.degree. C./5% CO.sub.2 environment) before mixing with
2.5.times.10.sup.5 PBMC in V bottomed 96 well plates. PBMC plus
FabA-X and/or FabB-Y combinations were then incubated together for
a further 90 minutes. After this time B cells were activated by the
addition of 12.5 .mu.g/mL of goat F(ab')2 anti-human IgM (Southern
Biotechnology) for 10 minutes at 37.degree. C. The signalling
reaction was then halted by adding an equal volume of Cytofix
buffer (BD Biosciences). Plates were then left at room temperature
for 15 minutes before centrifugation at 500.times.g for 5 minutes.
Excess supernatant was discarded from the cell pellet which was
resuspended in flow buffer (PBS+1% BSA+0.1% NaN.sub.3+2 mM EDTA)
and washed once more. Cells were then resuspended in ice cold Perm
Buffer III (BD Biosciences) for 30 minutes before being washed
twice in flow buffer.
[0769] Cells were then stained with a fluorescently labelled
anti-CD20 antibody (BD Biosciences), and an anti-phospho
PLC.gamma.2 antibody that recognises a modified tyrosine residue at
position 759. Plates were then resuspended and incubated for 1 hour
at room temperature in the dark. After this time plates were washed
a further two times and resuspended in 40 .mu.l of flow buffer.
Cellular expression of CD20 and PLC.gamma.2 was measured using an
Intellicyt HTFC flow cytometer.
[0770] Using the data analysis software package FORECYT
(Intellicyt) B cells were identified as distinct from other cell
populations and the geometric mean of PLC.gamma.2 levels were
calculated for each well. All data was then expressed as the
percentage inhibition of the maximal response (anti-IgM only) minus
the background (cells only).
[0771] As can be seen in FIG. 4 the data shows that the combination
of CD45 with CD79b with different antibody V regions can inhibit
phospho-PLC.gamma.2 expression in B cells stimulated with
anti-IgM.
Example 6: Grid Screening of Large Panels of Heterodimerically
Tethered Protein Complexes to Identify Novel Bispecific Antibody
Targets
[0772] Introduction:
[0773] Following the successful validation of the bispecific format
and screening method in the earlier examples the screening was
expanded to a larger number of antigen pairs. A panel of antibody
variable (V) region pairs to 23 different antigens expressed on B
cells was generated. Using the Fab-Kd-Fab [i.e. A-X:Y-B wherein A
and B are Fab fragments] format a grid of heterodimerically
tethered protein complexes was formed representing multiple V
region combinations of each of 315 different antigen pair
combinations. These combinations were screened for their ability to
modulate BCR (B cell receptor) signalling in a high through-put
flow cytometry assay to select novel target pairs for intervention
with a bispecific antibody.
[0774] Immunisation:
[0775] DNA encoding selected antigens was obtained by gene
synthesis or commercial sources & cloned into an expression
vector with a strong constitutive promoter. Plasmid DNA was then
transfected into Rab-9 rabbit fibroblast cells (ATCC CRL-1414)
using an in-house electroporation system. Twenty four hours later
cells were checked for antigen expression by flow cytometry &
frozen in aliquots in liquid nitrogen until use. Up to 6 antigens
were immunised per rabbit by either co-expression on the same cell
or making mixtures of singly or multiple transfected cells. Rabbits
were immunised with 3 doses of cells.
[0776] Antibody Discovery:
[0777] B cell cultures were prepared using a method similar to that
described by Zubler et al. (1985). Briefly, spleen or PBMC-derived
B cells from immunized rabbits were cultured at a density of
approximately 2000-5000 cells per well in bar-coded 96-well tissue
culture plates with 200 .mu.l/well RPMI 1640 medium (Gibco BRL)
supplemented with 10% FCS (PAA laboratories ltd), 2% HEPES (Sigma
Aldrich), 1% L-Glutamine (Gibco BRL), 1% penicillin/streptomycin
solution (Gibco BRL), 0.1% .beta.-mercaptoethanol (Gibco BRL), 3%
activated splenocyte culture supernatant and gamma-irradiated
mutant EL4 murine thymoma cells (5.times.10.sup.4/well) for seven
days at 37.degree. C. in an atmosphere of 5% CO.sub.2.
[0778] The presence of antigen-specific antibodies in B cell
culture supernatants was determined using a homogeneous
fluorescence-based binding assay using HEK293 cells co-transfected
with the antigens that the rabbits were immunized with. Screening
involved the transfer of 10 ul of supernatant from barcoded 96-well
tissue culture plates into barcoded 384-well black-walled assay
plates containing HEK293 cells transfected with target antigen
(approximately 3000 cells/well) using a Matrix Platemate liquid
handler. Binding was revealed with a goat anti-rabbit IgG
Fc.gamma.-specific Cy-5 conjugate (Jackson). Plates were read on an
Applied Biosystems 8200 cellular detection system.
[0779] Following primary screening, positive supernatants were
consolidated on 96-well bar-coded master plates using an Aviso Onyx
hit-picking robot and B cells in cell culture plates frozen at
-80.degree. C. Master plates were then screened in a homogeneous
fluorescence-based binding assay on HEK293 cells transfected with
antigens separately and SUPERAVIDIN beads (Bangs Laboratories)
coated with recombinant protein as a source of antigen. This was
done in order to determine the antigen specificity for each
well.
[0780] To allow recovery of antibody variable region genes from a
selection of wells of interest, a deconvolution step was performed
to enable identification of the antigen-specific B cells in a given
well that contained a heterogeneous population of B cells. This was
achieved using the Fluorescent foci method (Clargo et al.,
2014.Mabs 2014 Jan. 1: 6(1) 143-159; EP1570267B1). Briefly,
Immunoglobulin-secreting B cells from a positive well were mixed
with either HEK293 cells transfected with target antigen or
streptavidin beads (New England Biolabs) coated with biotinylated
target antigen and a 1:1200 final dilution of a goat anti-rabbit
Fc.gamma. fragment-specific FITC conjugate (Jackson). After static
incubation at 37.degree. C. for 1 hour, antigen-specific B cells
could be identified due to the presence of a fluorescent halo
surrounding that B cell. A number of these individual B cell
clones, identified using an Olympus microscope, were then picked
with an Eppendorf micromanipulator and deposited into a PCR tube.
The fluorescent foci method was also used to identify
antigen-specific B cells from a heterogeneous population of B cells
directly from the bone marrow of immunized rabbits.
[0781] Antibody variable region genes were recovered from single
cells by reverse transcription (RT)-PCR using heavy and light chain
variable region-specific primers. Two rounds of PCR were performed,
with the nested secondary PCR incorporating restriction sites at
the 3' and 5' ends allowing cloning of the variable region into
mouse Fab-X and Fab-Y (VH) or mouse kappa (VL) mammalian expression
vectors. Heavy and light chain constructs for the Fab-X and Fab-Y
expression vectors were co-transfected into HEK-293 cells using
Fectin 293 (Life Technologies) or Expi293 cells using Expifectamine
(Life Technologies) and recombinant antibody expressed in 6-well
tissue culture plates in a volume of 5 ml. After 5-7 days
expression, supernatants were harvested. Supernatants were tested
in a homogeneous fluorescence-based binding assay on HEK293 cells
transfected with antigen and SUPERAVIDIN beads (Bangs Laboratories)
coated with recombinant protein or antigen transfected HEK cells.
This was done to confirm the specificity of the cloned
antibodies.
[0782] Production of small scale Fab A-X and Fab B-Y (Small Scale
(50 mL) Expi293 Transfection) The Expi293 cells were routinely
sub-cultured in EXPI293 Expression Medium to a final concentration
of 0.5.times.10.sup.6 viable cells/mL and were incubated in an
orbital shaking incubator (Multitron, Infors HT) at 120 rpm 8%
CO.sub.2 and 37.degree. C.
[0783] On the day of transfection cell viability and concentration
were measured using an automated Cell Counter (Vi-CELL, Beckman
Coulter). To achieve a final cell concentration of
2.5.times.10.sup.6 viable cells/mL the appropriate volume of cell
suspension was added to a sterile 250 mL Erlenmeyer shake flask and
brought up to the volume of 42.5 mL by adding fresh, pre-warmed
EXPI293 Expression Medium for each 50 mL transfection.
[0784] To prepare the lipid-DNA complexes for each transfection a
total of 50 .mu.g of heavy chain and light chain plasmid DNAs were
diluted in OPTI-MEM I medium (LifeTechnologies) to a total volume
of 2.5 mL and 135 .mu.L of EXPIFECTAMINE 293 Reagent
(LifeTechnologies) was diluted in OPTI-MEM I medium to a total
volume of 2.5 mL. All dilutions were mixed gently and incubate for
no longer than 5 minutes at room temperature before each DNA
solution was added to the respective diluted EXPIFECTAMINE 293
Reagent to obtain a total volume of 5 mL. The DNA-EXPIFECTAMINE 293
Reagent mixtures were mixed gently and incubated for 20-30 minutes
at room temperature to allow the DNA-EXPIFECTAMINE 293 Reagent
complexes to form.
[0785] After the DNA-EXPIFECTAMINE 293 reagent complex incubation
was completed, the 5 mL of DNA-EXPIFECTAMINE 293 Reagent complex
was added to each shake flask. The shake flasks were incubated in
an orbital shaking incubator (Multitron, Infors HT) at 120 rpm, 8%
CO.sub.2 and 37.degree. C.
[0786] Approximately 16-18 hours post-transfection, 250 .mu.L of
EXPIFECTAMINE 293 Transfection Enhancer 1 (LifeTechnologies) and
2.5 mL of EXPIFECTAMINE 293 Transfection Enhancer 2
(LifeTechnologies) were added to each shake flask.
[0787] The cell cultures were harvested 7 days post transfection.
The cells were transferred into 50 mL spin tubes (Falcon) and spun
down for 30 min at 4000 rpm followed by sterile filtration through
a 0.22 um Stericup (Merck Millipore). The clarified and sterile
filtered supernatants were stored at 4.degree. C. Final expression
levels were determined by Protein G-HPLC. Small Scale (50 ml)
Purification:
[0788] Both Fab-X and Fab-Y were purified separately by affinity
capture using a small scale vacuum based purification system.
Briefly, the 50 ml of culture supernatants were 0.22 .mu.m sterile
filtered before 500 .mu.L of Ni Sepharose beads (GE Healthcare)
were added. The supernatant beads mixture was then tumbled for
about an hour before supernatant was removed by applying vacuum.
Beads were then washed with Wash 1 (50 mM Sodium Phosphate 1 M NaCl
pH 6.2) and Wash 2 (0.5 M NaCl). Elution was performed with 50 mM
sodium acetate, pH4.0+1M NaCl. The eluted fractions buffer
exchanged into PBS (Sigma), pH7.4 and 0.22 .mu.m filtered. Final
pools were assayed by A280 scan, SE-UPLC (BEH200 method), SDS-PAGE
(reduced & non-reduced) and for endotoxin using the PTS
Endosafe system.
Screening Assays
[0789] Donor PBMCs were rapidly thawed using a water bath set to
37.degree. C., and carefully transferred to a 50 ml Falcon tube.
They were then diluted dropwise to 5 ml in assay media to minimise
the osmotic shock. The cells were then diluted to 20 ml carefully
before adding the final media diluent to make the volume 50 ml. The
cells were then spun at 500 g for 5 minutes before removing the
supernatant and resuspending the cells in 1 ml media. The cells
were then counted and diluted to 1.66.times.10.sup.6 cells/ml
before dispensing 30 .mu.l per well into a V-bottom TC plate giving
a final assay concentration of 5.0.times.10.sup.4 cells/well. The
cell plate was then stored covered in a 37.degree. C., 5% CO.sub.2
incubator until they were required, giving them a minimum of 1 hour
to rest.
[0790] Fab-X and Fab-Y reagents were mixed in an equimolar ratio at
5.times. the final assay concentration in assay media and incubated
for 90 min at 37.degree. C., 5% CO.sub.2. Samples were prepared in
a 96-well U-bottom polypropylene plate and covered during the
incubation.
[0791] 10 .mu.l of 5.times. Fab-KD-Fab mixture was added to the
appropriate test wells containing cells and mixed by shaking at
1000 rpm for 30 sec prior to being incubated for 90 min at
37.degree. C., 5% CO.sub.2. The cells were then stimulated with 10
.mu.l of anti-human IgM. The final assay concentration of stimulus
varied depending on the assay panel readouts, the three antibody
cocktails A, B and C (detailed below) were stimulated at a final
assay concentration of either 50 .mu.g/ml (cocktail A & C) or
25 .mu.g/ml (cocktail B). The assay plates were then gently mixed
at 1000 rpm for 30 sec prior to incubation at 37.degree. C., 5%
CO.sub.2 for 5 min (antibody cocktail A & C) or 2 min (antibody
cocktail B). The assay was stopped by adding 150 .mu.l ice-cold BD
CytoFix to all wells and incubated for 15 min at RT. The fixed
cells were then spun at 500 g for 5 min to pellet the cells and
allow removal of the supernatant using a BioTek ELx405 plate
washer. The pellet was re-suspended by vortexing the plate at 2400
rpm for 30 sec. The cells were then permeabilised at 4.degree. C.
by adding 100 .mu.l ice-cold BD Cell Permeabilisation Buffer III
for 30 min. The cells were then washed in 100 .mu.l FACS buffer and
spun at 500 g for 5 min. Supernatant was again removed by the
ELx405 before using it to rapidly dispense 200 .mu.l FACS Buffer to
wash away any residual permeabilisation buffer. Cells were again
spun at 500 g and the supernatant removed by inversion. During the
preceding spin step the antibody cocktail was prepared in FACS
Buffer and kept shielded from the light. The cells were then
re-suspended by vortexing (2400 RPM, 30 sec) before 20 .mu.l of
antibody cocktail was added to all wells and the plate shaken for
30 sec at 1000 rpm. The cells were then incubated for 60 min at RT
in the dark.
[0792] The cells were then washed twice in 200 .mu.l FACS buffer
with a 500 g spin and supernatant removed after each step. Finally
the cells were re-suspended by vortexing for 30 sec at 2400 rpm
before adding a final 20 .mu.l FACS buffer. The plate(s) were then
read on the Intellicyt HTFC/iQue instrument.
FACS Buffer=PBS+1% BSA+0.05% NaN.sub.3+2 mM EDTA
Antibody Cocktail A=1:2 CD20 PerCp-Cy5.5 (BD Biosciences)+1:5
PLC.gamma.2 AF88+1:10 Akt AF647+1:50 ERK1/2 PE (diluted in FACS
buffer).
Antibody Cocktail B=1:2 CD20 PerCp-Cy5.5 (BD Biosciences)+1:5 Syk
PE+1:5 BLNK AF647 (diluted in FACS buffer)
Antibody Cocktail C=1:5 CD20 PerCp-Cy5.5 (Biolegend)+1:5
PLC.gamma.2 AF488+1:10 Akt AF647+1:5 Syk PE (diluted in FACS
buffer)
TABLE-US-00016 Reagent Supplier Catalogue number Anti-human IgM
Southern Biotech 2022-14 CytoFix BD Biosciences 554655 Perm Buffer
III BD Biosciences 558050 Anti Akt (pS473) AF647 BD Biosciences
561670 Anti SYK (pY348) PE BD Biosciences 558529 Anti PLC.gamma.2
(pY759) AF488 BD Biosciences 558507 Anti-BLNK(pY84) AF647 BD
Biosciences 558443 Anti ERK1/2 (pT202/pY204) PE BD Biosciences
561991 Anti-human CD20 PerCp-Cy5.5 BD Biosciences 558021 Anti-human
CD20 AF488 BD Biosciences 558056 Anti-human CD20 PerCp-Cy5.5
Biolegend 340508 Phosphate Buffer Saline (PBS) Fisher Scientific
10562765 RPMI 1640 Life Technologies 31870 Foetal Calf Serum (FCS)
Life Technologies 16140 Glutamax Life Technologies 35050
Penicillin/Streptomycin (P/S) Life Technologies 15070 EDTA Sigma
03690 Sodium Azide (NaN3) Sigma S2002 Bovine Serum Albumin (BSA)
Sigma A1470
[0793] Fab-X+Fab-Y combinations were screened with either antibody
cocktail A and B or C alone. All screens were conducted on cone
cells from 2 different blood donors. Data was captured and
evaluated using commercially available software tools. A total of
2500 Fab-X+Fab-Y combinations were screened to 315 different
antigen combinations.
Results
[0794] The percentage inhibition of the induction of
phosphorylation of BCR signalling cascade proteins by each
Fab-Kd-Fab [i.e. A-X:Y-B where A and B are Fab fragments]
combination was calculated, in this example looking for new
combinations of antigens that inhibit B cell function, the criteria
for a positive combination was set as at least 30% inhibition of at
least two phospho-readouts by at least one combination of V
regions. According to this threshold 11 new antigen pair
combinations out of 315 examined met the required criteria. This
represents a 3.5% hit rate demonstrating the importance of
screening large numbers of combinations to find those of desired
activity and how rare the activity of the combination of CD79b and
CD45 is.
[0795] FIGS. 6-8 show the data for the antigen grid cross
specificities. Values are percentage inhibition (negative value for
activation) of phosphorlylation of Syk, PLC.gamma.2 & AKT
respectively and represent the mean of multiple V-region
combinations evaluated. 315 different antigen combinations were
tested and as can be seen the effect on BCR signalling by different
combinations of antibody varied significantly from strong
inhibition e.g. antigen 2 (CD79b) on Fab-X combined with antigen 4
(CD45) on Fab-Y (70.4% inhibition of phospho Syk FIG. 6) to
activation e.g antigen 6 on X and antigen 11 on Y (minus 118.10%
phospho Syk FIG. 6). Each data point representing the mean % values
represented in FIGS. 6-8 is shown for antigen combination 2 (CD79b)
on Fab-X and antigen 4 (CD45) on Fab-Y in FIG. 9. In this case, 10
different combinations of different antibody V regions were
evaluated. The same antigen combination but in alternative
orientation, i.e. antigen 2 (CD79b) on Fab-Y and antigen 4 (CD45)
on Fab-X is shown in FIG. 10. In this case, 6 different
combinations of different antibody V regions were evaluated. Again,
all V regions show inhibition but optimal V region combinations can
be identified and selected using the method.
Example 7--Screening of Transiently Expressed V-Regions to Antigen
CD45 as Fab-X with Purified Anti-CD79b Fab-Y in Heterodimerically
Tethered Protein Complexes to Select Optimal Anti-CD45 Antibody
V-Regions
[0796] Introduction:
[0797] New V-regions to CD45 that inhibit B cell signalling as a
bispecific antibody in combination with CD79b specific V regions
were identified using grid screening of heterodimerically tethered
protein complexes. The CD45 V regions were expressed transiently as
Fab-X and combined with purified anti-CD79b Fab-Y. The inhibition
of activation of B cell signalling was measured to select the most
potent anti-CD45 and anti-CD79b V regions. The preparation of
antigen expressing cells and immunisation of rabbits was carried
out in the same way as described in Example 6.
[0798] Antibody Discovery:
[0799] B cell cultures were prepared in the same way as described
in Example 6. The screening of antigen-specific antibodies in B
cell culture supernatants and the deconvolution step for
identification of antigen specific B cells was determined in the
same way as Example 6.
[0800] Antibody variable region genes were recovered from single
cells by reverse transcription (RT)-PCR using heavy and light chain
variable region-specific primers. Two rounds of PCR were performed,
with the nested 2.degree. PCR incorporating restriction sites at
the 3' and 5' ends allowing cloning of the variable region into
mouse Fab-X and mouse kappa (VL) mammalian expression vector. These
vectors were then co-transfected in HEK-293 cells using 293Fectin
(Life Technologies) or in Expi293 cells using Expifectamine (Life
Technologies) and left to express for 6 days. Supernatants were
tested in a homogeneous fluorescence-based binding assay on HEK293
cells transfected with antigen and SUPERAVIDIN beads (Bangs
Laboratories) coated with recombinant protein or antigen
transfected HEK cells. This was done to confirm the specificity of
the cloned antibodies.
[0801] In addition to the Fab-X transient supernatants, negative
control Mock supernatants were prepared in the same way using an
irrelevant control DNA.
[0802] The expression levels of Fab-X were determined by Protein
G-HPLC.
[0803] Production of Purified Fab-X and Fab-Y:
[0804] Purified Fab-X and Fab-Y was prepared using the same method
described in Example 6.
[0805] PhosFlow Assay:
[0806] CD79b-specific Fab-Y and CD45-specific Fab-X, either
purified or in transient supernatant, were incubated together for
60 minutes (in a 37.degree. C. & 5% CO.sub.2 environment) at
equimolar concentration of 200 nM and 90 nM. A mock supernatant was
also included neat. In V-bottomed 96 well plates,
5.0.times.10.sup.4 PBMC were added to wells, to which were added
titrated Fab-X and Fab-Y combinations or mock supernatant. The
combinations and cells were then incubated together for a further
90 minutes. After this time B cells were activated by the addition
of 25 .mu.g/mL of goat F(ab').sub.2 anti-human IgM (Southern
Biotechnology) for 15 minutes at 37.degree. C. plus 5% CO.sub.2.
The signalling reaction was then halted by adding an equal volume
of Cytofix buffer (BD Biosciences). Plates were then left at room
temperature for 15 minutes before centrifugation at 500.times.g for
5 minutes. Excess supernatant was discarded from the cell pellet
which was resuspended in FACS buffer (PBS+1% BSA+0.01% NaN.sub.3+2
mM EDTA) and washed once more. Cells were then resuspended in ice
cold Perm Buffer III (BD Biosciences) for 30 minutes before being
washed twice in flow buffer. Cells were then stained as described
in Example 6, except that instead of 3 different antibody
cocktails, only one cocktail was used with the same assay
concentrations and incubation conditions as described for antibody
cocktail A in Example 6.
Antibody Cocktail=1:3 CD20 PerCp-Cy5.5+1:5 PLC.gamma.2 AF88+1:10
Akt AF647+1:5 p38 MAPK PE (diluted in FACS buffer).
Results
[0807] As can be seen in FIGS. 11 to 16, the data shows that the
combination of different transiently expressed antigen CD45 V
regions in Fab-X with 2 different purified antigen CD79b V regions
(VR447 and VR4450) in Fab-Y can inhibit B cell activation (as
measured by inhibition of PLC.gamma.2, p38 and Akt) to different
levels and screening in a bispecific format therefore facilitates
selection of optimal V region combinations. Combinations with
transient Fab-X are compared to a reference combination with a
purified CD45 Fab-X (VR4122).
Example 8--Effect of Co-Targeting the Antigen CD79b Plus Antigen
CD45 on Memory B Cell Function Using Molecularly Linked Bispecific
Bybes with or without Further Addition of an Anti-Albumin
[0808] Introduction:
[0809] To check that targeting CD79b/CD45 has a functional effect
on B cells in long term culture, IgG production from B cells in a
mixed PBMC culture was measured. The measurement of specific
antibodies to the recall antigen tetanus toxoid provides a read out
of memory B cell function.
[0810] Antigen CD79b specificity (VR4447) and antigen CD45
specificity (VR4248 and VR4133) were generated in a BYbe format
with or without addition of an anti-albumin fragment (VR0645). The
anti-albumin antibody fragment was fused to the light chain of the
antigen CD45 Fab of the BYbe format as described in Example 8.
[0811] Description of constructs used in this experiment.
TABLE-US-00017 Heavy Chain Light Chain Construct Name Fab
Specificity scFv sFv VR4447/VR4248 BYbe Antigen CD79b Antigen CD45
None VR4447/VR4248/VR645 Antigen CD79b Antigen CD45 Albumin
BYbe/Albumin VR4447/VR4133 BYbe Antigen CD79b Antigen CD45 None
VR4447/VR4133/VR645 Antigen CD79b Antigen CD45 Albumin
BYbe/Albumin
Methods
[0812] Purification of BYbes with/without Anti-Albumin Additional
Specificity
[0813] The BYbe (Fab-dsscFv [scFv off C-terminus of Fab heavy
chain]) and BYbe with anti-albimin (Fab-2.times.dsscFv [scFvs off
C-terminus of Fab heavy chain and light chain]) formats were
purified as follows. Clarified cell culture supernatants from
standard expiHEK or CHO expression were 0.22 .mu.m sterile
filtered. The filtered supernatants were loaded at 2 ml/min onto 50
ml GammabindPlus Sepharose XK26 columns (GE Healthcare)
equilibrated in PBS pH7.4 (Sigma Aldrich Chemicals). After loading
the columns were washed with PBS pH7.4 and then eluted with 0.1M
Glycine/HCl. pH 2.7. The elution was followed by absorbance at 280
nm, the elution peak collected, and then neutralised with
1/25.sup.th volume of 2 M Tris/HCl pH8.5. The neutralised samples
were concentrated using Amicon Ultra-15 concentrators with either a
10 kDa or 30 kDa molecular weight cut off membrane and
centrifugation at 4000.times.g in a swing out rotor. Concentrated
samples were applied to either a XK16/60 or XK26/60 Superdex 200
column (GE Healthcare) equilibrated in PBS, pH7.4. The columns were
developed with an isocratic gradient of PBS, pH7.4 at either 1
ml/min or 2.6 ml/min respectively. Fractions were collected and
analysed by size exclusion chromatography on a TSK gel G3000SWXL; 5
.mu.m, 7.8.times.300 mm column developed with an isocratic gradient
of 0.2 M phosphate, pH 7.0 at 1 ml/min, with detection by
absorbance at 280 nm. Selected monomer fractions were pooled and
concentrated to >1 mg/ml using an Amicon Ultra-15 concentrator
with a 10 kDa or 30 kDa molecular weight cut off membrane and
centrifugation at 4000.times.g in a swing out rotor. Final samples
were assayed; for concentration by A280 Scanning UV-visible
spectrophotometer (Cary 50Bio); for % monomer by size exclusion
chromatography on a TSK gel G3000SWXL; 5 .mu.m, 7.8.times.300 mm
column developed with an isocratic gradient of 0.2 M phosphate,
pH7.0 at 1 ml/min, with detection by absorbance at 280 nm; by
reducing and non-reducing SDS-PAGE run on 4-20% Tris-Glycine 1.5 mm
gels (Novex) at 50 mA (per gel) for 53 minutes; and for endotoxin
by Charles River's ENDOSAFE Portable Test System with Limulus
Amebocyte Lysate (LAL) test cartridges.
Activation of B Cells and Measurement of Tetanus Toxoid Specific
IgG
[0814] Human PBMCs were stimulated with 500 ng/ml CD40L, 1 ug/ml
CpG and 50 ng/ml IL-21 in 1640 media plus 10% foetal bovine serum
and 2 mM Glutamax (R10 medium) for 6 days. Constructs of purified
protein were added at a final concentration of 100 nM at day 0 and
remained in the culture medium for the duration of the assay. After
6 days the supernatants were harvested and the amount of tetanus
toxoid specific IgG was detected by ELISA. Briefly, Maxisorp
half-well ELISA plates (Nunc) were coated with 10 ug/ml tetanus
toxoid in PBS overnight at 4.degree. C. The plates were then
blocked in 5% Milk--in PBS containing 0.05% Tween 20 for 2 hours.
The supernatants were diluted and then added for 2 hours at room
temperature. The plates were washed with PBS-0.05% Tween20 and
tetanus bound antibody was detected using a peroxidase-goat
anti-human IgG(H+L) diluted to 1 ug/ml in 5% milk-PBS 0.05% Tween
20. Plates were developed using TMB substrate solution (KPL) and
absorbance was measured at 450 nM using a Synergy 2 micro-plate
reader (Biotek). Data was exported to Excel and percentage
inhibition was calculated relative to cells cultured without test
antibodies. The data was then imported into Graphpad PRISM and
plotted as bar charts.
[0815] FIG. 17 shows the inhibition of tetanus toxoid IgG
production from PBMCs cultured with VR4447/VR4248 BYbe,
VR4447/VR4133 BYbe, VR4447/VR4248/VR645 BYbe/Albumin and
VR4447/VR4133/VR645 BYbe/Albumin. Data shown is from a single
donor.
Sequence CWU 1 SEQUENCE LISTING <160> NUMBER OF SEQ ID
NOS: 143 <210> SEQ ID NO 1 <211> LENGTH: 44 <212>
TYPE: PRT <213> ORGANISM: Artificial Sequence <220>
FEATURE: <223> OTHER INFORMATION: GCN4(7P14P) sequence
<400> SEQUENCE: 1 Ala Ser Gly Gly Gly Arg Met Lys Gln Leu Glu
Pro Lys Val Glu Glu 1 5 10 15 Leu Leu Pro Lys Asn Tyr His Leu Glu
Asn Glu Val Ala Arg Leu Lys 20 25 30 Lys Leu Val Gly Glu Arg His
His His His His His 35 40 <210> SEQ ID NO 2 <211>
LENGTH: 132 <212> TYPE: DNA <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
GCN4(7P14P) sequence <400> SEQUENCE: 2 gctagcggag gcggaagaat
gaaacaactt gaacccaagg ttgaagaatt gcttccgaaa 60 aattatcact
tggaaaatga ggttgccaga ttaaagaaat tagttggcga acgccatcac 120
catcaccatc ac 132 <210> SEQ ID NO 3 <211> LENGTH: 262
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: 52SR4 ds scFv
sequence <400> SEQUENCE: 3 Asp Ala Val Val Thr Gln Glu Ser
Ala Leu Thr Ser Ser Pro Gly Glu 1 5 10 15 Thr Val Thr Leu Thr Cys
Arg Ser Ser Thr Gly Ala Val Thr Thr Ser 20 25 30 Asn Tyr Ala Ser
Trp Val Gln Glu Lys Pro Asp His Leu Phe Thr Gly 35 40 45 Leu Ile
Gly Gly Thr Asn Asn Arg Ala Pro Gly Val Pro Ala Arg Phe 50 55 60
Ser Gly Ser Leu Ile Gly Asp Lys Ala Ala Leu Thr Ile Thr Gly Ala 65
70 75 80 Gln Thr Glu Asp Glu Ala Ile Tyr Phe Cys Val Leu Trp Tyr
Ser Asp 85 90 95 His Trp Val Phe Gly Cys Gly Thr Lys Leu Thr Val
Leu Gly Gly Gly 100 105 110 Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly
Gly Gly Ser Gly Gly Gly 115 120 125 Gly Ser Asp Val Gln Leu Gln Gln
Ser Gly Pro Gly Leu Val Ala Pro 130 135 140 Ser Gln Ser Leu Ser Ile
Thr Cys Thr Val Ser Gly Phe Leu Leu Thr 145 150 155 160 Asp Tyr Gly
Val Asn Trp Val Arg Gln Ser Pro Gly Lys Cys Leu Glu 165 170 175 Trp
Leu Gly Val Ile Trp Gly Asp Gly Ile Thr Asp Tyr Asn Ser Ala 180 185
190 Leu Lys Ser Arg Leu Ser Val Thr Lys Asp Asn Ser Lys Ser Gln Val
195 200 205 Phe Leu Lys Met Asn Ser Leu Gln Ser Gly Asp Ser Ala Arg
Tyr Tyr 210 215 220 Cys Val Thr Gly Leu Phe Asp Tyr Trp Gly Gln Gly
Thr Thr Leu Thr 225 230 235 240 Val Ser Ser Ala Ala Ala His His His
His His His Glu Gln Lys Leu 245 250 255 Ile Ser Glu Glu Asp Leu 260
<210> SEQ ID NO 4 <211> LENGTH: 792 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: 52SR4 ds scFv sequence <400>
SEQUENCE: 4 gatgcggtgg tgacccagga aagcgcgctg accagcagcc cgggcgaaac
cgtgaccctg 60 acctgccgca gcagcaccgg cgcggtgacc accagcaact
atgcgagctg ggtgcaggaa 120 aaaccggatc atctgtttac cggcctgatt
ggcggcacca acaaccgcgc gccgggcgtg 180 ccggcgcgct ttagcggcag
cctgattggc gataaagcgg cgctgaccat taccggcgcg 240 cagaccgaag
atgaagcgat ttatttttgc gtgctgtggt atagcgacca ttgggtgttt 300
ggctgcggca ccaaactgac cgtgctgggt ggaggcggtg gctcaggcgg aggtggctca
360 ggcggtggcg ggtctggcgg cggcggcagc gatgtgcagc tgcagcagag
cggcccgggc 420 ctggtggcgc cgagccagag cctgagcatt acctgcaccg
tgagcggctt tctcctgacc 480 gattatggcg tgaactgggt gcgccagagc
ccgggcaaat gcctggaatg gctgggcgtg 540 atttggggcg atggcattac
cgattataac agcgcgctga aaagccgcct gagcgtgacc 600 aaagataaca
gcaaaagcca ggtgtttctg aaaatgaaca gcctgcagag cggcgatagc 660
gcgcgctatt attgcgtgac cggcctgttt gattattggg gccagggcac caccctgacc
720 gtgagcagcg cggccgccca tcaccatcac catcacgaac agaaactgat
tagcgaagaa 780 gatctgtaat ag 792 <210> SEQ ID NO 5
<211> LENGTH: 8 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Hinge linker <400> SEQUENCE: 5 Asp Lys Thr His
Thr Cys Ala Ala 1 5 <210> SEQ ID NO 6 <211> LENGTH: 11
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Hinge linker
<400> SEQUENCE: 6 Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala
1 5 10 <210> SEQ ID NO 7 <211> LENGTH: 18 <212>
TYPE: PRT <213> ORGANISM: Artificial Sequence <220>
FEATURE: <223> OTHER INFORMATION: Hinge linker <400>
SEQUENCE: 7 Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Thr Cys Pro
Pro Cys 1 5 10 15 Pro Ala <210> SEQ ID NO 8 <211>
LENGTH: 25 <212> TYPE: PRT <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION: Hinge
linker <400> SEQUENCE: 8 Asp Lys Thr His Thr Cys Pro Pro Cys
Pro Ala Thr Cys Pro Pro Cys 1 5 10 15 Pro Ala Thr Cys Pro Pro Cys
Pro Ala 20 25 <210> SEQ ID NO 9 <211> LENGTH: 30
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Hinge linker
<400> SEQUENCE: 9 Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala
Gly Lys Pro Thr Leu 1 5 10 15 Tyr Asn Ser Leu Val Met Ser Asp Thr
Ala Gly Thr Cys Tyr 20 25 30 <210> SEQ ID NO 10 <211>
LENGTH: 31 <212> TYPE: PRT <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION: Hinge
linker <400> SEQUENCE: 10 Asp Lys Thr His Thr Cys Pro Pro Cys
Pro Ala Gly Lys Pro Thr His 1 5 10 15 Val Asn Val Ser Val Val Met
Ala Glu Val Asp Gly Thr Cys Tyr 20 25 30 <210> SEQ ID NO 11
<211> LENGTH: 15 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Hinge linker <400> SEQUENCE: 11 Asp Lys Thr His
Thr Cys Cys Val Glu Cys Pro Pro Cys Pro Ala 1 5 10 15 <210>
SEQ ID NO 12 <211> LENGTH: 26 <212> TYPE: PRT
<213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Hinge linker <400> SEQUENCE:
12 Asp Lys Thr His Thr Cys Pro Arg Cys Pro Glu Pro Lys Ser Cys Asp
1 5 10 15 Thr Pro Pro Pro Cys Pro Arg Cys Pro Ala 20 25 <210>
SEQ ID NO 13 <211> LENGTH: 11 <212> TYPE: PRT
<213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Hinge linker <400> SEQUENCE:
13 Asp Lys Thr His Thr Cys Pro Ser Cys Pro Ala 1 5 10 <210>
SEQ ID NO 14 <211> LENGTH: 7 <212> TYPE: PRT
<213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Flexible linker <400>
SEQUENCE: 14 Ser Gly Gly Gly Gly Ser Glu 1 5 <210> SEQ ID NO
15 <211> LENGTH: 6 <212> TYPE: PRT <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Flexible linker <400> SEQUENCE: 15 Asp Lys
Thr His Thr Ser 1 5 <210> SEQ ID NO 16 <211> LENGTH: 6
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Flexible linker
<400> SEQUENCE: 16 Ser Gly Gly Gly Gly Ser 1 5 <210>
SEQ ID NO 17 <211> LENGTH: 11 <212> TYPE: PRT
<213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Flexible linker <400>
SEQUENCE: 17 Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser 1 5 10
<210> SEQ ID NO 18 <211> LENGTH: 16 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Flexible linker <400>
SEQUENCE: 18 Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly
Gly Gly Ser 1 5 10 15 <210> SEQ ID NO 19 <211> LENGTH:
21 <212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Flexible linker
<400> SEQUENCE: 19 Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly
Ser Gly Gly Gly Gly Ser 1 5 10 15 Gly Gly Gly Gly Ser 20
<210> SEQ ID NO 20 <211> LENGTH: 26 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Flexible linker <400>
SEQUENCE: 20 Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly
Gly Gly Ser 1 5 10 15 Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser 20 25
<210> SEQ ID NO 21 <211> LENGTH: 11 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Flexible linker <400>
SEQUENCE: 21 Ala Ala Ala Gly Ser Gly Gly Ala Ser Ala Ser 1 5 10
<210> SEQ ID NO 22 <211> LENGTH: 16 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Flexible linker <220> FEATURE:
<221> NAME/KEY: misc_feature <222> LOCATION: (7)..(7)
<223> OTHER INFORMATION: Xaa can be naturally occuring amino
acid <400> SEQUENCE: 22 Ala Ala Ala Gly Ser Gly Xaa Gly Gly
Gly Ser Gly Ala Ser Ala Ser 1 5 10 15 <210> SEQ ID NO 23
<211> LENGTH: 21 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Flexible linker <220> FEATURE: <221>
NAME/KEY: misc_feature <222> LOCATION: (7)..(7) <223>
OTHER INFORMATION: Xaa can be naturally occuring amino acid
<220> FEATURE: <221> NAME/KEY: misc_feature <222>
LOCATION: (12)..(12) <223> OTHER INFORMATION: Xaa can be
naturally occuring amino acid <400> SEQUENCE: 23 Ala Ala Ala
Gly Ser Gly Xaa Gly Gly Gly Ser Xaa Gly Gly Gly Ser 1 5 10 15 Gly
Ala Ser Ala Ser 20 <210> SEQ ID NO 24 <211> LENGTH: 26
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Flexible linker
<220> FEATURE: <221> NAME/KEY: misc_feature <222>
LOCATION: (7)..(7) <223> OTHER INFORMATION: Xaa can be
naturally occuring amino acid <220> FEATURE: <221>
NAME/KEY: misc_feature <222> LOCATION: (12)..(12) <223>
OTHER INFORMATION: Xaa can be naturally occuring amino acid
<220> FEATURE: <221> NAME/KEY: misc_feature <222>
LOCATION: (17)..(17) <223> OTHER INFORMATION: Xaa can be
naturally occuring amino acid <400> SEQUENCE: 24 Ala Ala Ala
Gly Ser Gly Xaa Gly Gly Gly Ser Xaa Gly Gly Gly Ser 1 5 10 15 Xaa
Gly Gly Gly Ser Gly Ala Ser Ala Ser 20 25 <210> SEQ ID NO 25
<211> LENGTH: 31 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Flexible linker <220> FEATURE: <221>
NAME/KEY: misc_feature <222> LOCATION: (7)..(7) <223>
OTHER INFORMATION: Xaa can be naturally occuring amino acid
<220> FEATURE: <221> NAME/KEY: misc_feature <222>
LOCATION: (12)..(12) <223> OTHER INFORMATION: Xaa can be
naturally occuring amino acid <220> FEATURE: <221>
NAME/KEY: misc_feature <222> LOCATION: (17)..(17) <223>
OTHER INFORMATION: Xaa can be naturally occuring amino acid
<220> FEATURE: <221> NAME/KEY: misc_feature <222>
LOCATION: (22)..(22) <223> OTHER INFORMATION: Xaa can be
naturally occuring amino acid <400> SEQUENCE: 25 Ala Ala Ala
Gly Ser Gly Xaa Gly Gly Gly Ser Xaa Gly Gly Gly Ser 1 5 10 15 Xaa
Gly Gly Gly Ser Xaa Gly Gly Gly Ser Gly Ala Ser Ala Ser 20 25 30
<210> SEQ ID NO 26 <211> LENGTH: 13 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Flexible linker <220> FEATURE:
<221> NAME/KEY: misc_feature <222> LOCATION: (7)..(7)
<223> OTHER INFORMATION: Xaa can be naturally occuring amino
acid <400> SEQUENCE: 26 Ala Ala Ala Gly Ser Gly Xaa Ser Gly
Ala Ser Ala Ser 1 5 10 <210> SEQ ID NO 27 <211> LENGTH:
28 <212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Flexible linker
<400> SEQUENCE: 27 Pro Gly Gly Asn Arg Gly Thr Thr Thr Thr
Arg Arg Pro Ala Thr Thr 1 5 10 15 Thr Gly Ser Ser Pro Gly Pro Thr
Gln Ser His Tyr 20 25 <210> SEQ ID NO 28 <211> LENGTH:
11 <212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Flexible linker
<400> SEQUENCE: 28 Ala Thr Thr Thr Gly Ser Ser Pro Gly Pro
Thr 1 5 10 <210> SEQ ID NO 29 <211> LENGTH: 6
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Flexible linker
<400> SEQUENCE: 29 Ala Thr Thr Thr Gly Ser 1 5 <210>
SEQ ID NO 30 <400> SEQUENCE: 30 000 <210> SEQ ID NO 31
<211> LENGTH: 21 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Flexible linker <400> SEQUENCE: 31 Glu Pro Ser
Gly Pro Ile Ser Thr Ile Asn Ser Pro Pro Ser Lys Glu 1 5 10 15 Ser
His Lys Ser Pro 20 <210> SEQ ID NO 32 <211> LENGTH: 15
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Flexible linker
<400> SEQUENCE: 32 Gly Thr Val Ala Ala Pro Ser Val Phe Ile
Phe Pro Pro Ser Asp 1 5 10 15 <210> SEQ ID NO 33 <211>
LENGTH: 15 <212> TYPE: PRT <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Flexible linker <400> SEQUENCE: 33 Gly Gly Gly Gly Ile Ala
Pro Ser Met Val Gly Gly Gly Gly Ser 1 5 10 15 <210> SEQ ID NO
34 <211> LENGTH: 15 <212> TYPE: PRT <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Flexible linker <400> SEQUENCE: 34 Gly Gly
Gly Gly Lys Val Glu Gly Ala Gly Gly Gly Gly Gly Ser 1 5 10 15
<210> SEQ ID NO 35 <211> LENGTH: 15 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Flexible linker <400>
SEQUENCE: 35 Gly Gly Gly Gly Ser Met Lys Ser His Asp Gly Gly Gly
Gly Ser 1 5 10 15 <210> SEQ ID NO 36 <211> LENGTH: 15
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Flexible linker
<400> SEQUENCE: 36 Gly Gly Gly Gly Asn Leu Ile Thr Ile Val
Gly Gly Gly Gly Ser 1 5 10 15 <210> SEQ ID NO 37 <211>
LENGTH: 15 <212> TYPE: PRT <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Flexible linker <400> SEQUENCE: 37 Gly Gly Gly Gly Val Val
Pro Ser Leu Pro Gly Gly Gly Gly Ser 1 5 10 15 <210> SEQ ID NO
38 <211> LENGTH: 12 <212> TYPE: PRT <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Flexible linker <400> SEQUENCE: 38 Gly Gly
Glu Lys Ser Ile Pro Gly Gly Gly Gly Ser 1 5 10 <210> SEQ ID
NO 39 <211> LENGTH: 18 <212> TYPE: PRT <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Flexible linker <400> SEQUENCE: 39 Arg Pro
Leu Ser Tyr Arg Pro Pro Phe Pro Phe Gly Phe Pro Ser Val 1 5 10 15
Arg Pro <210> SEQ ID NO 40 <211> LENGTH: 18 <212>
TYPE: PRT <213> ORGANISM: Artificial Sequence <220>
FEATURE: <223> OTHER INFORMATION: Flexible linker <400>
SEQUENCE: 40 Tyr Pro Arg Ser Ile Tyr Ile Arg Arg Arg His Pro Ser
Pro Ser Leu 1 5 10 15 Thr Thr <210> SEQ ID NO 41 <211>
LENGTH: 18 <212> TYPE: PRT <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Flexible linker <400> SEQUENCE: 41 Thr Pro Ser His Leu Ser
His Ile Leu Pro Ser Phe Gly Leu Pro Thr 1 5 10 15 Phe Asn
<210> SEQ ID NO 42 <211> LENGTH: 18 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Flexible linker <400>
SEQUENCE: 42 Arg Pro Val Ser Pro Phe Thr Phe Pro Arg Leu Ser Asn
Ser Trp Leu 1 5 10 15 Pro Ala <210> SEQ ID NO 43 <211>
LENGTH: 18 <212> TYPE: PRT <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Flexible linker <400> SEQUENCE: 43 Ser Pro Ala Ala His Phe
Pro Arg Ser Ile Pro Arg Pro Gly Pro Ile 1 5 10 15 Arg Thr
<210> SEQ ID NO 44 <211> LENGTH: 18 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Flexible linker <400>
SEQUENCE: 44 Ala Pro Gly Pro Ser Ala Pro Ser His Arg Ser Leu Pro
Ser Arg Ala 1 5 10 15 Phe Gly <210> SEQ ID NO 45 <211>
LENGTH: 18 <212> TYPE: PRT <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Flexible linker <400> SEQUENCE: 45 Pro Arg Asn Ser Ile His
Phe Leu His Pro Leu Leu Val Ala Pro Leu 1 5 10 15 Gly Ala
<210> SEQ ID NO 46 <211> LENGTH: 18 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Flexible linker <400>
SEQUENCE: 46 Met Pro Ser Leu Ser Gly Val Leu Gln Val Arg Tyr Leu
Ser Pro Pro 1 5 10 15 Asp Leu <210> SEQ ID NO 47 <211>
LENGTH: 18 <212> TYPE: PRT <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Flexible linker <400> SEQUENCE: 47 Ser Pro Gln Tyr Pro Ser
Pro Leu Thr Leu Thr Leu Pro Pro His Pro 1 5 10 15 Ser Leu
<210> SEQ ID NO 48 <211> LENGTH: 18 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Flexible linker <400>
SEQUENCE: 48 Asn Pro Ser Leu Asn Pro Pro Ser Tyr Leu His Arg Ala
Pro Ser Arg 1 5 10 15 Ile Ser <210> SEQ ID NO 49 <211>
LENGTH: 17 <212> TYPE: PRT <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Flexible linker <400> SEQUENCE: 49 Leu Pro Trp Arg Thr Ser
Leu Leu Pro Ser Leu Pro Leu Arg Arg Arg 1 5 10 15 Pro <210>
SEQ ID NO 50 <211> LENGTH: 18 <212> TYPE: PRT
<213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Flexible linker <400>
SEQUENCE: 50 Pro Pro Leu Phe Ala Lys Gly Pro Val Gly Leu Leu Ser
Arg Ser Phe 1 5 10 15 Pro Pro <210> SEQ ID NO 51 <211>
LENGTH: 18 <212> TYPE: PRT <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Flexible linker <400> SEQUENCE: 51 Val Pro Pro Ala Pro Val
Val Ser Leu Arg Ser Ala His Ala Arg Pro 1 5 10 15 Pro Tyr
<210> SEQ ID NO 52 <211> LENGTH: 17 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Flexible linker <400>
SEQUENCE: 52 Leu Arg Pro Thr Pro Pro Arg Val Arg Ser Tyr Thr Cys
Cys Pro Thr 1 5 10 15 Pro <210> SEQ ID NO 53 <211>
LENGTH: 18 <212> TYPE: PRT <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Flexible linker <400> SEQUENCE: 53 Pro Asn Val Ala His Val
Leu Pro Leu Leu Thr Val Pro Trp Asp Asn 1 5 10 15 Leu Arg
<210> SEQ ID NO 54 <211> LENGTH: 18 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Flexible linker <400>
SEQUENCE: 54 Cys Asn Pro Leu Leu Pro Leu Cys Ala Arg Ser Pro Ala
Val Arg Thr 1 5 10 15 Phe Pro <210> SEQ ID NO 55 <211>
LENGTH: 11 <212> TYPE: PRT <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
albumin binding peptide <400> SEQUENCE: 55 Asp Leu Cys Leu
Arg Asp Trp Gly Cys Leu Trp 1 5 10 <210> SEQ ID NO 56
<211> LENGTH: 11 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: albumin binding peptide <400> SEQUENCE: 56 Asp
Ile Cys Leu Pro Arg Trp Gly Cys Leu Trp 1 5 10 <210> SEQ ID
NO 57 <211> LENGTH: 15 <212> TYPE: PRT <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: albumin binding peptide <400> SEQUENCE: 57
Met Glu Asp Ile Cys Leu Pro Arg Trp Gly Cys Leu Trp Gly Asp 1 5 10
15 <210> SEQ ID NO 58 <211> LENGTH: 20 <212>
TYPE: PRT <213> ORGANISM: Artificial Sequence <220>
FEATURE: <223> OTHER INFORMATION: albumin binding peptide
<400> SEQUENCE: 58 Gln Arg Leu Met Glu Asp Ile Cys Leu Pro
Arg Trp Gly Cys Leu Trp 1 5 10 15 Glu Asp Asp Glu 20 <210>
SEQ ID NO 59 <211> LENGTH: 20 <212> TYPE: PRT
<213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: albumin binding peptide <400>
SEQUENCE: 59 Gln Gly Leu Ile Gly Asp Ile Cys Leu Pro Arg Trp Gly
Cys Leu Trp 1 5 10 15 Gly Arg Ser Val 20 <210> SEQ ID NO 60
<211> LENGTH: 21 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: albumin binding peptide <400> SEQUENCE: 60 Gln
Gly Leu Ile Gly Asp Ile Cys Leu Pro Arg Trp Gly Cys Leu Trp 1 5 10
15 Gly Arg Ser Val Lys 20 <210> SEQ ID NO 61 <211>
LENGTH: 15 <212> TYPE: PRT <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
albumin binding peptide <400> SEQUENCE: 61 Glu Asp Ile Cys
Leu Pro Arg Trp Gly Cys Leu Trp Glu Asp Asp 1 5 10 15 <210>
SEQ ID NO 62 <211> LENGTH: 18 <212> TYPE: PRT
<213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: albumin binding peptide <400>
SEQUENCE: 62 Arg Leu Met Glu Asp Ile Cys Leu Pro Arg Trp Gly Cys
Leu Trp Glu 1 5 10 15 Asp Asp <210> SEQ ID NO 63 <211>
LENGTH: 16 <212> TYPE: PRT <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
albumin binding peptide <400> SEQUENCE: 63 Met Glu Asp Ile
Cys Leu Pro Arg Trp Gly Cys Leu Trp Glu Asp Asp 1 5 10 15
<210> SEQ ID NO 64 <211> LENGTH: 15 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: albumin binding peptide <400>
SEQUENCE: 64 Met Glu Asp Ile Cys Leu Pro Arg Trp Gly Cys Leu Trp
Glu Asp 1 5 10 15 <210> SEQ ID NO 65 <211> LENGTH: 18
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: albumin binding
peptide <400> SEQUENCE: 65 Arg Leu Met Glu Asp Ile Cys Leu
Ala Arg Trp Gly Cys Leu Trp Glu 1 5 10 15 Asp Asp <210> SEQ
ID NO 66 <211> LENGTH: 20 <212> TYPE: PRT <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: albumin binding peptide <400> SEQUENCE: 66
Glu Val Arg Ser Phe Cys Thr Arg Trp Pro Ala Glu Lys Ser Cys Lys 1 5
10 15 Pro Leu Arg Gly 20 <210> SEQ ID NO 67 <211>
LENGTH: 20 <212> TYPE: PRT <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
albumin binding peptide <400> SEQUENCE: 67 Arg Ala Pro Glu
Ser Phe Val Cys Tyr Trp Glu Thr Ile Cys Phe Glu 1 5 10 15 Arg Ser
Glu Gln 20 <210> SEQ ID NO 68 <211> LENGTH: 11
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: albumin binding
peptide <400> SEQUENCE: 68 Glu Met Cys Tyr Phe Pro Gly Ile
Cys Trp Met 1 5 10 <210> SEQ ID NO 69 <211> LENGTH: 11
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: rigid linkers
<400> SEQUENCE: 69 Gly Ala Pro Ala Pro Ala Ala Pro Ala Pro
Ala 1 5 10 <210> SEQ ID NO 70 <211> LENGTH: 4
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: rigid linker
<400> SEQUENCE: 70 Pro Pro Pro Pro 1 <210> SEQ ID NO 71
<211> LENGTH: 113 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Rabbit Ab 4447 VL region <400> SEQUENCE: 71 Ala
Gln Val Leu Thr Gln Thr Pro Ser Pro Val Ser Ala Pro Val Gly 1 5 10
15 Gly Thr Val Thr Ile Asn Cys Gln Ala Ser Gln Ser Val Val Ser Gly
20 25 30 Asn Tyr Leu Ala Trp Leu Gln Gln Lys Pro Gly Gln Pro Pro
Lys Gln 35 40 45 Leu Ile His Ser Ala Ser Thr Leu Ala Ser Gly Val
Ser Ser Arg Phe 50 55 60 Ser Gly Ser Gly Ser Gly Thr Gln Phe Thr
Leu Thr Ile Ser Gly Val 65 70 75 80 Gln Cys Glu Asp Ala Ala Thr Tyr
Tyr Cys Leu Gly Glu Phe Ser Cys 85 90 95 Ser Ser His Asp Cys Asn
Ala Phe Gly Gly Gly Thr Glu Val Val Val 100 105 110 Lys <210>
SEQ ID NO 72 <211> LENGTH: 339 <212> TYPE: DNA
<213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Rabbit Ab 4447 VL region <400>
SEQUENCE: 72 gcccaagtgc tgacccagac tccgtcccct gtgtctgcac ctgtgggagg
cacagtcacc 60 atcaattgcc aggccagtca gagtgttgtt agtggcaatt
acctagcctg gcttcagcag 120 aaaccagggc agcctcccaa gcaactgatc
cattctgcat ccactctggc atctggggtc 180 tcatcgcggt tcagcggcag
tggatctggg acacaattca ctctcaccat cagcggcgtg 240 cagtgtgaag
atgctgccac ttactactgt ctaggcgaat ttagttgtag tagtcatgat 300
tgtaatgctt tcggcggagg gaccgaggtg gtggtcaaa 339 <210> SEQ ID
NO 73 <211> LENGTH: 123 <212> TYPE: PRT <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Rabbit Ab 4447 VH region <400> SEQUENCE:
73 Gln Ser Leu Glu Glu Ser Gly Gly Arg Leu Val Thr Pro Gly Thr Pro
1 5 10 15 Leu Thr Leu Thr Cys Thr Val Ser Gly Phe Ser Leu Ser Asn
Tyr Ala 20 25 30 Val Ser Trp Val Arg Gln Ala Pro Gly Glu Gly Leu
Glu Trp Ile Gly 35 40 45 Ile Ile Tyr Ile Glu Thr Gly Thr Thr Trp
Tyr Ala Asn Trp Ala Lys 50 55 60 Gly Arg Phe Thr Ile Ser Lys Thr
Ser Thr Thr Val Asp Leu Thr Ile 65 70 75 80 Thr Ser Pro Ser Thr Glu
Asp Thr Ala Thr Tyr Phe Cys Ala Arg Glu 85 90 95 Pro Tyr Glu Pro
Tyr Asp Asp Ser Asn Ile Tyr Tyr Gly Met Asp Pro 100 105 110 Trp Gly
Pro Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> SEQ ID NO
74 <211> LENGTH: 369 <212> TYPE: DNA <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Rabbit Ab 4447 VH region <400> SEQUENCE:
74 cagtcgctgg aggagtccgg gggtcgcctg gtcacgcctg ggacacccct
gacactcacc 60 tgcaccgtct ctggattctc cctcagtaac tatgcagtaa
gctgggtccg ccaggctcca 120 ggggagggac tggaatggat cgggatcatt
tatattgaaa ctggtaccac atggtacgcg 180 aactgggcga aaggccgatt
caccatctcc aaaacctcga ccacggtgga tctgacaatc 240 accagtccgt
caaccgagga cacggccacc tatttctgtg ccagagaacc ttatgaacct 300
tatgatgata gtaatattta ctacggcatg gacccctggg gcccaggcac cctcgtcacc
360 gtctcgagt 369 <210> SEQ ID NO 75 <211> LENGTH: 13
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: CDRL1
<400> SEQUENCE: 75 Gln Ala Ser Gln Ser Val Val Ser Gly Asn
Tyr Leu Ala 1 5 10 <210> SEQ ID NO 76 <211> LENGTH: 7
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: CDRL2
<400> SEQUENCE: 76 Ser Ala Ser Thr Leu Ala Ser 1 5
<210> SEQ ID NO 77 <211> LENGTH: 13 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: CDRL3 <400> SEQUENCE: 77 Leu
Gly Glu Phe Ser Cys Ser Ser His Asp Cys Asn Ala 1 5 10 <210>
SEQ ID NO 78 <211> LENGTH: 10 <212> TYPE: PRT
<213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: CDRH1 <400> SEQUENCE: 78 Gly
Phe Ser Leu Ser Asn Tyr Ala Val Ser 1 5 10 <210> SEQ ID NO 79
<211> LENGTH: 17 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: CDRH2 <400> SEQUENCE: 79 Ile Ile Tyr Ile Glu Thr
Gly Thr Thr Trp Tyr Ala Asn Trp Ala Lys 1 5 10 15 Gly <210>
SEQ ID NO 80 <211> LENGTH: 17 <212> TYPE: PRT
<213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: CDRH3 <400> SEQUENCE: 80 Glu
Pro Tyr Glu Pro Tyr Asp Asp Ser Asn Ile Tyr Tyr Gly Met Asp 1 5 10
15 Pro <210> SEQ ID NO 81 <211> LENGTH: 111 <212>
TYPE: PRT <213> ORGANISM: Artificial Sequence <220>
FEATURE: <223> OTHER INFORMATION: Rabbit Ab 4450 VL region
<400> SEQUENCE: 81 Ala Ile Asp Met Thr Gln Thr Pro Ser Pro
Val Ser Ala Ala Val Gly 1 5 10 15 Gly Thr Val Thr Ile Asn Cys Gln
Ser Ser Gln Ser Ile Tyr Asn Asn 20 25 30 Asn Asp Leu Ala Trp Tyr
Gln Gln Lys Pro Gly Gln Pro Pro Lys Leu 35 40 45 Leu Ile Tyr Glu
Ala Ser Lys Leu Ala Ser Gly Val Pro Ser Arg Phe 50 55 60 Lys Gly
Ser Gly Ser Gly Thr Gln Phe Thr Leu Thr Ile Ser Gly Val 65 70 75 80
Gln Cys Asp Asp Ala Ala Thr Tyr Tyr Cys Gln Gly Gly Gly Ser Gly 85
90 95 Gly Asp Gly Ile Ala Phe Gly Gly Gly Thr Lys Val Val Val Glu
100 105 110 <210> SEQ ID NO 82 <211> LENGTH: 333
<212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Rabbit Ab 4450
VL region <400> SEQUENCE: 82 gccattgata tgacccagac tccatccccc
gtgtctgcag ctgtgggagg cacagtcacc 60 atcaattgcc agtccagtca
gagtatttat aataataatg acttagcctg gtatcagcag 120 aaaccagggc
agcctcccaa gctcctgatc tacgaagcat ccaaactggc atctggggtc 180
ccatcgcggt tcaaaggcag tggatctggg acacagttca ctctcaccat cagtggcgtg
240 cagtgtgatg atgctgccac ttactactgt cagggcggtg gtagtggtgg
tgatggcatt 300 gctttcggcg gagggaccaa ggtggtcgtc gaa 333 <210>
SEQ ID NO 83 <211> LENGTH: 117 <212> TYPE: PRT
<213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Rabbit Ab 4450 VH region <400>
SEQUENCE: 83 Gln Ser Val Glu Glu Ser Gly Gly Arg Leu Val Thr Pro
Gly Ala Pro 1 5 10 15 Leu Thr Leu Thr Cys Thr Val Ser Gly Phe Ser
Leu Asn Asn Tyr Val 20 25 30 Met Val Trp Val Arg Gln Ala Pro Gly
Lys Gly Leu Glu Trp Ile Gly 35 40 45 Ile Ile Tyr Val Ser Gly Asn
Ala Tyr Tyr Ala Ser Trp Ala Lys Gly 50 55 60 Arg Phe Thr Ile Ser
Arg Thr Ser Thr Thr Val Asp Leu Lys Val Thr 65 70 75 80 Ser Leu Thr
Thr Glu Asp Thr Ala Thr Tyr Phe Cys Ala Arg Asp Ala 85 90 95 Gly
His Ser Asp Val Asp Val Leu Asp Ile Trp Gly Pro Gly Thr Leu 100 105
110 Val Thr Val Ser Ser 115 <210> SEQ ID NO 84 <211>
LENGTH: 351 <212> TYPE: DNA <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION: Rabbit
Ab 4450 VH region <400> SEQUENCE: 84 cagtcggtgg aggagtccgg
gggtcgcctg gtcacgcctg gggcacccct gacactcacc 60 tgcacagtct
ctggattctc cctcaataac tatgtaatgg tctgggtccg ccaggctcca 120
gggaaggggc tggaatggat cggaatcatt tatgttagtg gtaatgcata ctacgcgagc
180 tgggcaaaag gccgattcac catctccaga acctcgacca cggtggatct
gaaagtgacc 240 agtctgacaa ccgaggacac ggccacctat ttctgtgcca
gagatgctgg tcatagtgat 300 gtcgatgttt tggatatttg gggcccgggc
accctcgtca ccgtctcgag t 351 <210> SEQ ID NO 85 <211>
LENGTH: 13 <212> TYPE: PRT <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION: CDRL1
<400> SEQUENCE: 85 Gln Ser Ser Gln Ser Ile Tyr Asn Asn Asn
Asp Leu Ala 1 5 10 <210> SEQ ID NO 86 <211> LENGTH: 7
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: CDRL2
<400> SEQUENCE: 86 Glu Ala Ser Lys Leu Ala Ser 1 5
<210> SEQ ID NO 87 <211> LENGTH: 11 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: CDRL3 <400> SEQUENCE: 87 Gln
Gly Gly Gly Ser Gly Gly Asp Gly Ile Ala 1 5 10 <210> SEQ ID
NO 88 <211> LENGTH: 10 <212> TYPE: PRT <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: CDRH1 <400> SEQUENCE: 88 Gly Phe Ser Leu
Asn Asn Tyr Val Met Val 1 5 10 <210> SEQ ID NO 89 <211>
LENGTH: 16 <212> TYPE: PRT <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION: CDRH2
<400> SEQUENCE: 89 Ile Ile Tyr Val Ser Gly Asn Ala Tyr Tyr
Ala Ser Trp Ala Lys Gly 1 5 10 15 <210> SEQ ID NO 90
<211> LENGTH: 12 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: CDRH3 <400> SEQUENCE: 90 Asp Ala Gly His Ser Asp
Val Asp Val Leu Asp Ile 1 5 10 <210> SEQ ID NO 91 <211>
LENGTH: 111 <212> TYPE: PRT <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION: Rabbit
Ab 4122 VL region <400> SEQUENCE: 91 Asp Ile Val Met Thr Gln
Thr Pro Ala Ser Val Ser Glu Pro Val Gly 1 5 10 15 Gly Thr Val Thr
Ile Met Cys Gln Ala Ser Gln Ser Ile Ser Asn Trp 20 25 30 Leu Ala
Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro Lys Leu Leu Ile 35 40 45
Tyr Gln Ala Ser Lys Leu Ala Ser Gly Val Pro Ser Arg Phe Lys Gly 50
55 60 Ser Gly Ser Gly Thr Glu Tyr Thr Leu Thr Ile Ser Asp Leu Glu
Cys 65 70 75 80 Ala Asp Ala Ala Thr Tyr Tyr Cys Gln Ser Tyr Tyr Asp
Ser Gly Ser 85 90 95 Asn Val Phe Phe Ala Phe Gly Gly Gly Thr Lys
Val Val Val Glu 100 105 110 <210> SEQ ID NO 92 <211>
LENGTH: 333 <212> TYPE: DNA <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION: Rabbit
Ab 4122 VL region <400> SEQUENCE: 92 gacattgtga tgacccagac
tccagcctcc gtgtctgaac ctgtgggagg cacagtcacc 60 atcatgtgcc
aggccagtca gagcattagc aattggttag cctggtatca acagaaacca 120
gggcagcctc ccaagctcct gatctaccag gcatccaaac tggcatctgg ggtcccatcg
180 cggttcaaag gcagtggatc tgggacagag tacactctca ccatcagcga
cctggagtgt 240 gccgatgctg ccacttacta ctgtcaaagc tattatgata
gtggtagtaa tgtttttttt 300 gctttcggcg gagggaccaa ggtggtggtc gaa 333
<210> SEQ ID NO 93 <211> LENGTH: 119 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Rabbit Ab 4122 VH region <400>
SEQUENCE: 93 Leu Ser Leu Glu Glu Ser Gly Gly Asp Leu Val Lys Pro
Gly Ala Ser 1 5 10 15 Leu Thr Leu Thr Cys Thr Ala Ser Gly Phe Ser
Phe Ser Ala Gly Tyr 20 25 30 Trp Ile Cys Trp Val Arg Gln Ala Pro
Gly Lys Gly Leu Glu Trp Ile 35 40 45 Ala Cys Thr Tyr Ala Gly Arg
Ser Gly Ser Thr Tyr Tyr Ala Asn Trp 50 55 60 Val Asn Gly Arg Phe
Thr Ile Pro Lys Thr Ser Ser Thr Thr Val Thr 65 70 75 80 Leu Gln Met
Thr Ser Leu Ser Gly Ala Asp Thr Ala Ser Tyr Phe Cys 85 90 95 Ala
Arg Gly Asn Ala Gly Val Ala Val Gly Ala Leu Trp Gly Pro Gly 100 105
110 Thr Leu Val Thr Val Ser Ser 115 <210> SEQ ID NO 94
<211> LENGTH: 357 <212> TYPE: DNA <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Rabbit Ab 4122 VH region <400> SEQUENCE: 94
ctgtcgttgg aggagtccgg gggagacctg gtcaagcctg gggcatccct gacactcacc
60 tgcacagcct ctggattctc cttcagtgcc ggctattgga tatgttgggt
ccgccaggct 120 ccagggaagg ggctggagtg gatcgcatgc acttatgctg
gtcgtagtgg tagcacttac 180 tacgcgaact gggtgaatgg ccgattcacc
atccccaaaa cctcgtcgac cacggtgact 240 ctgcaaatga ccagtctgtc
aggcgcggac acggccagct atttctgtgc gagaggtaat 300 gctggtgttg
ctgttggtgc cttgtggggc ccaggcaccc tggtcaccgt ctcgagt 357 <210>
SEQ ID NO 95 <211> LENGTH: 11 <212> TYPE: PRT
<213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: CDRL1 <400> SEQUENCE: 95 Gln
Ala Ser Gln Ser Ile Ser Asn Trp Leu Ala 1 5 10 <210> SEQ ID
NO 96 <211> LENGTH: 7 <212> TYPE: PRT <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: CDRL2 <400> SEQUENCE: 96 Gln Ala Ser Lys
Leu Ala Ser 1 5 <210> SEQ ID NO 97 <211> LENGTH: 13
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: CDRL3
<400> SEQUENCE: 97 Gln Ser Tyr Tyr Asp Ser Gly Ser Asn Val
Phe Phe Ala 1 5 10 <210> SEQ ID NO 98 <211> LENGTH: 11
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: CDRH1
<400> SEQUENCE: 98 Gly Phe Ser Phe Ser Ala Gly Tyr Trp Ile
Cys 1 5 10 <210> SEQ ID NO 99 <211> LENGTH: 18
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: CDRH2
<400> SEQUENCE: 99 Cys Thr Tyr Ala Gly Arg Ser Gly Ser Thr
Tyr Tyr Ala Asn Trp Val 1 5 10 15 Asn Gly <210> SEQ ID NO 100
<211> LENGTH: 10 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: CDRH3 <400> SEQUENCE: 100 Gly Asn Ala Gly Val
Ala Val Gly Ala Leu 1 5 10 <210> SEQ ID NO 101 <211>
LENGTH: 111 <212> TYPE: PRT <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION: Rabbit
Ab 4129 VL region <400> SEQUENCE: 101 Asp Ile Val Met Thr Gln
Thr Pro Ala Ser Val Glu Ala Ala Val Gly 1 5 10 15 Gly Thr Val Thr
Ile Asn Cys Gln Ala Ser Gln Ser Ile Ser Ser Trp 20 25 30 Leu Ser
Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro Lys Leu Leu Ile 35 40 45
Tyr Gly Ala Ser Asn Leu Ala Ser Gly Val Pro Ser Arg Phe Ser Gly 50
55 60 Ser Gly Ser Gly Thr Gln Phe Ser Leu Thr Ile Ser Asp Leu Glu
Cys 65 70 75 80 Ala Asp Ala Ala Thr Tyr Tyr Cys Gln Ser Tyr Tyr Asp
Ser Gly Ser 85 90 95 Ser Val Phe Phe Asn Phe Gly Gly Gly Thr Lys
Val Val Val Lys 100 105 110 <210> SEQ ID NO 102 <211>
LENGTH: 333 <212> TYPE: DNA <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION: Rabbit
Ab 4129 VL region <400> SEQUENCE: 102 gacattgtga tgacccagac
tccagcctcc gtggaggcag ctgtgggagg cacagtcacc 60 atcaattgcc
aagccagtca gagcattagc agttggttat cctggtatca gcagaaacca 120
gggcagcctc ccaagctcct gatctatggt gcatccaatc tggcatctgg ggtcccatca
180 cggttcagcg gcagtggatc tgggacacag ttcagtctca ccatcagcga
cctggagtgt 240 gccgatgctg ccacttacta ctgtcaaagc tattatgata
gtggtagtag tgtttttttt 300 aatttcggcg gagggaccaa ggtggtcgtc aaa 333
<210> SEQ ID NO 103 <211> LENGTH: 119 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Rabbit Ab 4129 VH region <400>
SEQUENCE: 103 Gln Ser Leu Glu Glu Ser Gly Gly Asp Leu Val Lys Pro
Gly Ala Ser 1 5 10 15 Leu Thr Leu Thr Cys Thr Ala Ser Gly Phe Ser
Phe Ser Ala Gly Tyr 20 25 30 Trp Ile Cys Trp Val Arg Gln Ala Pro
Gly Lys Gly Leu Glu Trp Ile 35 40 45 Ala Cys Ile Tyr Ala Gly Ser
Ser Gly Ser Thr Tyr Tyr Ala Ser Trp 50 55 60 Ala Lys Gly Arg Phe
Thr Ile Pro Lys Thr Ser Ser Thr Thr Val Thr 65 70 75 80 Leu Gln Met
Thr Ser Leu Thr Gly Ala Asp Thr Ala Thr Tyr Phe Cys 85 90 95 Ala
Arg Gly Asn Ala Gly Val Ala Val Gly Ala Leu Trp Gly Pro Gly 100 105
110 Thr Leu Val Thr Val Ser Ser 115 <210> SEQ ID NO 104
<211> LENGTH: 357 <212> TYPE: DNA <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Rabbit Ab 4129 VH region <400> SEQUENCE: 104
cagtcgttgg aggagtccgg gggagacctg gttaagcctg gggcatccct gacactcacc
60 tgcacagcct ctggattctc cttcagtgcc ggctattgga tatgttgggt
ccgccaggct 120 ccagggaagg ggctggagtg gatcgcatgc atttatgctg
gtagtagtgg tagcacttac 180 tacgcgagct gggcgaaagg ccgattcacc
atccccaaaa cctcgtcgac cacggtgact 240 ctgcaaatga ccagtctgac
aggcgcggac acggccacct atttctgtgc gagaggtaat 300 gctggtgttg
ctgttggtgc cttgtggggc ccaggcaccc tcgtcaccgt ctcgagt 357 <210>
SEQ ID NO 105 <211> LENGTH: 11 <212> TYPE: PRT
<213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: CDRL1 <400> SEQUENCE: 105 Gln
Ala Ser Gln Ser Ile Ser Ser Trp Leu Ser 1 5 10 <210> SEQ ID
NO 106 <211> LENGTH: 7 <212> TYPE: PRT <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: CDRL2 <400> SEQUENCE: 106 Gly Ala Ser Asn
Leu Ala Ser 1 5 <210> SEQ ID NO 107 <211> LENGTH: 13
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: CDRL3
<400> SEQUENCE: 107 Gln Ser Tyr Tyr Asp Ser Gly Ser Ser Val
Phe Phe Asn 1 5 10 <210> SEQ ID NO 108 <211> LENGTH: 11
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: CDRH1
<400> SEQUENCE: 108 Gly Phe Ser Phe Ser Ala Gly Tyr Trp Ile
Cys 1 5 10 <210> SEQ ID NO 109 <211> LENGTH: 18
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: CDRH2
<400> SEQUENCE: 109 Cys Ile Tyr Ala Gly Ser Ser Gly Ser Thr
Tyr Tyr Ala Ser Trp Ala 1 5 10 15 Lys Gly <210> SEQ ID NO 110
<211> LENGTH: 10 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: CDRH3 <400> SEQUENCE: 110 Gly Asn Ala Gly Val
Ala Val Gly Ala Leu 1 5 10 <210> SEQ ID NO 111 <211>
LENGTH: 107 <212> TYPE: PRT <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION: Rabbit
Ab 4131 VL region <400> SEQUENCE: 111 Asp Ile Val Met Thr Gln
Thr Pro Ala Ser Val Ser Glu Pro Val Gly 1 5 10 15 Gly Ser Val Thr
Ile Lys Cys Gln Ala Ser Gln Ser Phe Tyr Asn Leu 20 25 30 Leu Ala
Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro Lys Leu Leu Ile 35 40 45
Tyr Asp Ala Ser Asp Leu Ala Ser Gly Val Pro Ser Arg Phe Lys Gly 50
55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Asp Leu Glu
Cys 65 70 75 80 Ala Asp Ala Ala Ala Tyr Tyr Cys Gln Ser Ala Asp Gly
Ser Ser Tyr 85 90 95 Ala Phe Gly Gly Gly Thr Glu Val Val Val Lys
100 105 <210> SEQ ID NO 112 <211> LENGTH: 321
<212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Rabbit Ab 4131
VL region <400> SEQUENCE: 112 gacattgtga tgacccagac
tccagcctcc gtgtctgaac ctgtgggagg ctcagtcacc 60 atcaagtgcc
aggccagtca gagcttttac aacctcttag cctggtatca gcagaaacca 120
gggcagcctc ccaaactcct gatctatgat gcatccgatc tggcatctgg ggtcccatcg
180 cggttcaaag gcagtggatc tgggactgat ttcactctca ccatcagcga
cctggagtgt 240 gccgatgctg ccgcttacta ctgtcaaagt gctgatggta
gtagttacgc tttcggcgga 300 gggaccgagg tggtcgtcaa a 321 <210>
SEQ ID NO 113 <211> LENGTH: 120 <212> TYPE: PRT
<213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Rabbit Ab 4131 VH region <400>
SEQUENCE: 113 Gln Glu Gln Leu Glu Glu Ser Gly Gly Gly Leu Val Lys
Pro Glu Gly 1 5 10 15 Ser Leu Thr Leu Thr Cys Thr Ala Ser Gly Val
Ser Phe Ser Ser Ser 20 25 30 Tyr Trp Ile Tyr Trp Val Arg Gln Ala
Pro Gly Lys Gly Leu Glu Trp 35 40 45 Ile Ala Cys Ile Tyr Thr Gly
Ser Ser Gly Ser Thr Tyr Tyr Ala Ser 50 55 60 Trp Ala Lys Gly Arg
Phe Thr Val Ser Glu Thr Ser Ser Thr Thr Val 65 70 75 80 Thr Leu Gln
Met Thr Ser Leu Thr Ala Ala Asp Thr Ala Thr Tyr Phe 85 90 95 Cys
Ala Arg Ala Ser Ala Trp Thr Tyr Gly Met Asp Leu Trp Gly Pro 100 105
110 Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> SEQ ID NO
114 <211> LENGTH: 360 <212> TYPE: DNA <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Rabbit Ab 4131 VH region <400> SEQUENCE:
114 caggagcaat tggaggagtc cgggggaggc ctggtcaagc ctgagggatc
cctgacactc 60 acctgcacag cctctggagt ctccttcagt agcagctatt
ggatatactg ggtccgccag 120 gctccaggga aggggctgga gtggatcgca
tgcatttata ctggtagtag tggtagcact 180 tactacgcga gctgggcgaa
aggccgattc accgtctccg aaacctcgtc gaccacggtg 240 actctgcaaa
tgaccagtct gacagccgcg gacacggcca cctatttctg tgcgagagca 300
agcgcttgga cctacggcat ggacctctgg ggcccgggca ccctcgtcac cgtctcgagt
360 <210> SEQ ID NO 115 <211> LENGTH: 11 <212>
TYPE: PRT <213> ORGANISM: Artificial Sequence <220>
FEATURE: <223> OTHER INFORMATION: CDRL1 <400> SEQUENCE:
115 Gln Ala Ser Gln Ser Phe Tyr Asn Leu Leu Ala 1 5 10 <210>
SEQ ID NO 116 <211> LENGTH: 7 <212> TYPE: PRT
<213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: CDRL2 <400> SEQUENCE: 116 Asp
Ala Ser Asp Leu Ala Ser 1 5 <210> SEQ ID NO 117 <211>
LENGTH: 9 <212> TYPE: PRT <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION: CDRL3
<400> SEQUENCE: 117 Gln Ser Ala Asp Gly Ser Ser Tyr Ala 1 5
<210> SEQ ID NO 118 <211> LENGTH: 11 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: CDRH1 <400> SEQUENCE: 118 Gly
Val Ser Phe Ser Ser Ser Tyr Trp Ile Tyr 1 5 10 <210> SEQ ID
NO 119 <211> LENGTH: 18 <212> TYPE: PRT <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: CDRH2 <400> SEQUENCE: 119 Cys Ile Tyr Thr
Gly Ser Ser Gly Ser Thr Tyr Tyr Ala Ser Trp Ala 1 5 10 15 Lys Gly
<210> SEQ ID NO 120 <211> LENGTH: 10 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: CDRH3 <400> SEQUENCE: 120 Ala
Ser Ala Trp Thr Tyr Gly Met Asp Leu 1 5 10 <210> SEQ ID NO
121 <211> LENGTH: 112 <212> TYPE: PRT <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Rabbit Ab 4133 VL region <400> SEQUENCE:
121 Ala Gln Val Leu Thr Gln Thr Pro Ser Pro Val Ser Ala Val Val Gly
1 5 10 15 Gly Thr Val Ser Ile Ser Cys Gln Ala Ser Gln Ser Val Tyr
Asn Asn 20 25 30 Asn Asn Leu Ser Trp Tyr Gln Gln Lys Pro Gly Gln
Pro Pro Lys Leu 35 40 45 Leu Ile Tyr Asp Ala Ser Lys Leu Ala Ser
Gly Val Pro Ser Arg Phe 50 55 60 Lys Gly Ser Gly Ser Gly Thr Gln
Phe Thr Leu Thr Ile Ser Gly Val 65 70 75 80 Gln Cys Asp Asp Ala Ala
Thr Tyr Tyr Cys Leu Gly Gly Tyr Tyr Ser 85 90 95 Ser Gly Trp Tyr
Phe Ala Phe Gly Gly Gly Thr Lys Val Val Val Lys 100 105 110
<210> SEQ ID NO 122 <211> LENGTH: 336 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Rabbit Ab 4133 VL region <400>
SEQUENCE: 122 gcgcaagtgc tgacccagac tccatctccc gtgtctgcag
ttgtgggagg cacagtcagc 60 atcagttgcc aggccagtca gagtgtttat
aataacaaca acttatcctg gtatcagcag 120 aaaccagggc agcctcccaa
gctcttgatc tacgatgcat ccaaattggc atctggggtc 180 ccatcccggt
tcaaaggcag tggatctggg acacagttca ctctcaccat cagcggcgtg 240
cagtgtgacg atgctgccac ttactactgt ctaggcggtt attatagtag tggttggtat
300 tttgctttcg gcggagggac caaggtggtg gtcaaa 336 <210> SEQ ID
NO 123 <211> LENGTH: 122 <212> TYPE: PRT <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Rabbit Ab 4133 VH region <400> SEQUENCE:
123 Gln Glu Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Glu Gly
1 5 10 15 Ser Leu Thr Leu Thr Cys Thr Ala Ser Gly Phe Ser Phe Ser
Gly Asn 20 25 30 Tyr Tyr Met Cys Trp Val Arg Gln Ala Pro Gly Lys
Gly Leu Glu Trp 35 40 45 Ile Gly Cys Leu Tyr Thr Gly Ser Ser Gly
Ser Thr Tyr Tyr Ala Ser 50 55 60 Trp Ala Lys Gly Arg Phe Thr Ile
Ser Lys Thr Ser Ser Thr Thr Val 65 70 75 80 Thr Leu Gln Met Thr Ser
Leu Thr Ala Ala Asp Thr Ala Thr Tyr Phe 85 90 95 Cys Ala Arg Asp
Leu Gly Tyr Glu Ile Asp Gly Tyr Gly Gly Leu Trp 100 105 110 Gly Gln
Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> SEQ ID NO 124
<211> LENGTH: 366 <212> TYPE: DNA <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Rabbit Ab 4133 VH region <400> SEQUENCE: 124
caggagcagc tggtggagtc cgggggaggc ctggtccagc ctgagggatc cctgacacta
60 acctgcacag cttctggatt ctccttcagt ggcaactact acatgtgctg
ggtccgccag 120 gctccaggga aggggctgga gtggatcgga tgcctttata
ctggtagtag tggtagcaca 180 tattacgcga gctgggcgaa aggccgattc
accatctcca aaacctcgtc gaccacggtg 240 actctgcaaa tgaccagtct
gacagccgcg gacacggcca cctatttctg tgcgagagat 300 ctaggttatg
aaattgatgg ttatgggggc ttgtggggcc agggcaccct cgtcaccgtc 360 tcgagt
366 <210> SEQ ID NO 125 <211> LENGTH: 13 <212>
TYPE: PRT <213> ORGANISM: Artificial Sequence <220>
FEATURE: <223> OTHER INFORMATION: CDRL1 <400> SEQUENCE:
125 Gln Ala Ser Gln Ser Val Tyr Asn Asn Asn Asn Leu Ser 1 5 10
<210> SEQ ID NO 126 <211> LENGTH: 7 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: CDRL2 <400> SEQUENCE: 126 Asp
Ala Ser Lys Leu Ala Ser 1 5 <210> SEQ ID NO 127 <211>
LENGTH: 12 <212> TYPE: PRT <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION: CDRL3
<400> SEQUENCE: 127 Leu Gly Gly Tyr Tyr Ser Ser Gly Trp Tyr
Phe Ala 1 5 10 <210> SEQ ID NO 128 <211> LENGTH: 11
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: CDRH1
<400> SEQUENCE: 128 Gly Phe Ser Phe Ser Gly Asn Tyr Tyr Met
Cys 1 5 10 <210> SEQ ID NO 129 <211> LENGTH: 18
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: CDRH2
<400> SEQUENCE: 129 Cys Leu Tyr Thr Gly Ser Ser Gly Ser Thr
Tyr Tyr Ala Ser Trp Ala 1 5 10 15 Lys Gly <210> SEQ ID NO 130
<211> LENGTH: 12 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: CDRH3 <400> SEQUENCE: 130 Asp Leu Gly Tyr Glu
Ile Asp Gly Tyr Gly Gly Leu 1 5 10 <210> SEQ ID NO 131
<211> LENGTH: 10 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: CDRH1 dAbH1 <400> SEQUENCE: 131 Gly Ile Asp Leu
Ser Asn Tyr Ala Ile Asn 1 5 10 <210> SEQ ID NO 132
<211> LENGTH: 16 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: CDRH2 dAbH1 <400> SEQUENCE: 132 Ile Ile Trp Ala
Ser Gly Thr Thr Phe Tyr Ala Thr Trp Ala Lys Gly 1 5 10 15
<210> SEQ ID NO 133 <211> LENGTH: 13 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: CDRH3 dAbH1 <400> SEQUENCE:
133 Thr Val Pro Gly Tyr Ser Thr Ala Pro Tyr Phe Asp Leu 1 5 10
<210> SEQ ID NO 134 <211> LENGTH: 12 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: CDRL1 dAbL1 <400> SEQUENCE:
134 Gln Ser Ser Pro Ser Val Trp Ser Asn Phe Leu Ser 1 5 10
<210> SEQ ID NO 135 <211> LENGTH: 7 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: CDRL2 dAbL1 <400> SEQUENCE:
135 Glu Ala Ser Lys Leu Thr Ser 1 5 <210> SEQ ID NO 136
<211> LENGTH: 11 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: CDRL3 dAbL1 <400> SEQUENCE: 136 Gly Gly Gly Tyr
Ser Ser Ile Ser Asp Thr Thr 1 5 10 <210> SEQ ID NO 137
<211> LENGTH: 121 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Heavy chain variable domain of anti-albumin antibody
(no ds) <400> SEQUENCE: 137 Glu Val Gln Leu Leu Glu Ser Gly
Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys
Ala Val Ser Gly Ile Asp Leu Ser Asn Tyr 20 25 30 Ala Ile Asn Trp
Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Ile 35 40 45 Gly Ile
Ile Trp Ala Ser Gly Thr Thr Phe Tyr Ala Thr Trp Ala Lys 50 55 60
Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Val Tyr Leu 65
70 75 80 Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr
Cys Ala 85 90 95 Arg Thr Val Pro Gly Tyr Ser Thr Ala Pro Tyr Phe
Asp Leu Trp Gly 100 105 110 Gln Gly Thr Leu Val Thr Val Ser Ser 115
120 <210> SEQ ID NO 138 <211> LENGTH: 121 <212>
TYPE: PRT <213> ORGANISM: Artificial Sequence <220>
FEATURE: <223> OTHER INFORMATION: Heavy chain variable domain
of anti-albumin antibody (ds) <400> SEQUENCE: 138 Glu Val Gln
Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser
Leu Arg Leu Ser Cys Ala Val Ser Gly Ile Asp Leu Ser Asn Tyr 20 25
30 Ala Ile Asn Trp Val Arg Gln Ala Pro Gly Lys Cys Leu Glu Trp Ile
35 40 45 Gly Ile Ile Trp Ala Ser Gly Thr Thr Phe Tyr Ala Thr Trp
Ala Lys 50 55 60 Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn
Thr Val Tyr Leu 65 70 75 80 Gln Met Asn Ser Leu Arg Ala Glu Asp Thr
Ala Val Tyr Tyr Cys Ala 85 90 95 Arg Thr Val Pro Gly Tyr Ser Thr
Ala Pro Tyr Phe Asp Leu Trp Gly 100 105 110 Gln Gly Thr Leu Val Thr
Val Ser Ser 115 120 <210> SEQ ID NO 139 <211> LENGTH:
112 <212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Light chain
variable domain of anti-albumin antibody (no ds) <400>
SEQUENCE: 139 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Val Ser Ala
Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Gln Ser Ser Pro
Ser Val Trp Ser Asn 20 25 30 Phe Leu Ser Trp Tyr Gln Gln Lys Pro
Gly Lys Ala Pro Lys Leu Leu 35 40 45 Ile Tyr Glu Ala Ser Lys Leu
Thr Ser Gly Val Pro Ser Arg Phe Ser 50 55 60 Gly Ser Gly Ser Gly
Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln 65 70 75 80 Pro Glu Asp
Phe Ala Thr Tyr Tyr Cys Gly Gly Gly Tyr Ser Ser Ile 85 90 95 Ser
Asp Thr Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Arg Thr 100 105
110 <210> SEQ ID NO 140 <211> LENGTH: 112 <212>
TYPE: PRT <213> ORGANISM: Artificial Sequence <220>
FEATURE: <223> OTHER INFORMATION: Light chain variable domain
of anti-albumin antibody (ds) <400> SEQUENCE: 140 Asp Ile Gln
Met Thr Gln Ser Pro Ser Ser Val Ser Ala Ser Val Gly 1 5 10 15 Asp
Arg Val Thr Ile Thr Cys Gln Ser Ser Pro Ser Val Trp Ser Asn 20 25
30 Phe Leu Ser Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu
35 40 45 Ile Tyr Glu Ala Ser Lys Leu Thr Ser Gly Val Pro Ser Arg
Phe Ser 50 55 60 Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile
Ser Ser Leu Gln 65 70 75 80 Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gly
Gly Gly Tyr Ser Ser Ile 85 90 95 Ser Asp Thr Thr Phe Gly Cys Gly
Thr Tyr Val Glu Ile Lys Arg Thr 100 105 110 <210> SEQ ID NO
141 <211> LENGTH: 226 <212> TYPE: PRT <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Human CD79a <400> SEQUENCE: 141 Met Pro
Gly Gly Pro Gly Val Leu Gln Ala Leu Pro Ala Thr Ile Phe 1 5 10 15
Leu Leu Phe Leu Leu Ser Ala Val Tyr Leu Gly Pro Gly Cys Gln Ala 20
25 30 Leu Trp Met His Lys Val Pro Ala Ser Leu Met Val Ser Leu Gly
Glu 35 40 45 Asp Ala His Phe Gln Cys Pro His Asn Ser Ser Asn Asn
Ala Asn Val 50 55 60 Thr Trp Trp Arg Val Leu His Gly Asn Tyr Thr
Trp Pro Pro Glu Phe 65 70 75 80 Leu Gly Pro Gly Glu Asp Pro Asn Gly
Thr Leu Ile Ile Gln Asn Val 85 90 95 Asn Lys Ser His Gly Gly Ile
Tyr Val Cys Arg Val Gln Glu Gly Asn 100 105 110 Glu Ser Tyr Gln Gln
Ser Cys Gly Thr Tyr Leu Arg Val Arg Gln Pro 115 120 125 Pro Pro Arg
Pro Phe Leu Asp Met Gly Glu Gly Thr Lys Asn Arg Ile 130 135 140 Ile
Thr Ala Glu Gly Ile Ile Leu Leu Phe Cys Ala Val Val Pro Gly 145 150
155 160 Thr Leu Leu Leu Phe Arg Lys Arg Trp Gln Asn Glu Lys Leu Gly
Leu 165 170 175 Asp Ala Gly Asp Glu Tyr Glu Asp Glu Asn Leu Tyr Glu
Gly Leu Asn 180 185 190 Leu Asp Asp Cys Ser Met Tyr Glu Asp Ile Ser
Arg Gly Leu Gln Gly 195 200 205 Thr Tyr Gln Asp Val Gly Ser Leu Asn
Ile Gly Asp Val Gln Leu Glu 210 215 220 Lys Pro 225 <210> SEQ
ID NO 142 <211> LENGTH: 229 <212> TYPE: PRT <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Human CD79b <400> SEQUENCE: 142 Met Ala
Arg Leu Ala Leu Ser Pro Val Pro Ser His Trp Met Val Ala 1 5 10 15
Leu Leu Leu Leu Leu Ser Ala Glu Pro Val Pro Ala Ala Arg Ser Glu 20
25 30 Asp Arg Tyr Arg Asn Pro Lys Gly Ser Ala Cys Ser Arg Ile Trp
Gln 35 40 45 Ser Pro Arg Phe Ile Ala Arg Lys Arg Gly Phe Thr Val
Lys Met His 50 55 60 Cys Tyr Met Asn Ser Ala Ser Gly Asn Val Ser
Trp Leu Trp Lys Gln 65 70 75 80 Glu Met Asp Glu Asn Pro Gln Gln Leu
Lys Leu Glu Lys Gly Arg Met 85 90 95 Glu Glu Ser Gln Asn Glu Ser
Leu Ala Thr Leu Thr Ile Gln Gly Ile 100 105 110 Arg Phe Glu Asp Asn
Gly Ile Tyr Phe Cys Gln Gln Lys Cys Asn Asn 115 120 125 Thr Ser Glu
Val Tyr Gln Gly Cys Gly Thr Glu Leu Arg Val Met Gly 130 135 140 Phe
Ser Thr Leu Ala Gln Leu Lys Gln Arg Asn Thr Leu Lys Asp Gly 145 150
155 160 Ile Ile Met Ile Gln Thr Leu Leu Ile Ile Leu Phe Ile Ile Val
Pro 165 170 175 Ile Phe Leu Leu Leu Asp Lys Asp Asp Ser Lys Ala Gly
Met Glu Glu 180 185 190 Asp His Thr Tyr Glu Gly Leu Asp Ile Asp Gln
Thr Ala Thr Tyr Glu 195 200 205 Asp Ile Val Thr Leu Arg Thr Gly Glu
Val Lys Trp Ser Val Gly Glu 210 215 220 His Pro Gly Gln Glu 225
<210> SEQ ID NO 143 <211> LENGTH: 1304 <212>
TYPE: PRT <213> ORGANISM: Artificial Sequence <220>
FEATURE: <223> OTHER INFORMATION: Human CD45 <400>
SEQUENCE: 143 Met Tyr Leu Trp Leu Lys Leu Leu Ala Phe Gly Phe Ala
Phe Leu Asp 1 5 10 15 Thr Glu Val Phe Val Thr Gly Gln Ser Pro Thr
Pro Ser Pro Thr Gly 20 25 30 Leu Thr Thr Ala Lys Met Pro Ser Val
Pro Leu Ser Ser Asp Pro Leu 35 40 45 Pro Thr His Thr Thr Ala Phe
Ser Pro Ala Ser Thr Phe Glu Arg Glu 50 55 60 Asn Asp Phe Ser Glu
Thr Thr Thr Ser Leu Ser Pro Asp Asn Thr Ser 65 70 75 80 Thr Gln Val
Ser Pro Asp Ser Leu Asp Asn Ala Ser Ala Phe Asn Thr 85 90 95 Thr
Gly Val Ser Ser Val Gln Thr Pro His Leu Pro Thr His Ala Asp 100 105
110 Ser Gln Thr Pro Ser Ala Gly Thr Asp Thr Gln Thr Phe Ser Gly Ser
115 120 125 Ala Ala Asn Ala Lys Leu Asn Pro Thr Pro Gly Ser Asn Ala
Ile Ser 130 135 140 Asp Val Pro Gly Glu Arg Ser Thr Ala Ser Thr Phe
Pro Thr Asp Pro 145 150 155 160 Val Ser Pro Leu Thr Thr Thr Leu Ser
Leu Ala His His Ser Ser Ala 165 170 175 Ala Leu Pro Ala Arg Thr Ser
Asn Thr Thr Ile Thr Ala Asn Thr Ser 180 185 190 Asp Ala Tyr Leu Asn
Ala Ser Glu Thr Thr Thr Leu Ser Pro Ser Gly 195 200 205 Ser Ala Val
Ile Ser Thr Thr Thr Ile Ala Thr Thr Pro Ser Lys Pro 210 215 220 Thr
Cys Asp Glu Lys Tyr Ala Asn Ile Thr Val Asp Tyr Leu Tyr Asn 225 230
235 240 Lys Glu Thr Lys Leu Phe Thr Ala Lys Leu Asn Val Asn Glu Asn
Val 245 250 255 Glu Cys Gly Asn Asn Thr Cys Thr Asn Asn Glu Val His
Asn Leu Thr 260 265 270 Glu Cys Lys Asn Ala Ser Val Ser Ile Ser His
Asn Ser Cys Thr Ala 275 280 285 Pro Asp Lys Thr Leu Ile Leu Asp Val
Pro Pro Gly Val Glu Lys Phe 290 295 300 Gln Leu His Asp Cys Thr Gln
Val Glu Lys Ala Asp Thr Thr Ile Cys 305 310 315 320 Leu Lys Trp Lys
Asn Ile Glu Thr Phe Thr Cys Asp Thr Gln Asn Ile 325 330 335 Thr Tyr
Arg Phe Gln Cys Gly Asn Met Ile Phe Asp Asn Lys Glu Ile 340 345 350
Lys Leu Glu Asn Leu Glu Pro Glu His Glu Tyr Lys Cys Asp Ser Glu 355
360 365 Ile Leu Tyr Asn Asn His Lys Phe Thr Asn Ala Ser Lys Ile Ile
Lys 370 375 380 Thr Asp Phe Gly Ser Pro Gly Glu Pro Gln Ile Ile Phe
Cys Arg Ser 385 390 395 400 Glu Ala Ala His Gln Gly Val Ile Thr Trp
Asn Pro Pro Gln Arg Ser 405 410 415 Phe His Asn Phe Thr Leu Cys Tyr
Ile Lys Glu Thr Glu Lys Asp Cys 420 425 430 Leu Asn Leu Asp Lys Asn
Leu Ile Lys Tyr Asp Leu Gln Asn Leu Lys 435 440 445 Pro Tyr Thr Lys
Tyr Val Leu Ser Leu His Ala Tyr Ile Ile Ala Lys 450 455 460 Val Gln
Arg Asn Gly Ser Ala Ala Met Cys His Phe Thr Thr Lys Ser 465 470 475
480 Ala Pro Pro Ser Gln Val Trp Asn Met Thr Val Ser Met Thr Ser Asp
485 490 495 Asn Ser Met His Val Lys Cys Arg Pro Pro Arg Asp Arg Asn
Gly Pro 500 505 510 His Glu Arg Tyr His Leu Glu Val Glu Ala Gly Asn
Thr Leu Val Arg 515 520 525 Asn Glu Ser His Lys Asn Cys Asp Phe Arg
Val Lys Asp Leu Gln Tyr 530 535 540 Ser Thr Asp Tyr Thr Phe Lys Ala
Tyr Phe His Asn Gly Asp Tyr Pro 545 550 555 560 Gly Glu Pro Phe Ile
Leu His His Ser Thr Ser Tyr Asn Ser Lys Ala 565 570 575 Leu Ile Ala
Phe Leu Ala Phe Leu Ile Ile Val Thr Ser Ile Ala Leu 580 585 590 Leu
Val Val Leu Tyr Lys Ile Tyr Asp Leu His Lys Lys Arg Ser Cys 595 600
605 Asn Leu Asp Glu Gln Gln Glu Leu Val Glu Arg Asp Asp Glu Lys Gln
610 615 620 Leu Met Asn Val Glu Pro Ile His Ala Asp Ile Leu Leu Glu
Thr Tyr 625 630 635 640 Lys Arg Lys Ile Ala Asp Glu Gly Arg Leu Phe
Leu Ala Glu Phe Gln 645 650 655 Ser Ile Pro Arg Val Phe Ser Lys Phe
Pro Ile Lys Glu Ala Arg Lys 660 665 670 Pro Phe Asn Gln Asn Lys Asn
Arg Tyr Val Asp Ile Leu Pro Tyr Asp 675 680 685 Tyr Asn Arg Val Glu
Leu Ser Glu Ile Asn Gly Asp Ala Gly Ser Asn 690 695 700 Tyr Ile Asn
Ala Ser Tyr Ile Asp Gly Phe Lys Glu Pro Arg Lys Tyr 705 710 715 720
Ile Ala Ala Gln Gly Pro Arg Asp Glu Thr Val Asp Asp Phe Trp Arg 725
730 735 Met Ile Trp Glu Gln Lys Ala Thr Val Ile Val Met Val Thr Arg
Cys 740 745 750 Glu Glu Gly Asn Arg Asn Lys Cys Ala Glu Tyr Trp Pro
Ser Met Glu 755 760 765 Glu Gly Thr Arg Ala Phe Gly Asp Val Val Val
Lys Ile Asn Gln His 770 775 780 Lys Arg Cys Pro Asp Tyr Ile Ile Gln
Lys Leu Asn Ile Val Asn Lys 785 790 795 800 Lys Glu Lys Ala Thr Gly
Arg Glu Val Thr His Ile Gln Phe Thr Ser 805 810 815 Trp Pro Asp His
Gly Val Pro Glu Asp Pro His Leu Leu Leu Lys Leu 820 825 830 Arg Arg
Arg Val Asn Ala Phe Ser Asn Phe Phe Ser Gly Pro Ile Val 835 840 845
Val His Cys Ser Ala Gly Val Gly Arg Thr Gly Thr Tyr Ile Gly Ile 850
855 860 Asp Ala Met Leu Glu Gly Leu Glu Ala Glu Asn Lys Val Asp Val
Tyr 865 870 875 880 Gly Tyr Val Val Lys Leu Arg Arg Gln Arg Cys Leu
Met Val Gln Val 885 890 895 Glu Ala Gln Tyr Ile Leu Ile His Gln Ala
Leu Val Glu Tyr Asn Gln 900 905 910 Phe Gly Glu Thr Glu Val Asn Leu
Ser Glu Leu His Pro Tyr Leu His 915 920 925 Asn Met Lys Lys Arg Asp
Pro Pro Ser Glu Pro Ser Pro Leu Glu Ala 930 935 940 Glu Phe Gln Arg
Leu Pro Ser Tyr Arg Ser Trp Arg Thr Gln His Ile 945 950 955 960 Gly
Asn Gln Glu Glu Asn Lys Ser Lys Asn Arg Asn Ser Asn Val Ile 965 970
975 Pro Tyr Asp Tyr Asn Arg Val Pro Leu Lys His Glu Leu Glu Met Ser
980 985 990 Lys Glu Ser Glu His Asp Ser Asp Glu Ser Ser Asp Asp Asp
Ser Asp 995 1000 1005 Ser Glu Glu Pro Ser Lys Tyr Ile Asn Ala Ser
Phe Ile Met Ser 1010 1015 1020 Tyr Trp Lys Pro Glu Val Met Ile Ala
Ala Gln Gly Pro Leu Lys 1025 1030 1035 Glu Thr Ile Gly Asp Phe Trp
Gln Met Ile Phe Gln Arg Lys Val 1040 1045 1050 Lys Val Ile Val Met
Leu Thr Glu Leu Lys His Gly Asp Gln Glu 1055 1060 1065 Ile Cys Ala
Gln Tyr Trp Gly Glu Gly Lys Gln Thr Tyr Gly Asp 1070 1075 1080 Ile
Glu Val Asp Leu Lys Asp Thr Asp Lys Ser Ser Thr Tyr Thr 1085 1090
1095 Leu Arg Val Phe Glu Leu Arg His Ser Lys Arg Lys Asp Ser Arg
1100 1105 1110 Thr Val Tyr Gln Tyr Gln Tyr Thr Asn Trp Ser Val Glu
Gln Leu 1115 1120 1125 Pro Ala Glu Pro Lys Glu Leu Ile Ser Met Ile
Gln Val Val Lys 1130 1135 1140 Gln Lys Leu Pro Gln Lys Asn Ser Ser
Glu Gly Asn Lys His His 1145 1150 1155 Lys Ser Thr Pro Leu Leu Ile
His Cys Arg Asp Gly Ser Gln Gln 1160 1165 1170 Thr Gly Ile Phe Cys
Ala Leu Leu Asn Leu Leu Glu Ser Ala Glu 1175 1180 1185 Thr Glu Glu
Val Val Asp Ile Phe Gln Val Val Lys Ala Leu Arg 1190 1195 1200 Lys
Ala Arg Pro Gly Met Val Ser Thr Phe Glu Gln Tyr Gln Phe 1205 1210
1215 Leu Tyr Asp Val Ile Ala Ser Thr Tyr Pro Ala Gln Asn Gly Gln
1220 1225 1230 Val Lys Lys Asn Asn His Gln Glu Asp Lys Ile Glu Phe
Asp Asn 1235 1240 1245 Glu Val Asp Lys Val Lys Gln Asp Ala Asn Cys
Val Asn Pro Leu 1250 1255 1260 Gly Ala Pro Glu Lys Leu Pro Glu Ala
Lys Glu Gln Ala Glu Gly 1265 1270 1275 Ser Glu Pro Thr Ser Gly Thr
Glu Gly Pro Glu His Ser Val Asn 1280 1285 1290 Gly Pro Ala Ser Pro
Ala Leu Asn Gln Gly Ser 1295 1300
1 SEQUENCE LISTING <160> NUMBER OF SEQ ID NOS: 143
<210> SEQ ID NO 1 <211> LENGTH: 44 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: GCN4(7P14P) sequence <400>
SEQUENCE: 1 Ala Ser Gly Gly Gly Arg Met Lys Gln Leu Glu Pro Lys Val
Glu Glu 1 5 10 15 Leu Leu Pro Lys Asn Tyr His Leu Glu Asn Glu Val
Ala Arg Leu Lys 20 25 30 Lys Leu Val Gly Glu Arg His His His His
His His 35 40 <210> SEQ ID NO 2 <211> LENGTH: 132
<212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: GCN4(7P14P)
sequence <400> SEQUENCE: 2 gctagcggag gcggaagaat gaaacaactt
gaacccaagg ttgaagaatt gcttccgaaa 60 aattatcact tggaaaatga
ggttgccaga ttaaagaaat tagttggcga acgccatcac 120 catcaccatc ac 132
<210> SEQ ID NO 3 <211> LENGTH: 262 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: 52SR4 ds scFv sequence <400>
SEQUENCE: 3 Asp Ala Val Val Thr Gln Glu Ser Ala Leu Thr Ser Ser Pro
Gly Glu 1 5 10 15 Thr Val Thr Leu Thr Cys Arg Ser Ser Thr Gly Ala
Val Thr Thr Ser 20 25 30 Asn Tyr Ala Ser Trp Val Gln Glu Lys Pro
Asp His Leu Phe Thr Gly 35 40 45 Leu Ile Gly Gly Thr Asn Asn Arg
Ala Pro Gly Val Pro Ala Arg Phe 50 55 60 Ser Gly Ser Leu Ile Gly
Asp Lys Ala Ala Leu Thr Ile Thr Gly Ala 65 70 75 80 Gln Thr Glu Asp
Glu Ala Ile Tyr Phe Cys Val Leu Trp Tyr Ser Asp 85 90 95 His Trp
Val Phe Gly Cys Gly Thr Lys Leu Thr Val Leu Gly Gly Gly 100 105 110
Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly 115
120 125 Gly Ser Asp Val Gln Leu Gln Gln Ser Gly Pro Gly Leu Val Ala
Pro 130 135 140 Ser Gln Ser Leu Ser Ile Thr Cys Thr Val Ser Gly Phe
Leu Leu Thr 145 150 155 160 Asp Tyr Gly Val Asn Trp Val Arg Gln Ser
Pro Gly Lys Cys Leu Glu 165 170 175 Trp Leu Gly Val Ile Trp Gly Asp
Gly Ile Thr Asp Tyr Asn Ser Ala 180 185 190 Leu Lys Ser Arg Leu Ser
Val Thr Lys Asp Asn Ser Lys Ser Gln Val 195 200 205 Phe Leu Lys Met
Asn Ser Leu Gln Ser Gly Asp Ser Ala Arg Tyr Tyr 210 215 220 Cys Val
Thr Gly Leu Phe Asp Tyr Trp Gly Gln Gly Thr Thr Leu Thr 225 230 235
240 Val Ser Ser Ala Ala Ala His His His His His His Glu Gln Lys Leu
245 250 255 Ile Ser Glu Glu Asp Leu 260 <210> SEQ ID NO 4
<211> LENGTH: 792 <212> TYPE: DNA <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: 52SR4 ds scFv sequence <400> SEQUENCE: 4
gatgcggtgg tgacccagga aagcgcgctg accagcagcc cgggcgaaac cgtgaccctg
60 acctgccgca gcagcaccgg cgcggtgacc accagcaact atgcgagctg
ggtgcaggaa 120 aaaccggatc atctgtttac cggcctgatt ggcggcacca
acaaccgcgc gccgggcgtg 180 ccggcgcgct ttagcggcag cctgattggc
gataaagcgg cgctgaccat taccggcgcg 240 cagaccgaag atgaagcgat
ttatttttgc gtgctgtggt atagcgacca ttgggtgttt 300 ggctgcggca
ccaaactgac cgtgctgggt ggaggcggtg gctcaggcgg aggtggctca 360
ggcggtggcg ggtctggcgg cggcggcagc gatgtgcagc tgcagcagag cggcccgggc
420 ctggtggcgc cgagccagag cctgagcatt acctgcaccg tgagcggctt
tctcctgacc 480 gattatggcg tgaactgggt gcgccagagc ccgggcaaat
gcctggaatg gctgggcgtg 540 atttggggcg atggcattac cgattataac
agcgcgctga aaagccgcct gagcgtgacc 600 aaagataaca gcaaaagcca
ggtgtttctg aaaatgaaca gcctgcagag cggcgatagc 660 gcgcgctatt
attgcgtgac cggcctgttt gattattggg gccagggcac caccctgacc 720
gtgagcagcg cggccgccca tcaccatcac catcacgaac agaaactgat tagcgaagaa
780 gatctgtaat ag 792 <210> SEQ ID NO 5 <211> LENGTH: 8
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Hinge linker
<400> SEQUENCE: 5 Asp Lys Thr His Thr Cys Ala Ala 1 5
<210> SEQ ID NO 6 <211> LENGTH: 11 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Hinge linker <400> SEQUENCE: 6
Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala 1 5 10 <210> SEQ
ID NO 7 <211> LENGTH: 18 <212> TYPE: PRT <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Hinge linker <400> SEQUENCE: 7 Asp Lys Thr
His Thr Cys Pro Pro Cys Pro Ala Thr Cys Pro Pro Cys 1 5 10 15 Pro
Ala <210> SEQ ID NO 8 <211> LENGTH: 25 <212>
TYPE: PRT <213> ORGANISM: Artificial Sequence <220>
FEATURE: <223> OTHER INFORMATION: Hinge linker <400>
SEQUENCE: 8 Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Thr Cys Pro
Pro Cys 1 5 10 15 Pro Ala Thr Cys Pro Pro Cys Pro Ala 20 25
<210> SEQ ID NO 9 <211> LENGTH: 30 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Hinge linker <400> SEQUENCE: 9
Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Gly Lys Pro Thr Leu 1 5
10 15 Tyr Asn Ser Leu Val Met Ser Asp Thr Ala Gly Thr Cys Tyr 20 25
30 <210> SEQ ID NO 10 <211> LENGTH: 31 <212>
TYPE: PRT <213> ORGANISM: Artificial Sequence <220>
FEATURE: <223> OTHER INFORMATION: Hinge linker <400>
SEQUENCE: 10 Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Gly Lys
Pro Thr His 1 5 10 15 Val Asn Val Ser Val Val Met Ala Glu Val Asp
Gly Thr Cys Tyr 20 25 30 <210> SEQ ID NO 11 <211>
LENGTH: 15 <212> TYPE: PRT <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION: Hinge
linker <400> SEQUENCE: 11 Asp Lys Thr His Thr Cys Cys Val Glu
Cys Pro Pro Cys Pro Ala 1 5 10 15 <210> SEQ ID NO 12
<211> LENGTH: 26 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Hinge linker <400> SEQUENCE: 12
Asp Lys Thr His Thr Cys Pro Arg Cys Pro Glu Pro Lys Ser Cys Asp 1 5
10 15 Thr Pro Pro Pro Cys Pro Arg Cys Pro Ala 20 25 <210> SEQ
ID NO 13 <211> LENGTH: 11 <212> TYPE: PRT <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Hinge linker <400> SEQUENCE: 13 Asp Lys
Thr His Thr Cys Pro Ser Cys Pro Ala 1 5 10 <210> SEQ ID NO 14
<211> LENGTH: 7 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Flexible linker <400> SEQUENCE: 14 Ser Gly Gly
Gly Gly Ser Glu 1 5 <210> SEQ ID NO 15 <211> LENGTH: 6
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Flexible linker
<400> SEQUENCE: 15 Asp Lys Thr His Thr Ser 1 5 <210>
SEQ ID NO 16 <211> LENGTH: 6 <212> TYPE: PRT
<213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Flexible linker <400>
SEQUENCE: 16 Ser Gly Gly Gly Gly Ser 1 5 <210> SEQ ID NO 17
<211> LENGTH: 11 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Flexible linker <400> SEQUENCE: 17 Ser Gly Gly
Gly Gly Ser Gly Gly Gly Gly Ser 1 5 10 <210> SEQ ID NO 18
<211> LENGTH: 16 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Flexible linker <400> SEQUENCE: 18 Ser Gly Gly
Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser 1 5 10 15
<210> SEQ ID NO 19 <211> LENGTH: 21 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Flexible linker <400>
SEQUENCE: 19 Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly
Gly Gly Ser 1 5 10 15 Gly Gly Gly Gly Ser 20 <210> SEQ ID NO
20 <211> LENGTH: 26 <212> TYPE: PRT <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Flexible linker <400> SEQUENCE: 20 Ser Gly
Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser 1 5 10 15
Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser 20 25 <210> SEQ ID NO
21 <211> LENGTH: 11 <212> TYPE: PRT <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Flexible linker <400> SEQUENCE: 21 Ala Ala
Ala Gly Ser Gly Gly Ala Ser Ala Ser 1 5 10 <210> SEQ ID NO 22
<211> LENGTH: 16 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Flexible linker <220> FEATURE: <221>
NAME/KEY: misc_feature <222> LOCATION: (7)..(7) <223>
OTHER INFORMATION: Xaa can be naturally occuring amino acid
<400> SEQUENCE: 22 Ala Ala Ala Gly Ser Gly Xaa Gly Gly Gly
Ser Gly Ala Ser Ala Ser 1 5 10 15 <210> SEQ ID NO 23
<211> LENGTH: 21 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Flexible linker <220> FEATURE: <221>
NAME/KEY: misc_feature <222> LOCATION: (7)..(7) <223>
OTHER INFORMATION: Xaa can be naturally occuring amino acid
<220> FEATURE: <221> NAME/KEY: misc_feature <222>
LOCATION: (12)..(12) <223> OTHER INFORMATION: Xaa can be
naturally occuring amino acid <400> SEQUENCE: 23 Ala Ala Ala
Gly Ser Gly Xaa Gly Gly Gly Ser Xaa Gly Gly Gly Ser 1 5 10 15 Gly
Ala Ser Ala Ser 20 <210> SEQ ID NO 24 <211> LENGTH: 26
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Flexible linker
<220> FEATURE: <221> NAME/KEY: misc_feature <222>
LOCATION: (7)..(7) <223> OTHER INFORMATION: Xaa can be
naturally occuring amino acid <220> FEATURE: <221>
NAME/KEY: misc_feature <222> LOCATION: (12)..(12) <223>
OTHER INFORMATION: Xaa can be naturally occuring amino acid
<220> FEATURE: <221> NAME/KEY: misc_feature <222>
LOCATION: (17)..(17) <223> OTHER INFORMATION: Xaa can be
naturally occuring amino acid <400> SEQUENCE: 24 Ala Ala Ala
Gly Ser Gly Xaa Gly Gly Gly Ser Xaa Gly Gly Gly Ser 1 5 10 15 Xaa
Gly Gly Gly Ser Gly Ala Ser Ala Ser 20 25 <210> SEQ ID NO 25
<211> LENGTH: 31 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Flexible linker <220> FEATURE: <221>
NAME/KEY: misc_feature <222> LOCATION: (7)..(7) <223>
OTHER INFORMATION: Xaa can be naturally occuring amino acid
<220> FEATURE: <221> NAME/KEY: misc_feature <222>
LOCATION: (12)..(12) <223> OTHER INFORMATION: Xaa can be
naturally occuring amino acid <220> FEATURE: <221>
NAME/KEY: misc_feature <222> LOCATION: (17)..(17) <223>
OTHER INFORMATION: Xaa can be naturally occuring amino acid
<220> FEATURE: <221> NAME/KEY: misc_feature <222>
LOCATION: (22)..(22) <223> OTHER INFORMATION: Xaa can be
naturally occuring amino acid <400> SEQUENCE: 25 Ala Ala Ala
Gly Ser Gly Xaa Gly Gly Gly Ser Xaa Gly Gly Gly Ser 1 5 10 15 Xaa
Gly Gly Gly Ser Xaa Gly Gly Gly Ser Gly Ala Ser Ala Ser 20 25 30
<210> SEQ ID NO 26 <211> LENGTH: 13 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Flexible linker <220> FEATURE:
<221> NAME/KEY: misc_feature <222> LOCATION: (7)..(7)
<223> OTHER INFORMATION: Xaa can be naturally occuring amino
acid <400> SEQUENCE: 26 Ala Ala Ala Gly Ser Gly Xaa Ser Gly
Ala Ser Ala Ser 1 5 10 <210> SEQ ID NO 27 <211> LENGTH:
28
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Flexible linker
<400> SEQUENCE: 27 Pro Gly Gly Asn Arg Gly Thr Thr Thr Thr
Arg Arg Pro Ala Thr Thr 1 5 10 15 Thr Gly Ser Ser Pro Gly Pro Thr
Gln Ser His Tyr 20 25 <210> SEQ ID NO 28 <211> LENGTH:
11 <212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Flexible linker
<400> SEQUENCE: 28 Ala Thr Thr Thr Gly Ser Ser Pro Gly Pro
Thr 1 5 10 <210> SEQ ID NO 29 <211> LENGTH: 6
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Flexible linker
<400> SEQUENCE: 29 Ala Thr Thr Thr Gly Ser 1 5 <210>
SEQ ID NO 30 <400> SEQUENCE: 30 000 <210> SEQ ID NO 31
<211> LENGTH: 21 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Flexible linker <400> SEQUENCE: 31 Glu Pro Ser
Gly Pro Ile Ser Thr Ile Asn Ser Pro Pro Ser Lys Glu 1 5 10 15 Ser
His Lys Ser Pro 20 <210> SEQ ID NO 32 <211> LENGTH: 15
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Flexible linker
<400> SEQUENCE: 32 Gly Thr Val Ala Ala Pro Ser Val Phe Ile
Phe Pro Pro Ser Asp 1 5 10 15 <210> SEQ ID NO 33 <211>
LENGTH: 15 <212> TYPE: PRT <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Flexible linker <400> SEQUENCE: 33 Gly Gly Gly Gly Ile Ala
Pro Ser Met Val Gly Gly Gly Gly Ser 1 5 10 15 <210> SEQ ID NO
34 <211> LENGTH: 15 <212> TYPE: PRT <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Flexible linker <400> SEQUENCE: 34 Gly Gly
Gly Gly Lys Val Glu Gly Ala Gly Gly Gly Gly Gly Ser 1 5 10 15
<210> SEQ ID NO 35 <211> LENGTH: 15 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Flexible linker <400>
SEQUENCE: 35 Gly Gly Gly Gly Ser Met Lys Ser His Asp Gly Gly Gly
Gly Ser 1 5 10 15 <210> SEQ ID NO 36 <211> LENGTH: 15
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Flexible linker
<400> SEQUENCE: 36 Gly Gly Gly Gly Asn Leu Ile Thr Ile Val
Gly Gly Gly Gly Ser 1 5 10 15 <210> SEQ ID NO 37 <211>
LENGTH: 15 <212> TYPE: PRT <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Flexible linker <400> SEQUENCE: 37 Gly Gly Gly Gly Val Val
Pro Ser Leu Pro Gly Gly Gly Gly Ser 1 5 10 15 <210> SEQ ID NO
38 <211> LENGTH: 12 <212> TYPE: PRT <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Flexible linker <400> SEQUENCE: 38 Gly Gly
Glu Lys Ser Ile Pro Gly Gly Gly Gly Ser 1 5 10 <210> SEQ ID
NO 39 <211> LENGTH: 18 <212> TYPE: PRT <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Flexible linker <400> SEQUENCE: 39 Arg Pro
Leu Ser Tyr Arg Pro Pro Phe Pro Phe Gly Phe Pro Ser Val 1 5 10 15
Arg Pro <210> SEQ ID NO 40 <211> LENGTH: 18 <212>
TYPE: PRT <213> ORGANISM: Artificial Sequence <220>
FEATURE: <223> OTHER INFORMATION: Flexible linker <400>
SEQUENCE: 40 Tyr Pro Arg Ser Ile Tyr Ile Arg Arg Arg His Pro Ser
Pro Ser Leu 1 5 10 15 Thr Thr <210> SEQ ID NO 41 <211>
LENGTH: 18 <212> TYPE: PRT <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Flexible linker <400> SEQUENCE: 41 Thr Pro Ser His Leu Ser
His Ile Leu Pro Ser Phe Gly Leu Pro Thr 1 5 10 15 Phe Asn
<210> SEQ ID NO 42 <211> LENGTH: 18 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Flexible linker <400>
SEQUENCE: 42 Arg Pro Val Ser Pro Phe Thr Phe Pro Arg Leu Ser Asn
Ser Trp Leu 1 5 10 15 Pro Ala <210> SEQ ID NO 43 <211>
LENGTH: 18 <212> TYPE: PRT <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Flexible linker <400> SEQUENCE: 43 Ser Pro Ala Ala His Phe
Pro Arg Ser Ile Pro Arg Pro Gly Pro Ile 1 5 10 15 Arg Thr
<210> SEQ ID NO 44 <211> LENGTH: 18 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Flexible linker <400>
SEQUENCE: 44 Ala Pro Gly Pro Ser Ala Pro Ser His Arg Ser Leu Pro
Ser Arg Ala 1 5 10 15 Phe Gly <210> SEQ ID NO 45 <211>
LENGTH: 18 <212> TYPE: PRT <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Flexible linker
<400> SEQUENCE: 45 Pro Arg Asn Ser Ile His Phe Leu His Pro
Leu Leu Val Ala Pro Leu 1 5 10 15 Gly Ala <210> SEQ ID NO 46
<211> LENGTH: 18 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Flexible linker <400> SEQUENCE: 46 Met Pro Ser
Leu Ser Gly Val Leu Gln Val Arg Tyr Leu Ser Pro Pro 1 5 10 15 Asp
Leu <210> SEQ ID NO 47 <211> LENGTH: 18 <212>
TYPE: PRT <213> ORGANISM: Artificial Sequence <220>
FEATURE: <223> OTHER INFORMATION: Flexible linker <400>
SEQUENCE: 47 Ser Pro Gln Tyr Pro Ser Pro Leu Thr Leu Thr Leu Pro
Pro His Pro 1 5 10 15 Ser Leu <210> SEQ ID NO 48 <211>
LENGTH: 18 <212> TYPE: PRT <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Flexible linker <400> SEQUENCE: 48 Asn Pro Ser Leu Asn Pro
Pro Ser Tyr Leu His Arg Ala Pro Ser Arg 1 5 10 15 Ile Ser
<210> SEQ ID NO 49 <211> LENGTH: 17 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Flexible linker <400>
SEQUENCE: 49 Leu Pro Trp Arg Thr Ser Leu Leu Pro Ser Leu Pro Leu
Arg Arg Arg 1 5 10 15 Pro <210> SEQ ID NO 50 <211>
LENGTH: 18 <212> TYPE: PRT <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Flexible linker <400> SEQUENCE: 50 Pro Pro Leu Phe Ala Lys
Gly Pro Val Gly Leu Leu Ser Arg Ser Phe 1 5 10 15 Pro Pro
<210> SEQ ID NO 51 <211> LENGTH: 18 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Flexible linker <400>
SEQUENCE: 51 Val Pro Pro Ala Pro Val Val Ser Leu Arg Ser Ala His
Ala Arg Pro 1 5 10 15 Pro Tyr <210> SEQ ID NO 52 <211>
LENGTH: 17 <212> TYPE: PRT <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Flexible linker <400> SEQUENCE: 52 Leu Arg Pro Thr Pro Pro
Arg Val Arg Ser Tyr Thr Cys Cys Pro Thr 1 5 10 15 Pro <210>
SEQ ID NO 53 <211> LENGTH: 18 <212> TYPE: PRT
<213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Flexible linker <400>
SEQUENCE: 53 Pro Asn Val Ala His Val Leu Pro Leu Leu Thr Val Pro
Trp Asp Asn 1 5 10 15 Leu Arg <210> SEQ ID NO 54 <211>
LENGTH: 18 <212> TYPE: PRT <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Flexible linker <400> SEQUENCE: 54 Cys Asn Pro Leu Leu Pro
Leu Cys Ala Arg Ser Pro Ala Val Arg Thr 1 5 10 15 Phe Pro
<210> SEQ ID NO 55 <211> LENGTH: 11 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: albumin binding peptide <400>
SEQUENCE: 55 Asp Leu Cys Leu Arg Asp Trp Gly Cys Leu Trp 1 5 10
<210> SEQ ID NO 56 <211> LENGTH: 11 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: albumin binding peptide <400>
SEQUENCE: 56 Asp Ile Cys Leu Pro Arg Trp Gly Cys Leu Trp 1 5 10
<210> SEQ ID NO 57 <211> LENGTH: 15 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: albumin binding peptide <400>
SEQUENCE: 57 Met Glu Asp Ile Cys Leu Pro Arg Trp Gly Cys Leu Trp
Gly Asp 1 5 10 15 <210> SEQ ID NO 58 <211> LENGTH: 20
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: albumin binding
peptide <400> SEQUENCE: 58 Gln Arg Leu Met Glu Asp Ile Cys
Leu Pro Arg Trp Gly Cys Leu Trp 1 5 10 15 Glu Asp Asp Glu 20
<210> SEQ ID NO 59 <211> LENGTH: 20 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: albumin binding peptide <400>
SEQUENCE: 59 Gln Gly Leu Ile Gly Asp Ile Cys Leu Pro Arg Trp Gly
Cys Leu Trp 1 5 10 15 Gly Arg Ser Val 20 <210> SEQ ID NO 60
<211> LENGTH: 21 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: albumin binding peptide <400> SEQUENCE: 60 Gln
Gly Leu Ile Gly Asp Ile Cys Leu Pro Arg Trp Gly Cys Leu Trp 1 5 10
15 Gly Arg Ser Val Lys 20 <210> SEQ ID NO 61 <211>
LENGTH: 15 <212> TYPE: PRT <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
albumin binding peptide <400> SEQUENCE: 61 Glu Asp Ile Cys
Leu Pro Arg Trp Gly Cys Leu Trp Glu Asp Asp 1 5 10 15 <210>
SEQ ID NO 62 <211> LENGTH: 18 <212> TYPE: PRT
<213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: albumin binding peptide <400>
SEQUENCE: 62
Arg Leu Met Glu Asp Ile Cys Leu Pro Arg Trp Gly Cys Leu Trp Glu 1 5
10 15 Asp Asp <210> SEQ ID NO 63 <211> LENGTH: 16
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: albumin binding
peptide <400> SEQUENCE: 63 Met Glu Asp Ile Cys Leu Pro Arg
Trp Gly Cys Leu Trp Glu Asp Asp 1 5 10 15 <210> SEQ ID NO 64
<211> LENGTH: 15 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: albumin binding peptide <400> SEQUENCE: 64 Met
Glu Asp Ile Cys Leu Pro Arg Trp Gly Cys Leu Trp Glu Asp 1 5 10 15
<210> SEQ ID NO 65 <211> LENGTH: 18 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: albumin binding peptide <400>
SEQUENCE: 65 Arg Leu Met Glu Asp Ile Cys Leu Ala Arg Trp Gly Cys
Leu Trp Glu 1 5 10 15 Asp Asp <210> SEQ ID NO 66 <211>
LENGTH: 20 <212> TYPE: PRT <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
albumin binding peptide <400> SEQUENCE: 66 Glu Val Arg Ser
Phe Cys Thr Arg Trp Pro Ala Glu Lys Ser Cys Lys 1 5 10 15 Pro Leu
Arg Gly 20 <210> SEQ ID NO 67 <211> LENGTH: 20
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: albumin binding
peptide <400> SEQUENCE: 67 Arg Ala Pro Glu Ser Phe Val Cys
Tyr Trp Glu Thr Ile Cys Phe Glu 1 5 10 15 Arg Ser Glu Gln 20
<210> SEQ ID NO 68 <211> LENGTH: 11 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: albumin binding peptide <400>
SEQUENCE: 68 Glu Met Cys Tyr Phe Pro Gly Ile Cys Trp Met 1 5 10
<210> SEQ ID NO 69 <211> LENGTH: 11 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: rigid linkers <400> SEQUENCE:
69 Gly Ala Pro Ala Pro Ala Ala Pro Ala Pro Ala 1 5 10 <210>
SEQ ID NO 70 <211> LENGTH: 4 <212> TYPE: PRT
<213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: rigid linker <400> SEQUENCE:
70 Pro Pro Pro Pro 1 <210> SEQ ID NO 71 <211> LENGTH:
113 <212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Rabbit Ab 4447
VL region <400> SEQUENCE: 71 Ala Gln Val Leu Thr Gln Thr Pro
Ser Pro Val Ser Ala Pro Val Gly 1 5 10 15 Gly Thr Val Thr Ile Asn
Cys Gln Ala Ser Gln Ser Val Val Ser Gly 20 25 30 Asn Tyr Leu Ala
Trp Leu Gln Gln Lys Pro Gly Gln Pro Pro Lys Gln 35 40 45 Leu Ile
His Ser Ala Ser Thr Leu Ala Ser Gly Val Ser Ser Arg Phe 50 55 60
Ser Gly Ser Gly Ser Gly Thr Gln Phe Thr Leu Thr Ile Ser Gly Val 65
70 75 80 Gln Cys Glu Asp Ala Ala Thr Tyr Tyr Cys Leu Gly Glu Phe
Ser Cys 85 90 95 Ser Ser His Asp Cys Asn Ala Phe Gly Gly Gly Thr
Glu Val Val Val 100 105 110 Lys <210> SEQ ID NO 72
<211> LENGTH: 339 <212> TYPE: DNA <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Rabbit Ab 4447 VL region <400> SEQUENCE: 72
gcccaagtgc tgacccagac tccgtcccct gtgtctgcac ctgtgggagg cacagtcacc
60 atcaattgcc aggccagtca gagtgttgtt agtggcaatt acctagcctg
gcttcagcag 120 aaaccagggc agcctcccaa gcaactgatc cattctgcat
ccactctggc atctggggtc 180 tcatcgcggt tcagcggcag tggatctggg
acacaattca ctctcaccat cagcggcgtg 240 cagtgtgaag atgctgccac
ttactactgt ctaggcgaat ttagttgtag tagtcatgat 300 tgtaatgctt
tcggcggagg gaccgaggtg gtggtcaaa 339 <210> SEQ ID NO 73
<211> LENGTH: 123 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Rabbit Ab 4447 VH region <400> SEQUENCE: 73 Gln
Ser Leu Glu Glu Ser Gly Gly Arg Leu Val Thr Pro Gly Thr Pro 1 5 10
15 Leu Thr Leu Thr Cys Thr Val Ser Gly Phe Ser Leu Ser Asn Tyr Ala
20 25 30 Val Ser Trp Val Arg Gln Ala Pro Gly Glu Gly Leu Glu Trp
Ile Gly 35 40 45 Ile Ile Tyr Ile Glu Thr Gly Thr Thr Trp Tyr Ala
Asn Trp Ala Lys 50 55 60 Gly Arg Phe Thr Ile Ser Lys Thr Ser Thr
Thr Val Asp Leu Thr Ile 65 70 75 80 Thr Ser Pro Ser Thr Glu Asp Thr
Ala Thr Tyr Phe Cys Ala Arg Glu 85 90 95 Pro Tyr Glu Pro Tyr Asp
Asp Ser Asn Ile Tyr Tyr Gly Met Asp Pro 100 105 110 Trp Gly Pro Gly
Thr Leu Val Thr Val Ser Ser 115 120 <210> SEQ ID NO 74
<211> LENGTH: 369 <212> TYPE: DNA <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Rabbit Ab 4447 VH region <400> SEQUENCE: 74
cagtcgctgg aggagtccgg gggtcgcctg gtcacgcctg ggacacccct gacactcacc
60 tgcaccgtct ctggattctc cctcagtaac tatgcagtaa gctgggtccg
ccaggctcca 120 ggggagggac tggaatggat cgggatcatt tatattgaaa
ctggtaccac atggtacgcg 180 aactgggcga aaggccgatt caccatctcc
aaaacctcga ccacggtgga tctgacaatc 240 accagtccgt caaccgagga
cacggccacc tatttctgtg ccagagaacc ttatgaacct 300 tatgatgata
gtaatattta ctacggcatg gacccctggg gcccaggcac cctcgtcacc 360
gtctcgagt 369 <210> SEQ ID NO 75 <211> LENGTH: 13
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: CDRL1
<400> SEQUENCE: 75 Gln Ala Ser Gln Ser Val Val Ser Gly Asn
Tyr Leu Ala 1 5 10 <210> SEQ ID NO 76 <211> LENGTH: 7
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: CDRL2
<400> SEQUENCE: 76 Ser Ala Ser Thr Leu Ala Ser 1 5
<210> SEQ ID NO 77 <211> LENGTH: 13 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: CDRL3 <400> SEQUENCE: 77 Leu
Gly Glu Phe Ser Cys Ser Ser His Asp Cys Asn Ala 1 5 10 <210>
SEQ ID NO 78 <211> LENGTH: 10 <212> TYPE: PRT
<213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: CDRH1 <400> SEQUENCE: 78 Gly
Phe Ser Leu Ser Asn Tyr Ala Val Ser 1 5 10 <210> SEQ ID NO 79
<211> LENGTH: 17 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: CDRH2 <400> SEQUENCE: 79 Ile Ile Tyr Ile Glu Thr
Gly Thr Thr Trp Tyr Ala Asn Trp Ala Lys 1 5 10 15 Gly <210>
SEQ ID NO 80 <211> LENGTH: 17 <212> TYPE: PRT
<213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: CDRH3 <400> SEQUENCE: 80 Glu
Pro Tyr Glu Pro Tyr Asp Asp Ser Asn Ile Tyr Tyr Gly Met Asp 1 5 10
15 Pro <210> SEQ ID NO 81 <211> LENGTH: 111 <212>
TYPE: PRT <213> ORGANISM: Artificial Sequence <220>
FEATURE: <223> OTHER INFORMATION: Rabbit Ab 4450 VL region
<400> SEQUENCE: 81 Ala Ile Asp Met Thr Gln Thr Pro Ser Pro
Val Ser Ala Ala Val Gly 1 5 10 15 Gly Thr Val Thr Ile Asn Cys Gln
Ser Ser Gln Ser Ile Tyr Asn Asn 20 25 30 Asn Asp Leu Ala Trp Tyr
Gln Gln Lys Pro Gly Gln Pro Pro Lys Leu 35 40 45 Leu Ile Tyr Glu
Ala Ser Lys Leu Ala Ser Gly Val Pro Ser Arg Phe 50 55 60 Lys Gly
Ser Gly Ser Gly Thr Gln Phe Thr Leu Thr Ile Ser Gly Val 65 70 75 80
Gln Cys Asp Asp Ala Ala Thr Tyr Tyr Cys Gln Gly Gly Gly Ser Gly 85
90 95 Gly Asp Gly Ile Ala Phe Gly Gly Gly Thr Lys Val Val Val Glu
100 105 110 <210> SEQ ID NO 82 <211> LENGTH: 333
<212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Rabbit Ab 4450
VL region <400> SEQUENCE: 82 gccattgata tgacccagac tccatccccc
gtgtctgcag ctgtgggagg cacagtcacc 60 atcaattgcc agtccagtca
gagtatttat aataataatg acttagcctg gtatcagcag 120 aaaccagggc
agcctcccaa gctcctgatc tacgaagcat ccaaactggc atctggggtc 180
ccatcgcggt tcaaaggcag tggatctggg acacagttca ctctcaccat cagtggcgtg
240 cagtgtgatg atgctgccac ttactactgt cagggcggtg gtagtggtgg
tgatggcatt 300 gctttcggcg gagggaccaa ggtggtcgtc gaa 333 <210>
SEQ ID NO 83 <211> LENGTH: 117 <212> TYPE: PRT
<213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Rabbit Ab 4450 VH region <400>
SEQUENCE: 83 Gln Ser Val Glu Glu Ser Gly Gly Arg Leu Val Thr Pro
Gly Ala Pro 1 5 10 15 Leu Thr Leu Thr Cys Thr Val Ser Gly Phe Ser
Leu Asn Asn Tyr Val 20 25 30 Met Val Trp Val Arg Gln Ala Pro Gly
Lys Gly Leu Glu Trp Ile Gly 35 40 45 Ile Ile Tyr Val Ser Gly Asn
Ala Tyr Tyr Ala Ser Trp Ala Lys Gly 50 55 60 Arg Phe Thr Ile Ser
Arg Thr Ser Thr Thr Val Asp Leu Lys Val Thr 65 70 75 80 Ser Leu Thr
Thr Glu Asp Thr Ala Thr Tyr Phe Cys Ala Arg Asp Ala 85 90 95 Gly
His Ser Asp Val Asp Val Leu Asp Ile Trp Gly Pro Gly Thr Leu 100 105
110 Val Thr Val Ser Ser 115 <210> SEQ ID NO 84 <211>
LENGTH: 351 <212> TYPE: DNA <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION: Rabbit
Ab 4450 VH region <400> SEQUENCE: 84 cagtcggtgg aggagtccgg
gggtcgcctg gtcacgcctg gggcacccct gacactcacc 60 tgcacagtct
ctggattctc cctcaataac tatgtaatgg tctgggtccg ccaggctcca 120
gggaaggggc tggaatggat cggaatcatt tatgttagtg gtaatgcata ctacgcgagc
180 tgggcaaaag gccgattcac catctccaga acctcgacca cggtggatct
gaaagtgacc 240 agtctgacaa ccgaggacac ggccacctat ttctgtgcca
gagatgctgg tcatagtgat 300 gtcgatgttt tggatatttg gggcccgggc
accctcgtca ccgtctcgag t 351 <210> SEQ ID NO 85 <211>
LENGTH: 13 <212> TYPE: PRT <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION: CDRL1
<400> SEQUENCE: 85 Gln Ser Ser Gln Ser Ile Tyr Asn Asn Asn
Asp Leu Ala 1 5 10 <210> SEQ ID NO 86 <211> LENGTH: 7
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: CDRL2
<400> SEQUENCE: 86 Glu Ala Ser Lys Leu Ala Ser 1 5
<210> SEQ ID NO 87 <211> LENGTH: 11 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: CDRL3 <400> SEQUENCE: 87 Gln
Gly Gly Gly Ser Gly Gly Asp Gly Ile Ala 1 5 10 <210> SEQ ID
NO 88 <211> LENGTH: 10 <212> TYPE: PRT <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: CDRH1 <400> SEQUENCE: 88 Gly Phe Ser Leu
Asn Asn Tyr Val Met Val 1 5 10 <210> SEQ ID NO 89 <211>
LENGTH: 16 <212> TYPE: PRT <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION: CDRH2
<400> SEQUENCE: 89 Ile Ile Tyr Val Ser Gly Asn Ala Tyr Tyr
Ala Ser Trp Ala Lys Gly 1 5 10 15 <210> SEQ ID NO 90
<211> LENGTH: 12 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: CDRH3 <400> SEQUENCE: 90 Asp Ala Gly His Ser Asp
Val Asp Val Leu Asp Ile 1 5 10 <210> SEQ ID NO 91
<211> LENGTH: 111 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Rabbit Ab 4122 VL region <400> SEQUENCE: 91 Asp
Ile Val Met Thr Gln Thr Pro Ala Ser Val Ser Glu Pro Val Gly 1 5 10
15 Gly Thr Val Thr Ile Met Cys Gln Ala Ser Gln Ser Ile Ser Asn Trp
20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro Lys Leu
Leu Ile 35 40 45 Tyr Gln Ala Ser Lys Leu Ala Ser Gly Val Pro Ser
Arg Phe Lys Gly 50 55 60 Ser Gly Ser Gly Thr Glu Tyr Thr Leu Thr
Ile Ser Asp Leu Glu Cys 65 70 75 80 Ala Asp Ala Ala Thr Tyr Tyr Cys
Gln Ser Tyr Tyr Asp Ser Gly Ser 85 90 95 Asn Val Phe Phe Ala Phe
Gly Gly Gly Thr Lys Val Val Val Glu 100 105 110 <210> SEQ ID
NO 92 <211> LENGTH: 333 <212> TYPE: DNA <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Rabbit Ab 4122 VL region <400> SEQUENCE:
92 gacattgtga tgacccagac tccagcctcc gtgtctgaac ctgtgggagg
cacagtcacc 60 atcatgtgcc aggccagtca gagcattagc aattggttag
cctggtatca acagaaacca 120 gggcagcctc ccaagctcct gatctaccag
gcatccaaac tggcatctgg ggtcccatcg 180 cggttcaaag gcagtggatc
tgggacagag tacactctca ccatcagcga cctggagtgt 240 gccgatgctg
ccacttacta ctgtcaaagc tattatgata gtggtagtaa tgtttttttt 300
gctttcggcg gagggaccaa ggtggtggtc gaa 333 <210> SEQ ID NO 93
<211> LENGTH: 119 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Rabbit Ab 4122 VH region <400> SEQUENCE: 93 Leu
Ser Leu Glu Glu Ser Gly Gly Asp Leu Val Lys Pro Gly Ala Ser 1 5 10
15 Leu Thr Leu Thr Cys Thr Ala Ser Gly Phe Ser Phe Ser Ala Gly Tyr
20 25 30 Trp Ile Cys Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu
Trp Ile 35 40 45 Ala Cys Thr Tyr Ala Gly Arg Ser Gly Ser Thr Tyr
Tyr Ala Asn Trp 50 55 60 Val Asn Gly Arg Phe Thr Ile Pro Lys Thr
Ser Ser Thr Thr Val Thr 65 70 75 80 Leu Gln Met Thr Ser Leu Ser Gly
Ala Asp Thr Ala Ser Tyr Phe Cys 85 90 95 Ala Arg Gly Asn Ala Gly
Val Ala Val Gly Ala Leu Trp Gly Pro Gly 100 105 110 Thr Leu Val Thr
Val Ser Ser 115 <210> SEQ ID NO 94 <211> LENGTH: 357
<212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Rabbit Ab 4122
VH region <400> SEQUENCE: 94 ctgtcgttgg aggagtccgg gggagacctg
gtcaagcctg gggcatccct gacactcacc 60 tgcacagcct ctggattctc
cttcagtgcc ggctattgga tatgttgggt ccgccaggct 120 ccagggaagg
ggctggagtg gatcgcatgc acttatgctg gtcgtagtgg tagcacttac 180
tacgcgaact gggtgaatgg ccgattcacc atccccaaaa cctcgtcgac cacggtgact
240 ctgcaaatga ccagtctgtc aggcgcggac acggccagct atttctgtgc
gagaggtaat 300 gctggtgttg ctgttggtgc cttgtggggc ccaggcaccc
tggtcaccgt ctcgagt 357 <210> SEQ ID NO 95 <211> LENGTH:
11 <212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: CDRL1
<400> SEQUENCE: 95 Gln Ala Ser Gln Ser Ile Ser Asn Trp Leu
Ala 1 5 10 <210> SEQ ID NO 96 <211> LENGTH: 7
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: CDRL2
<400> SEQUENCE: 96 Gln Ala Ser Lys Leu Ala Ser 1 5
<210> SEQ ID NO 97 <211> LENGTH: 13 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: CDRL3 <400> SEQUENCE: 97 Gln
Ser Tyr Tyr Asp Ser Gly Ser Asn Val Phe Phe Ala 1 5 10 <210>
SEQ ID NO 98 <211> LENGTH: 11 <212> TYPE: PRT
<213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: CDRH1 <400> SEQUENCE: 98 Gly
Phe Ser Phe Ser Ala Gly Tyr Trp Ile Cys 1 5 10 <210> SEQ ID
NO 99 <211> LENGTH: 18 <212> TYPE: PRT <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: CDRH2 <400> SEQUENCE: 99 Cys Thr Tyr Ala
Gly Arg Ser Gly Ser Thr Tyr Tyr Ala Asn Trp Val 1 5 10 15 Asn Gly
<210> SEQ ID NO 100 <211> LENGTH: 10 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: CDRH3 <400> SEQUENCE: 100 Gly
Asn Ala Gly Val Ala Val Gly Ala Leu 1 5 10 <210> SEQ ID NO
101 <211> LENGTH: 111 <212> TYPE: PRT <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Rabbit Ab 4129 VL region <400> SEQUENCE:
101 Asp Ile Val Met Thr Gln Thr Pro Ala Ser Val Glu Ala Ala Val Gly
1 5 10 15 Gly Thr Val Thr Ile Asn Cys Gln Ala Ser Gln Ser Ile Ser
Ser Trp 20 25 30 Leu Ser Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro
Lys Leu Leu Ile 35 40 45 Tyr Gly Ala Ser Asn Leu Ala Ser Gly Val
Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Gln Phe Ser
Leu Thr Ile Ser Asp Leu Glu Cys 65 70 75 80 Ala Asp Ala Ala Thr Tyr
Tyr Cys Gln Ser Tyr Tyr Asp Ser Gly Ser 85 90 95 Ser Val Phe Phe
Asn Phe Gly Gly Gly Thr Lys Val Val Val Lys 100 105 110 <210>
SEQ ID NO 102 <211> LENGTH: 333 <212> TYPE: DNA
<213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Rabbit Ab 4129 VL region <400>
SEQUENCE: 102 gacattgtga tgacccagac tccagcctcc gtggaggcag
ctgtgggagg cacagtcacc 60 atcaattgcc aagccagtca gagcattagc
agttggttat cctggtatca gcagaaacca 120 gggcagcctc ccaagctcct
gatctatggt gcatccaatc tggcatctgg ggtcccatca 180 cggttcagcg
gcagtggatc tgggacacag ttcagtctca ccatcagcga cctggagtgt 240
gccgatgctg ccacttacta ctgtcaaagc tattatgata gtggtagtag tgtttttttt
300 aatttcggcg gagggaccaa ggtggtcgtc aaa 333 <210> SEQ ID NO
103 <211> LENGTH: 119 <212> TYPE: PRT <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Rabbit Ab 4129 VH region <400> SEQUENCE:
103 Gln Ser Leu Glu Glu Ser Gly Gly Asp Leu Val Lys Pro Gly Ala
Ser
1 5 10 15 Leu Thr Leu Thr Cys Thr Ala Ser Gly Phe Ser Phe Ser Ala
Gly Tyr 20 25 30 Trp Ile Cys Trp Val Arg Gln Ala Pro Gly Lys Gly
Leu Glu Trp Ile 35 40 45 Ala Cys Ile Tyr Ala Gly Ser Ser Gly Ser
Thr Tyr Tyr Ala Ser Trp 50 55 60 Ala Lys Gly Arg Phe Thr Ile Pro
Lys Thr Ser Ser Thr Thr Val Thr 65 70 75 80 Leu Gln Met Thr Ser Leu
Thr Gly Ala Asp Thr Ala Thr Tyr Phe Cys 85 90 95 Ala Arg Gly Asn
Ala Gly Val Ala Val Gly Ala Leu Trp Gly Pro Gly 100 105 110 Thr Leu
Val Thr Val Ser Ser 115 <210> SEQ ID NO 104 <211>
LENGTH: 357 <212> TYPE: DNA <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION: Rabbit
Ab 4129 VH region <400> SEQUENCE: 104 cagtcgttgg aggagtccgg
gggagacctg gttaagcctg gggcatccct gacactcacc 60 tgcacagcct
ctggattctc cttcagtgcc ggctattgga tatgttgggt ccgccaggct 120
ccagggaagg ggctggagtg gatcgcatgc atttatgctg gtagtagtgg tagcacttac
180 tacgcgagct gggcgaaagg ccgattcacc atccccaaaa cctcgtcgac
cacggtgact 240 ctgcaaatga ccagtctgac aggcgcggac acggccacct
atttctgtgc gagaggtaat 300 gctggtgttg ctgttggtgc cttgtggggc
ccaggcaccc tcgtcaccgt ctcgagt 357 <210> SEQ ID NO 105
<211> LENGTH: 11 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: CDRL1 <400> SEQUENCE: 105 Gln Ala Ser Gln Ser
Ile Ser Ser Trp Leu Ser 1 5 10 <210> SEQ ID NO 106
<211> LENGTH: 7 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: CDRL2 <400> SEQUENCE: 106 Gly Ala Ser Asn Leu
Ala Ser 1 5 <210> SEQ ID NO 107 <211> LENGTH: 13
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: CDRL3
<400> SEQUENCE: 107 Gln Ser Tyr Tyr Asp Ser Gly Ser Ser Val
Phe Phe Asn 1 5 10 <210> SEQ ID NO 108 <211> LENGTH: 11
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: CDRH1
<400> SEQUENCE: 108 Gly Phe Ser Phe Ser Ala Gly Tyr Trp Ile
Cys 1 5 10 <210> SEQ ID NO 109 <211> LENGTH: 18
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: CDRH2
<400> SEQUENCE: 109 Cys Ile Tyr Ala Gly Ser Ser Gly Ser Thr
Tyr Tyr Ala Ser Trp Ala 1 5 10 15 Lys Gly <210> SEQ ID NO 110
<211> LENGTH: 10 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: CDRH3 <400> SEQUENCE: 110 Gly Asn Ala Gly Val
Ala Val Gly Ala Leu 1 5 10 <210> SEQ ID NO 111 <211>
LENGTH: 107 <212> TYPE: PRT <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION: Rabbit
Ab 4131 VL region <400> SEQUENCE: 111 Asp Ile Val Met Thr Gln
Thr Pro Ala Ser Val Ser Glu Pro Val Gly 1 5 10 15 Gly Ser Val Thr
Ile Lys Cys Gln Ala Ser Gln Ser Phe Tyr Asn Leu 20 25 30 Leu Ala
Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro Lys Leu Leu Ile 35 40 45
Tyr Asp Ala Ser Asp Leu Ala Ser Gly Val Pro Ser Arg Phe Lys Gly 50
55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Asp Leu Glu
Cys 65 70 75 80 Ala Asp Ala Ala Ala Tyr Tyr Cys Gln Ser Ala Asp Gly
Ser Ser Tyr 85 90 95 Ala Phe Gly Gly Gly Thr Glu Val Val Val Lys
100 105 <210> SEQ ID NO 112 <211> LENGTH: 321
<212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Rabbit Ab 4131
VL region <400> SEQUENCE: 112 gacattgtga tgacccagac
tccagcctcc gtgtctgaac ctgtgggagg ctcagtcacc 60 atcaagtgcc
aggccagtca gagcttttac aacctcttag cctggtatca gcagaaacca 120
gggcagcctc ccaaactcct gatctatgat gcatccgatc tggcatctgg ggtcccatcg
180 cggttcaaag gcagtggatc tgggactgat ttcactctca ccatcagcga
cctggagtgt 240 gccgatgctg ccgcttacta ctgtcaaagt gctgatggta
gtagttacgc tttcggcgga 300 gggaccgagg tggtcgtcaa a 321 <210>
SEQ ID NO 113 <211> LENGTH: 120 <212> TYPE: PRT
<213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Rabbit Ab 4131 VH region <400>
SEQUENCE: 113 Gln Glu Gln Leu Glu Glu Ser Gly Gly Gly Leu Val Lys
Pro Glu Gly 1 5 10 15 Ser Leu Thr Leu Thr Cys Thr Ala Ser Gly Val
Ser Phe Ser Ser Ser 20 25 30 Tyr Trp Ile Tyr Trp Val Arg Gln Ala
Pro Gly Lys Gly Leu Glu Trp 35 40 45 Ile Ala Cys Ile Tyr Thr Gly
Ser Ser Gly Ser Thr Tyr Tyr Ala Ser 50 55 60 Trp Ala Lys Gly Arg
Phe Thr Val Ser Glu Thr Ser Ser Thr Thr Val 65 70 75 80 Thr Leu Gln
Met Thr Ser Leu Thr Ala Ala Asp Thr Ala Thr Tyr Phe 85 90 95 Cys
Ala Arg Ala Ser Ala Trp Thr Tyr Gly Met Asp Leu Trp Gly Pro 100 105
110 Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> SEQ ID NO
114 <211> LENGTH: 360 <212> TYPE: DNA <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Rabbit Ab 4131 VH region <400> SEQUENCE:
114 caggagcaat tggaggagtc cgggggaggc ctggtcaagc ctgagggatc
cctgacactc 60 acctgcacag cctctggagt ctccttcagt agcagctatt
ggatatactg ggtccgccag 120 gctccaggga aggggctgga gtggatcgca
tgcatttata ctggtagtag tggtagcact 180 tactacgcga gctgggcgaa
aggccgattc accgtctccg aaacctcgtc gaccacggtg 240 actctgcaaa
tgaccagtct gacagccgcg gacacggcca cctatttctg tgcgagagca 300
agcgcttgga cctacggcat ggacctctgg ggcccgggca ccctcgtcac cgtctcgagt
360 <210> SEQ ID NO 115 <211> LENGTH: 11 <212>
TYPE: PRT <213> ORGANISM: Artificial Sequence <220>
FEATURE: <223> OTHER INFORMATION: CDRL1 <400> SEQUENCE:
115 Gln Ala Ser Gln Ser Phe Tyr Asn Leu Leu Ala 1 5 10 <210>
SEQ ID NO 116 <211> LENGTH: 7 <212> TYPE: PRT
<213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: CDRL2 <400> SEQUENCE: 116 Asp
Ala Ser Asp Leu Ala Ser 1 5 <210> SEQ ID NO 117 <211>
LENGTH: 9 <212> TYPE: PRT <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION: CDRL3
<400> SEQUENCE: 117 Gln Ser Ala Asp Gly Ser Ser Tyr Ala 1 5
<210> SEQ ID NO 118 <211> LENGTH: 11 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: CDRH1 <400> SEQUENCE: 118 Gly
Val Ser Phe Ser Ser Ser Tyr Trp Ile Tyr 1 5 10 <210> SEQ ID
NO 119 <211> LENGTH: 18 <212> TYPE: PRT <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: CDRH2 <400> SEQUENCE: 119 Cys Ile Tyr Thr
Gly Ser Ser Gly Ser Thr Tyr Tyr Ala Ser Trp Ala 1 5 10 15 Lys Gly
<210> SEQ ID NO 120 <211> LENGTH: 10 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: CDRH3 <400> SEQUENCE: 120 Ala
Ser Ala Trp Thr Tyr Gly Met Asp Leu 1 5 10 <210> SEQ ID NO
121 <211> LENGTH: 112 <212> TYPE: PRT <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Rabbit Ab 4133 VL region <400> SEQUENCE:
121 Ala Gln Val Leu Thr Gln Thr Pro Ser Pro Val Ser Ala Val Val Gly
1 5 10 15 Gly Thr Val Ser Ile Ser Cys Gln Ala Ser Gln Ser Val Tyr
Asn Asn 20 25 30 Asn Asn Leu Ser Trp Tyr Gln Gln Lys Pro Gly Gln
Pro Pro Lys Leu 35 40 45 Leu Ile Tyr Asp Ala Ser Lys Leu Ala Ser
Gly Val Pro Ser Arg Phe 50 55 60 Lys Gly Ser Gly Ser Gly Thr Gln
Phe Thr Leu Thr Ile Ser Gly Val 65 70 75 80 Gln Cys Asp Asp Ala Ala
Thr Tyr Tyr Cys Leu Gly Gly Tyr Tyr Ser 85 90 95 Ser Gly Trp Tyr
Phe Ala Phe Gly Gly Gly Thr Lys Val Val Val Lys 100 105 110
<210> SEQ ID NO 122 <211> LENGTH: 336 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Rabbit Ab 4133 VL region <400>
SEQUENCE: 122 gcgcaagtgc tgacccagac tccatctccc gtgtctgcag
ttgtgggagg cacagtcagc 60 atcagttgcc aggccagtca gagtgtttat
aataacaaca acttatcctg gtatcagcag 120 aaaccagggc agcctcccaa
gctcttgatc tacgatgcat ccaaattggc atctggggtc 180 ccatcccggt
tcaaaggcag tggatctggg acacagttca ctctcaccat cagcggcgtg 240
cagtgtgacg atgctgccac ttactactgt ctaggcggtt attatagtag tggttggtat
300 tttgctttcg gcggagggac caaggtggtg gtcaaa 336 <210> SEQ ID
NO 123 <211> LENGTH: 122 <212> TYPE: PRT <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Rabbit Ab 4133 VH region <400> SEQUENCE:
123 Gln Glu Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Glu Gly
1 5 10 15 Ser Leu Thr Leu Thr Cys Thr Ala Ser Gly Phe Ser Phe Ser
Gly Asn 20 25 30 Tyr Tyr Met Cys Trp Val Arg Gln Ala Pro Gly Lys
Gly Leu Glu Trp 35 40 45 Ile Gly Cys Leu Tyr Thr Gly Ser Ser Gly
Ser Thr Tyr Tyr Ala Ser 50 55 60 Trp Ala Lys Gly Arg Phe Thr Ile
Ser Lys Thr Ser Ser Thr Thr Val 65 70 75 80 Thr Leu Gln Met Thr Ser
Leu Thr Ala Ala Asp Thr Ala Thr Tyr Phe 85 90 95 Cys Ala Arg Asp
Leu Gly Tyr Glu Ile Asp Gly Tyr Gly Gly Leu Trp 100 105 110 Gly Gln
Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> SEQ ID NO 124
<211> LENGTH: 366 <212> TYPE: DNA <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Rabbit Ab 4133 VH region <400> SEQUENCE: 124
caggagcagc tggtggagtc cgggggaggc ctggtccagc ctgagggatc cctgacacta
60 acctgcacag cttctggatt ctccttcagt ggcaactact acatgtgctg
ggtccgccag 120 gctccaggga aggggctgga gtggatcgga tgcctttata
ctggtagtag tggtagcaca 180 tattacgcga gctgggcgaa aggccgattc
accatctcca aaacctcgtc gaccacggtg 240 actctgcaaa tgaccagtct
gacagccgcg gacacggcca cctatttctg tgcgagagat 300 ctaggttatg
aaattgatgg ttatgggggc ttgtggggcc agggcaccct cgtcaccgtc 360 tcgagt
366 <210> SEQ ID NO 125 <211> LENGTH: 13 <212>
TYPE: PRT <213> ORGANISM: Artificial Sequence <220>
FEATURE: <223> OTHER INFORMATION: CDRL1 <400> SEQUENCE:
125 Gln Ala Ser Gln Ser Val Tyr Asn Asn Asn Asn Leu Ser 1 5 10
<210> SEQ ID NO 126 <211> LENGTH: 7 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: CDRL2 <400> SEQUENCE: 126 Asp
Ala Ser Lys Leu Ala Ser 1 5 <210> SEQ ID NO 127 <211>
LENGTH: 12 <212> TYPE: PRT <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION: CDRL3
<400> SEQUENCE: 127 Leu Gly Gly Tyr Tyr Ser Ser Gly Trp Tyr
Phe Ala 1 5 10 <210> SEQ ID NO 128 <211> LENGTH: 11
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: CDRH1
<400> SEQUENCE: 128 Gly Phe Ser Phe Ser Gly Asn Tyr Tyr Met
Cys 1 5 10 <210> SEQ ID NO 129 <211> LENGTH: 18
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: CDRH2
<400> SEQUENCE: 129 Cys Leu Tyr Thr Gly Ser Ser Gly Ser Thr
Tyr Tyr Ala Ser Trp Ala 1 5 10 15 Lys Gly <210> SEQ ID NO 130
<211> LENGTH: 12 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: CDRH3 <400> SEQUENCE: 130
Asp Leu Gly Tyr Glu Ile Asp Gly Tyr Gly Gly Leu 1 5 10 <210>
SEQ ID NO 131 <211> LENGTH: 10 <212> TYPE: PRT
<213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: CDRH1 dAbH1 <400> SEQUENCE:
131 Gly Ile Asp Leu Ser Asn Tyr Ala Ile Asn 1 5 10 <210> SEQ
ID NO 132 <211> LENGTH: 16 <212> TYPE: PRT <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: CDRH2 dAbH1 <400> SEQUENCE: 132 Ile Ile
Trp Ala Ser Gly Thr Thr Phe Tyr Ala Thr Trp Ala Lys Gly 1 5 10 15
<210> SEQ ID NO 133 <211> LENGTH: 13 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: CDRH3 dAbH1 <400> SEQUENCE:
133 Thr Val Pro Gly Tyr Ser Thr Ala Pro Tyr Phe Asp Leu 1 5 10
<210> SEQ ID NO 134 <211> LENGTH: 12 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: CDRL1 dAbL1 <400> SEQUENCE:
134 Gln Ser Ser Pro Ser Val Trp Ser Asn Phe Leu Ser 1 5 10
<210> SEQ ID NO 135 <211> LENGTH: 7 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: CDRL2 dAbL1 <400> SEQUENCE:
135 Glu Ala Ser Lys Leu Thr Ser 1 5 <210> SEQ ID NO 136
<211> LENGTH: 11 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: CDRL3 dAbL1 <400> SEQUENCE: 136 Gly Gly Gly Tyr
Ser Ser Ile Ser Asp Thr Thr 1 5 10 <210> SEQ ID NO 137
<211> LENGTH: 121 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Heavy chain variable domain of anti-albumin antibody
(no ds) <400> SEQUENCE: 137 Glu Val Gln Leu Leu Glu Ser Gly
Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys
Ala Val Ser Gly Ile Asp Leu Ser Asn Tyr 20 25 30 Ala Ile Asn Trp
Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Ile 35 40 45 Gly Ile
Ile Trp Ala Ser Gly Thr Thr Phe Tyr Ala Thr Trp Ala Lys 50 55 60
Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Val Tyr Leu 65
70 75 80 Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr
Cys Ala 85 90 95 Arg Thr Val Pro Gly Tyr Ser Thr Ala Pro Tyr Phe
Asp Leu Trp Gly 100 105 110 Gln Gly Thr Leu Val Thr Val Ser Ser 115
120 <210> SEQ ID NO 138 <211> LENGTH: 121 <212>
TYPE: PRT <213> ORGANISM: Artificial Sequence <220>
FEATURE: <223> OTHER INFORMATION: Heavy chain variable domain
of anti-albumin antibody (ds) <400> SEQUENCE: 138 Glu Val Gln
Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser
Leu Arg Leu Ser Cys Ala Val Ser Gly Ile Asp Leu Ser Asn Tyr 20 25
30 Ala Ile Asn Trp Val Arg Gln Ala Pro Gly Lys Cys Leu Glu Trp Ile
35 40 45 Gly Ile Ile Trp Ala Ser Gly Thr Thr Phe Tyr Ala Thr Trp
Ala Lys 50 55 60 Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn
Thr Val Tyr Leu 65 70 75 80 Gln Met Asn Ser Leu Arg Ala Glu Asp Thr
Ala Val Tyr Tyr Cys Ala 85 90 95 Arg Thr Val Pro Gly Tyr Ser Thr
Ala Pro Tyr Phe Asp Leu Trp Gly 100 105 110 Gln Gly Thr Leu Val Thr
Val Ser Ser 115 120 <210> SEQ ID NO 139 <211> LENGTH:
112 <212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Light chain
variable domain of anti-albumin antibody (no ds) <400>
SEQUENCE: 139 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Val Ser Ala
Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Gln Ser Ser Pro
Ser Val Trp Ser Asn 20 25 30 Phe Leu Ser Trp Tyr Gln Gln Lys Pro
Gly Lys Ala Pro Lys Leu Leu 35 40 45 Ile Tyr Glu Ala Ser Lys Leu
Thr Ser Gly Val Pro Ser Arg Phe Ser 50 55 60 Gly Ser Gly Ser Gly
Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln 65 70 75 80 Pro Glu Asp
Phe Ala Thr Tyr Tyr Cys Gly Gly Gly Tyr Ser Ser Ile 85 90 95 Ser
Asp Thr Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Arg Thr 100 105
110 <210> SEQ ID NO 140 <211> LENGTH: 112 <212>
TYPE: PRT <213> ORGANISM: Artificial Sequence <220>
FEATURE: <223> OTHER INFORMATION: Light chain variable domain
of anti-albumin antibody (ds) <400> SEQUENCE: 140 Asp Ile Gln
Met Thr Gln Ser Pro Ser Ser Val Ser Ala Ser Val Gly 1 5 10 15 Asp
Arg Val Thr Ile Thr Cys Gln Ser Ser Pro Ser Val Trp Ser Asn 20 25
30 Phe Leu Ser Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu
35 40 45 Ile Tyr Glu Ala Ser Lys Leu Thr Ser Gly Val Pro Ser Arg
Phe Ser 50 55 60 Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile
Ser Ser Leu Gln 65 70 75 80 Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gly
Gly Gly Tyr Ser Ser Ile 85 90 95 Ser Asp Thr Thr Phe Gly Cys Gly
Thr Tyr Val Glu Ile Lys Arg Thr 100 105 110 <210> SEQ ID NO
141 <211> LENGTH: 226 <212> TYPE: PRT <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Human CD79a <400> SEQUENCE: 141 Met Pro
Gly Gly Pro Gly Val Leu Gln Ala Leu Pro Ala Thr Ile Phe 1 5 10 15
Leu Leu Phe Leu Leu Ser Ala Val Tyr Leu Gly Pro Gly Cys Gln Ala 20
25 30 Leu Trp Met His Lys Val Pro Ala Ser Leu Met Val Ser Leu Gly
Glu 35 40 45 Asp Ala His Phe Gln Cys Pro His Asn Ser Ser Asn Asn
Ala Asn Val 50 55 60 Thr Trp Trp Arg Val Leu His Gly Asn Tyr Thr
Trp Pro Pro Glu Phe 65 70 75 80 Leu Gly Pro Gly Glu Asp Pro Asn Gly
Thr Leu Ile Ile Gln Asn Val 85 90 95 Asn Lys Ser His Gly Gly Ile
Tyr Val Cys Arg Val Gln Glu Gly Asn 100 105 110 Glu Ser Tyr Gln Gln
Ser Cys Gly Thr Tyr Leu Arg Val Arg Gln Pro
115 120 125 Pro Pro Arg Pro Phe Leu Asp Met Gly Glu Gly Thr Lys Asn
Arg Ile 130 135 140 Ile Thr Ala Glu Gly Ile Ile Leu Leu Phe Cys Ala
Val Val Pro Gly 145 150 155 160 Thr Leu Leu Leu Phe Arg Lys Arg Trp
Gln Asn Glu Lys Leu Gly Leu 165 170 175 Asp Ala Gly Asp Glu Tyr Glu
Asp Glu Asn Leu Tyr Glu Gly Leu Asn 180 185 190 Leu Asp Asp Cys Ser
Met Tyr Glu Asp Ile Ser Arg Gly Leu Gln Gly 195 200 205 Thr Tyr Gln
Asp Val Gly Ser Leu Asn Ile Gly Asp Val Gln Leu Glu 210 215 220 Lys
Pro 225 <210> SEQ ID NO 142 <211> LENGTH: 229
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Human CD79b
<400> SEQUENCE: 142 Met Ala Arg Leu Ala Leu Ser Pro Val Pro
Ser His Trp Met Val Ala 1 5 10 15 Leu Leu Leu Leu Leu Ser Ala Glu
Pro Val Pro Ala Ala Arg Ser Glu 20 25 30 Asp Arg Tyr Arg Asn Pro
Lys Gly Ser Ala Cys Ser Arg Ile Trp Gln 35 40 45 Ser Pro Arg Phe
Ile Ala Arg Lys Arg Gly Phe Thr Val Lys Met His 50 55 60 Cys Tyr
Met Asn Ser Ala Ser Gly Asn Val Ser Trp Leu Trp Lys Gln 65 70 75 80
Glu Met Asp Glu Asn Pro Gln Gln Leu Lys Leu Glu Lys Gly Arg Met 85
90 95 Glu Glu Ser Gln Asn Glu Ser Leu Ala Thr Leu Thr Ile Gln Gly
Ile 100 105 110 Arg Phe Glu Asp Asn Gly Ile Tyr Phe Cys Gln Gln Lys
Cys Asn Asn 115 120 125 Thr Ser Glu Val Tyr Gln Gly Cys Gly Thr Glu
Leu Arg Val Met Gly 130 135 140 Phe Ser Thr Leu Ala Gln Leu Lys Gln
Arg Asn Thr Leu Lys Asp Gly 145 150 155 160 Ile Ile Met Ile Gln Thr
Leu Leu Ile Ile Leu Phe Ile Ile Val Pro 165 170 175 Ile Phe Leu Leu
Leu Asp Lys Asp Asp Ser Lys Ala Gly Met Glu Glu 180 185 190 Asp His
Thr Tyr Glu Gly Leu Asp Ile Asp Gln Thr Ala Thr Tyr Glu 195 200 205
Asp Ile Val Thr Leu Arg Thr Gly Glu Val Lys Trp Ser Val Gly Glu 210
215 220 His Pro Gly Gln Glu 225 <210> SEQ ID NO 143
<211> LENGTH: 1304 <212> TYPE: PRT <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Human CD45 <400> SEQUENCE: 143 Met Tyr Leu
Trp Leu Lys Leu Leu Ala Phe Gly Phe Ala Phe Leu Asp 1 5 10 15 Thr
Glu Val Phe Val Thr Gly Gln Ser Pro Thr Pro Ser Pro Thr Gly 20 25
30 Leu Thr Thr Ala Lys Met Pro Ser Val Pro Leu Ser Ser Asp Pro Leu
35 40 45 Pro Thr His Thr Thr Ala Phe Ser Pro Ala Ser Thr Phe Glu
Arg Glu 50 55 60 Asn Asp Phe Ser Glu Thr Thr Thr Ser Leu Ser Pro
Asp Asn Thr Ser 65 70 75 80 Thr Gln Val Ser Pro Asp Ser Leu Asp Asn
Ala Ser Ala Phe Asn Thr 85 90 95 Thr Gly Val Ser Ser Val Gln Thr
Pro His Leu Pro Thr His Ala Asp 100 105 110 Ser Gln Thr Pro Ser Ala
Gly Thr Asp Thr Gln Thr Phe Ser Gly Ser 115 120 125 Ala Ala Asn Ala
Lys Leu Asn Pro Thr Pro Gly Ser Asn Ala Ile Ser 130 135 140 Asp Val
Pro Gly Glu Arg Ser Thr Ala Ser Thr Phe Pro Thr Asp Pro 145 150 155
160 Val Ser Pro Leu Thr Thr Thr Leu Ser Leu Ala His His Ser Ser Ala
165 170 175 Ala Leu Pro Ala Arg Thr Ser Asn Thr Thr Ile Thr Ala Asn
Thr Ser 180 185 190 Asp Ala Tyr Leu Asn Ala Ser Glu Thr Thr Thr Leu
Ser Pro Ser Gly 195 200 205 Ser Ala Val Ile Ser Thr Thr Thr Ile Ala
Thr Thr Pro Ser Lys Pro 210 215 220 Thr Cys Asp Glu Lys Tyr Ala Asn
Ile Thr Val Asp Tyr Leu Tyr Asn 225 230 235 240 Lys Glu Thr Lys Leu
Phe Thr Ala Lys Leu Asn Val Asn Glu Asn Val 245 250 255 Glu Cys Gly
Asn Asn Thr Cys Thr Asn Asn Glu Val His Asn Leu Thr 260 265 270 Glu
Cys Lys Asn Ala Ser Val Ser Ile Ser His Asn Ser Cys Thr Ala 275 280
285 Pro Asp Lys Thr Leu Ile Leu Asp Val Pro Pro Gly Val Glu Lys Phe
290 295 300 Gln Leu His Asp Cys Thr Gln Val Glu Lys Ala Asp Thr Thr
Ile Cys 305 310 315 320 Leu Lys Trp Lys Asn Ile Glu Thr Phe Thr Cys
Asp Thr Gln Asn Ile 325 330 335 Thr Tyr Arg Phe Gln Cys Gly Asn Met
Ile Phe Asp Asn Lys Glu Ile 340 345 350 Lys Leu Glu Asn Leu Glu Pro
Glu His Glu Tyr Lys Cys Asp Ser Glu 355 360 365 Ile Leu Tyr Asn Asn
His Lys Phe Thr Asn Ala Ser Lys Ile Ile Lys 370 375 380 Thr Asp Phe
Gly Ser Pro Gly Glu Pro Gln Ile Ile Phe Cys Arg Ser 385 390 395 400
Glu Ala Ala His Gln Gly Val Ile Thr Trp Asn Pro Pro Gln Arg Ser 405
410 415 Phe His Asn Phe Thr Leu Cys Tyr Ile Lys Glu Thr Glu Lys Asp
Cys 420 425 430 Leu Asn Leu Asp Lys Asn Leu Ile Lys Tyr Asp Leu Gln
Asn Leu Lys 435 440 445 Pro Tyr Thr Lys Tyr Val Leu Ser Leu His Ala
Tyr Ile Ile Ala Lys 450 455 460 Val Gln Arg Asn Gly Ser Ala Ala Met
Cys His Phe Thr Thr Lys Ser 465 470 475 480 Ala Pro Pro Ser Gln Val
Trp Asn Met Thr Val Ser Met Thr Ser Asp 485 490 495 Asn Ser Met His
Val Lys Cys Arg Pro Pro Arg Asp Arg Asn Gly Pro 500 505 510 His Glu
Arg Tyr His Leu Glu Val Glu Ala Gly Asn Thr Leu Val Arg 515 520 525
Asn Glu Ser His Lys Asn Cys Asp Phe Arg Val Lys Asp Leu Gln Tyr 530
535 540 Ser Thr Asp Tyr Thr Phe Lys Ala Tyr Phe His Asn Gly Asp Tyr
Pro 545 550 555 560 Gly Glu Pro Phe Ile Leu His His Ser Thr Ser Tyr
Asn Ser Lys Ala 565 570 575 Leu Ile Ala Phe Leu Ala Phe Leu Ile Ile
Val Thr Ser Ile Ala Leu 580 585 590 Leu Val Val Leu Tyr Lys Ile Tyr
Asp Leu His Lys Lys Arg Ser Cys 595 600 605 Asn Leu Asp Glu Gln Gln
Glu Leu Val Glu Arg Asp Asp Glu Lys Gln 610 615 620 Leu Met Asn Val
Glu Pro Ile His Ala Asp Ile Leu Leu Glu Thr Tyr 625 630 635 640 Lys
Arg Lys Ile Ala Asp Glu Gly Arg Leu Phe Leu Ala Glu Phe Gln 645 650
655 Ser Ile Pro Arg Val Phe Ser Lys Phe Pro Ile Lys Glu Ala Arg Lys
660 665 670 Pro Phe Asn Gln Asn Lys Asn Arg Tyr Val Asp Ile Leu Pro
Tyr Asp 675 680 685 Tyr Asn Arg Val Glu Leu Ser Glu Ile Asn Gly Asp
Ala Gly Ser Asn 690 695 700 Tyr Ile Asn Ala Ser Tyr Ile Asp Gly Phe
Lys Glu Pro Arg Lys Tyr 705 710 715 720 Ile Ala Ala Gln Gly Pro Arg
Asp Glu Thr Val Asp Asp Phe Trp Arg 725 730 735 Met Ile Trp Glu Gln
Lys Ala Thr Val Ile Val Met Val Thr Arg Cys 740 745 750 Glu Glu Gly
Asn Arg Asn Lys Cys Ala Glu Tyr Trp Pro Ser Met Glu 755 760 765 Glu
Gly Thr Arg Ala Phe Gly Asp Val Val Val Lys Ile Asn Gln His 770 775
780 Lys Arg Cys Pro Asp Tyr Ile Ile Gln Lys Leu Asn Ile Val Asn Lys
785 790 795 800 Lys Glu Lys Ala Thr Gly Arg Glu Val Thr His Ile Gln
Phe Thr Ser 805 810 815 Trp Pro Asp His Gly Val Pro Glu Asp Pro His
Leu Leu Leu Lys Leu 820 825 830 Arg Arg Arg Val Asn Ala Phe Ser Asn
Phe Phe Ser Gly Pro Ile Val 835 840 845 Val His Cys Ser Ala Gly Val
Gly Arg Thr Gly Thr Tyr Ile Gly Ile 850 855 860 Asp Ala Met Leu Glu
Gly Leu Glu Ala Glu Asn Lys Val Asp Val Tyr
865 870 875 880 Gly Tyr Val Val Lys Leu Arg Arg Gln Arg Cys Leu Met
Val Gln Val 885 890 895 Glu Ala Gln Tyr Ile Leu Ile His Gln Ala Leu
Val Glu Tyr Asn Gln 900 905 910 Phe Gly Glu Thr Glu Val Asn Leu Ser
Glu Leu His Pro Tyr Leu His 915 920 925 Asn Met Lys Lys Arg Asp Pro
Pro Ser Glu Pro Ser Pro Leu Glu Ala 930 935 940 Glu Phe Gln Arg Leu
Pro Ser Tyr Arg Ser Trp Arg Thr Gln His Ile 945 950 955 960 Gly Asn
Gln Glu Glu Asn Lys Ser Lys Asn Arg Asn Ser Asn Val Ile 965 970 975
Pro Tyr Asp Tyr Asn Arg Val Pro Leu Lys His Glu Leu Glu Met Ser 980
985 990 Lys Glu Ser Glu His Asp Ser Asp Glu Ser Ser Asp Asp Asp Ser
Asp 995 1000 1005 Ser Glu Glu Pro Ser Lys Tyr Ile Asn Ala Ser Phe
Ile Met Ser 1010 1015 1020 Tyr Trp Lys Pro Glu Val Met Ile Ala Ala
Gln Gly Pro Leu Lys 1025 1030 1035 Glu Thr Ile Gly Asp Phe Trp Gln
Met Ile Phe Gln Arg Lys Val 1040 1045 1050 Lys Val Ile Val Met Leu
Thr Glu Leu Lys His Gly Asp Gln Glu 1055 1060 1065 Ile Cys Ala Gln
Tyr Trp Gly Glu Gly Lys Gln Thr Tyr Gly Asp 1070 1075 1080 Ile Glu
Val Asp Leu Lys Asp Thr Asp Lys Ser Ser Thr Tyr Thr 1085 1090 1095
Leu Arg Val Phe Glu Leu Arg His Ser Lys Arg Lys Asp Ser Arg 1100
1105 1110 Thr Val Tyr Gln Tyr Gln Tyr Thr Asn Trp Ser Val Glu Gln
Leu 1115 1120 1125 Pro Ala Glu Pro Lys Glu Leu Ile Ser Met Ile Gln
Val Val Lys 1130 1135 1140 Gln Lys Leu Pro Gln Lys Asn Ser Ser Glu
Gly Asn Lys His His 1145 1150 1155 Lys Ser Thr Pro Leu Leu Ile His
Cys Arg Asp Gly Ser Gln Gln 1160 1165 1170 Thr Gly Ile Phe Cys Ala
Leu Leu Asn Leu Leu Glu Ser Ala Glu 1175 1180 1185 Thr Glu Glu Val
Val Asp Ile Phe Gln Val Val Lys Ala Leu Arg 1190 1195 1200 Lys Ala
Arg Pro Gly Met Val Ser Thr Phe Glu Gln Tyr Gln Phe 1205 1210 1215
Leu Tyr Asp Val Ile Ala Ser Thr Tyr Pro Ala Gln Asn Gly Gln 1220
1225 1230 Val Lys Lys Asn Asn His Gln Glu Asp Lys Ile Glu Phe Asp
Asn 1235 1240 1245 Glu Val Asp Lys Val Lys Gln Asp Ala Asn Cys Val
Asn Pro Leu 1250 1255 1260 Gly Ala Pro Glu Lys Leu Pro Glu Ala Lys
Glu Gln Ala Glu Gly 1265 1270 1275 Ser Glu Pro Thr Ser Gly Thr Glu
Gly Pro Glu His Ser Val Asn 1280 1285 1290 Gly Pro Ala Ser Pro Ala
Leu Asn Gln Gly Ser 1295 1300
* * * * *
References