NZ616433B2 - Bcma (cd269/tnfrsf17) -binding proteins - Google Patents
Bcma (cd269/tnfrsf17) -binding proteins Download PDFInfo
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- NZ616433B2 NZ616433B2 NZ616433A NZ61643312A NZ616433B2 NZ 616433 B2 NZ616433 B2 NZ 616433B2 NZ 616433 A NZ616433 A NZ 616433A NZ 61643312 A NZ61643312 A NZ 61643312A NZ 616433 B2 NZ616433 B2 NZ 616433B2
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- New Zealand
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- seq
- antigen binding
- binding protein
- antibody
- bcma
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- A61K47/6835—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site
- A61K47/6849—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site the antibody targeting a receptor, a cell surface antigen or a cell surface determinant
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- C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
- C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
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- C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
- C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
- C07K16/2878—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the NGF-receptor/TNF-receptor superfamily, e.g. CD27, CD30, CD40, CD95
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- C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
- C07K2317/24—Immunoglobulins specific features characterized by taxonomic origin containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered
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- C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
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Abstract
Discloses antigen binding proteins and fragments thereof which specifically bind B Cell Maturation Antigen (BCMA), particularly human BCMA (hBCMA) and which inhibit the binding of BAFF and APRIL to the BCMA receptor, and wherein the antigen binding protein comprises CDRs of SEQ ID NOs: 1-6 or variants thereof, wherein the sequences are as defined in the complete specification. Further disclosed are pharmaceutical compositions, screening and medical treatment methods. ts thereof, wherein the sequences are as defined in the complete specification. Further disclosed are pharmaceutical compositions, screening and medical treatment methods.
Description
BCMA (CD269/TNFRSF1'7) -BINDING PROTEINS
Field of the invention
The present ion relates to antigen binding proteins and fragments thereof that specifically bind
B cell maturation antigen (BCMA) and in particular human BCMA (hBCMA).
The present invention also concerns methods of treating diseases or disorders with said antigen
binding nts, pharmaceutical compositions sing said antigen binding fragments and
methods of manufacture. Other embodiments of the present invention will be apparent from the
description below.
Background of the invention
BCMA (CD269 or TNFRSF17) is a member of the TNF receptor superfamily. It is a non-glycosylated
integral membrane receptor for the ligands BAFF and APRIL. BCMA’s s can also bind
additional receptors: TACI (Transmembrane Activator and Calcium tor and cyclophilin ligand
lnteractor), which binds APRIL and BAFF; as well as BAFF-R (BAFF Receptor or BR3), which shows
restricted but high ty for BAFF. Together, these receptors and their corresponding s
regulate different aspects of humoral immunity, B-cell development and homeostasis.
BCMA’s expression is typically restricted to the B-cell lineage and is reported to increase in terminal
B-cell differentiation. BCMA is expressed by human plasma blasts, plasma cells from tonsils, spleen
and bone marrow, but also by tonsillar memory B cells and by germinal centre B cells, which have a
TACI-BAFFR low ype (Darce et al, 2007). BCMA is virtually absent on naive and memory B-
cells (Novak et al., 2004a and b). The BCMA antigen is expressed on the cell surface so is accessible
to the antibody, but is also expressed in the golgi. As suggested by its expression profile, BCMA
signalling, lly linked with B-cell survival and proliferation, is important in the late stages of B-cell
differentiation, as well as the survival of long lived bone marrow plasma cells (O’Connor et al., 2004)
and plasmablasts (Avery et al., 2003). rmore, as BCMA binds APRIL with high affinity, the
BCMA-APRIL signalling axis is suggested to predominate at the later stages of B-cell differentiation,
perhaps being the most physiologically relevant interaction.
Multiple a (MM) is a clonal B-cell malignancy that occurs in multiple sites within the bone
marrow before ing to the circulation; either de novo, or as a progression from monoclonal
gammopathy of undetermined significance (MGUS). It is commonly characterised by increases in
paraprotein and osteoclast activity, as well as hypercalcaemia, cytopenia, renal ction,
hyperviscosity and peripheral neuropathy. Decreases in both normal antibody levels and s of
neutrophils are also , leading to a life threatening susceptibility to infection. BCMA has been
implicated in the growth and survival of a cell lines in vitro (Novak et al., 2004a and b;
Moreaux et al., 2004).
BCMA expression (both transcript and protein) is reported to correlate with disease progression in
MM. Using Affymetrix rrays, it was demonstrated that the TAC/ and BCMA genes were over-
expressed in Multiple Myeloma Cells (MMC) compared with their normal rparts (Moreaux et al,
2004). Gene expression analysis has been used to compare human myeloma cells with ed
plasma cells from patients with MGUS and from normal bone marrow as well as with primary tumour
cells from B-cell lineage leukaemias (Bellucci et al, 2005). The BCMA gene was highly expressed in
all myeloma samples. Although purified plasma cells from patients with MGUS had lower expression
of BCMA, there was no significant difference when compared with the expression found in normal
plasma cells or myeloma cells. In contrast, BCMA expression was significantly lower in B-cell Chronic
Lymphocytic Leukaemia (CLL), pre-B Acute Lymphocytic Leukaemia (ALL) and T-cell ALL (T-ALL).
Mouse models that transgenically over-express BAFF or APRIL have a significant se in B-cell
lymphomas (Batten et al., 2004 — BAFF; les et al., 2004 — APRIL). In humans, excess BAFF
and APRIL have been detected in the sera and micro-environments of patients with a number of B-
cell malignancies, as well as other B-cell disorders.
All patent and literature references disclosed within the present ication are expressly and
entirely incorporated herein by reference.
Brief Description of Figures
Figure 1: FMAT Binding Assay - Figure showing the results of the FMAT assay for CA8 antibody
binding to human and cyno BCMA expressing HEK293 cells. Human chimeric CA8 binds well to
human and cyno BCMA expressing cells.
Figure 2: ELISA Binding Assay - Figure showing the ELISA results for CA8 antibodies binding to
human and cyno BCMA inant proteins. This clearly shows that human chimeric CA8
antibodies bind to human and cyno BCMA proteins equally.
Figure 3: BiaCore Binding Assay - Figure g the binding of CA8 to BCMA-Fc, TACI-Fc and
BAFF-R-Fc proteins in the Biacore ment. CA8 chimera antibody does not bind to TACI or
BAFF-R proteins.
Figure 4: Cell binding assay - Figure showing binding of murine S307118G03, S3222110D07,
S332121F02 and 6E04 to H929 le myeloma cells and 10D07, S332121F02 and
S332126E04 to the BCMA transfected ARH77 cells as determined by FACS.
Multiple myeloma cell line H929 or ARH77-hBCMA 10B5 BCMA expressing transfectant cells were
stained with either murine anti BCMA antibodies (solid histogram) or murine lgGZa isotype control
(open histograms). Cells were analysed by FACS to detect antibody bound to the cells.
Figure 5: Cell binding assay - Figure showing binding of chimeric CA8 to a panel of multiple
myeloma cell lines as determined by FACS. g to H929, OPM-2, JJN-3 and U266 was tested by
flow cytometry and mean fluorescence intensity (MFI) values measured to determine binding. Synagis
was used as an vant e control.
Figure 6: Cell binding assay - Figure showing binding curves of sed CA8 variants to BCMA
transfected ARH77 cells (A) and multiple myeloma H929 cells (B) as determined by FACS.
Humanised variants J6M0, J6M1, J6M2, J9M0, J9M1 and J9M2 were tested by flow cytometry and
mean fluorescence intensity (MFI) values measured to determine binding ed to the CA8
chimera.
Figure 7: Ligand lisation assays —
(A and B) Figure showing the ability of CA8 and J6M0 to neutralise binding of recombinant BAFF or
APRIL to recombinant BCMA coated on an ELISA plate. OD values were used to calculate the
antibody mediated inhibition of the maximal signal achieved by the relevant ligand alone binding to
recombinant BCMA. Data is reported as percentage inhibition of the maximal signal. Antibodies tested
were ic CA8 and humanised CA8 version J6M0 in both wild type and afucosylated (Potelligent)
form.
40 (A) Neutralisation of BAFF ligand binding; (B)- Neutralisation APRIL ligand binding.
(C) — Figure showing the ability of J6M0 BCMA antibody in inhibition of BAFF or APRIL induced
phosphorylation of NFKappaB in H929 cells. H-929 cells were washed 3 times to remove any sBCMA
and resuspended in serum free medium. J6M0 potelligent antibody was added to a 96 well plate to
give a final well concentrations up to 100ug/ml along with BAFF or APRIL ligand to give a final well
concentration of 0.6 or 0.2 ug/ml respectively. H-929 cells were then plated at 7.5x104cells/well in
serum free medium. 30 s later the cells were lysed and phosphorylated NFkappaB levels
measured using a MSD pNFkappaB assay. MSD reader 502819.This is data from one independent
experiments. Each data point is the d of two replicates.
Figure 8: ADCC assay — Figure showing ADCC ty of chimeric CA8 and defucosylated (Fc
enhanced) CA8 with target cells expressing BCMA.
Human NK cells were incubated with europium ed ARH77 1OB5 BCMA transfected target cells
in the presence of varying concentrations of antibody. um e from the target cells was
measured and specific lysis calculated. (A) ADCC dose response curves of chimeric CA8 compared
to e control . (B) ADCC dose response curves for chimeric CA8 and defucosylated chimeric
CA8 (Fc enhanced), against the BCMA expressing cell line ARH77 1085.
Figure 9: ADCC assay — Figure showing ADCC assay on CA8 humanised antibodies using ARH77
BCMA expressing target cells.
Human PBMC were incubated with europium labelled ARH77 BCMA transfected target cells in the
presence of a range of concentrations of the J5, J6, J7, J8 or J9 series of humanised CA8 antibodies.
Europium release from the target cells was measured and ic lysis calculated. EC50 values are
shown in ug/ml.
Figure 10: ADCC assay — Figure showing ADCC activity of chimeric, S332121F02 (A),
83322110D07 (B) S307118G03 (C) and humanised S307118G03 H3L0 (D) against ARH771OB5
target cells with purified NK cells as effector cells. Human NK target cells were incubated with
europium labelled ARH77 1OB5 BCMA transfected target cells in the presence of varying
concentrations of antibody. um release from the target cells was measured and specific lysis
calculated.
Figure 11: Viability assay dose response curves — Figure g dose response curves in a cell
viability assay for chimeric CA8 antibody, chimeric CA8-chMAE and chimeric CA8-mcMMAF
antibody-drug conjugates in human multiple myeloma cell lines (A) NCl-H929 (B) U266-B1 (C) JJN3
and (D) OPM2. Antibody was added to the cells and the number of viable cells after 96 hours
measured using CelltiterGlo.Data points represent the mean of triplicate CellTiterGlo ements.
Error bars represent standard error.
Figure 12: Impact of CA8 ic antibody on cell cycle.
WO 63805
(A) Cell cycle histograms of NCl-H929 cells treated with unconjugated chimeric CA8, chimeric CA8-
chMAE ADC or chimeric CA8-mcMMAF ADC at 50ng/mL for the timepoints indicated. Pactitaxel
(100nM) was used as a positive control for GZ/M cell cycle arrest and cell death. Control human lgG1
was used as a negative l. Cell cycle analysis was carried out at the times shown on the .
(B) Quantification of the 4N DNA cell population indicative of GZ/M arrest and (C) sub-2N DNA cell
tion tive of cell death for each of the treatments indicated. Cells were seeded in 12-well
plates (2x105 cells per well in 1mL of RPMI + 10% FBS). Antibody or ADC was added 6 hours after
cell seeding.
Figure 13: Impact of chimeric CA8 on o-histone H3.
Chimeric CA8 ADC treatment results in increased phospho-Histone H3 staining of NCl-H929 cells.
(A,B) Dot plots of cells stained with propidium iodide to measure DNA content (FL3-H) X-axis and
anti-phospho-Histone H3 (Thr11) antibody (FL1-H) y-axis after treatment with either Control lgG (A) or
chimeric CA8-mcMMAF (B). (C) Quantification of phospho-Histone H3 positive NCl-H929 cells after a
48 hour treatment with the indicated concentrations of chimeric CA8 ADCs. Pactitaxel ) was
used as a positive l for mitotic arrest and control chimera lgG1 was used as a negative control.
Cells were seeded in 12-well plates (2x105 cells per well in 1mL of RPMI + 10% FBS). dy or
ADC was added 6 hours after cell seeding.
Figure 14: Impact of chimeric CA8 on Annexin-V.
Chimeric CA8 ADC treatment results in increased Annexin-V staining of NCl-H929 cells.
(A) rams of Annexin-V-FITC (FL1-H; top panels) and Live cell propidium iodide staining (FL3-H;
bottom panels) after treatment with increasing concentrations of chimeric CA8 ADCs (B)
Quantification of Annexin-V positive NCl-H929 cells after a 96 hour ent with the indicated
concentrations of chimeric CA8 ADCs. Pactitaxel (100nM) was used as a positive control for
apoptosis and control chimera lgG1 was used as a negative l. Cells were seeded in 12-well
plates (2x105 cells per well in 1mL of RPMI + 10% FBS). Antibody or ADC was added 6 hours after
cell seeding.
Figure 15: Viability assay dose response curves - Figure showing dose response curves for the
unconjugated (Naked) and chMAE and mcMMAF antibody-drug conjugates of chimeric CA8 or
zed J6M0 antibodies. Antibody drug conjugates were tested against human multiple myeloma
cell lines NCl-H929 and OPM2.
Figure 16: Viability assay dose response curves - Figure showing dose response curves for the
ugated antibodies, chMAE and mcMMAF dy-drug conjugates of murine anti-BCMA
antibodies S332121F02, S322110D07, S332126E04 and S307118G03 in human multiple myeloma
cell lines NCI-H929 and U266-B1.
Figure 17 ADCC ty of ADC J6M0 molecules — Figure showing ADCC assay on J6M0
antibodies using ARH77 BCMA expressing target cells. Human PBMC were incubated with europium
labelled ARH77 BCMA transfected target cells in the presence of a range of concentrations of J6M0
WT and potelligent BCMA antibodies conjugated to MMAE, MMAF, or unconjugated Europium
release was monitored on the Victor 2 1420 multilabel reader.
Figure 18 ADCC dose response curves of CA8 J6M0 Potelligent against a panel of 5
multiple myeloma lines - Human PBMC were incubated with multiple myeloma target cells in the
presence of varying concentrations of CA8 J6M0 potelligent antibody at an E:T ratio of 50:1 for 18
hours. The percentage of target cells remaining in the effecter plus target mixture was then measured
by FACS using a fluorescently labelled anti-CD138 antibody to detect the target cells and the t
cytotoxicity calculated. A) Example dose se curves for CA8 J6M0 potelligent against the five
multiple myeloma cell lines tested. Each data point is from a cate value.
Figure 19 Effect of dose escalation of J6M0 and drug conjugated J6M0 on the growth and
establishment of NCl-H929 cells in CB.17 SCID mice Calculated tumour volumes of NCl-H929
s in CB17 SCID mice following twice weekly intraperitoneal dosing of either 50 or 100ug J6M0
anti-BCMA or lgG1 isotype l unconjugated, or conjugated to MMAE or MMAF for 2 weeks.
Data points represent mean tumour volume of n=5 per group
Figure 20- Determination of soluble BCMA levels in serum from y volunteers and
myeloma patients. Serum samples were collected from MM patient samples were from a variety of
stages (progressive e, remission, relapsed, newly diagnosed, and others). The samples shown
in the figure are those from serum diluted 1/500 prior to the assay.
A Human BCMA/TNFRSF17 sandwich ELISA kit from R& D Systems which measures soluble human
BCMA levels was used to detect BCMA following the rd protocol provided with the kit.
Summary of the ion
The present invention es antigen binding ns which bind to membrane bound targets and
wherein the antigen binding protein is capable of internalisation. In a further embodiment there is
provided an immunoconjugate comprising the antigen binding protein of the t invention and a
cytotoxic agent. In a further embodiment the antigen binding protein has ADCC effector function for
example the antigen binding protein has enhanced ADCC effector function.
The t invention provides antigen binding proteins which specifically bind to BCMA, for example
dies which ically bind to BCMA and which inhibit the binding of BAFF and/or APRIL to the
BCMA receptor. The present invention also provides antigen binding proteins which specifically bind
to BCMA and which inhibits the binding of BAFF and/or APRIL to BCMA wherein the antigen binding
protein is capable of binding to A or is capable of chRlllA mediated effector function.
The antigen binding ns of the present invention specifically bind to BCMA and t the binding
of BAFF and/or APRIL to BCMA wherein the antigen binding protein has enhanced g to
chRlllA or has enhanced A mediated effector function. In one embodiment the antigen
binding protein is capable of internalisation.
In one aspect of the invention there is ed an antigen binding protein which binds to non-
membrane bound BCMA, for example to serum BCMA.
In one embodiment of the present ion there is provided an immunoconjugate comprising the
antigen binding protein of the present invention and a cytotoxic agent.
In a further embodiment the antigen binding proteins are conjugated to a toxin such as an auristatin.
In yet a further embodiment the drug conjugate is chMAE or mcMMAF. In one embodiment the
immunoconjugate is also ADCC enhanced.
The antigen binding proteins may be related to, or d from a murine monoclonal antibody CA8.
The CA8 murine heavy chain variable region amino acid sequence is provided as SEQ ID NO. 7 and
the CA8 murine light chain variable region amino acid sequence is provided as SEQ ID NO. 9.
The antigen binding proteins may be related to, or derived from a murine monoclonal antibody
S336105A07. The S336105A07 murine heavy chain variable region amino acid sequence is provided
as SEQ ID NO. 140 and the S336105A07 murine light chain variable region amino acid sequence is
ed as SEQ ID NO. 144.
Other murine monoclonal dies from which antigen binding proteins of the present invention may
also be derived are included in Table C.
In a particular aspect the present invention provides an antigen binding protein which specifically
binds to BCMA and which inhibits the binding of BAFF and/or APRIL to BCMA, wherein the
antigen binding protein is capable of g to FcγRIIIA or is capable of FcγRIIIA mediated effector
function, and wherein the antigen binding protein is capable of internalisation and wherein the antigen
binding protein comprises CDRH3 of SEQ ID NO.3 or a variant of SEQ ID NO. 3, CDR H1 of SEQ.
ID. NO: 1, CDRH2: SEQ. ID. NO: 2, CDRL1: SEQ. ID. NO: 4, CDRL2: SEQ. ID. NO: 5 and
CDRL3: SEQ. ID. NO: 6. Also provided are immunoconjugates thereof.
In a still further aspect, the t invention provides for the use of an antigen binding protein or an
immunoconjugate as described herein in the manufacture of a medicament for treating a human
patient afflicted with a B cell ma or an inflammatory disease or disorder.
(followed by 9A)
wed by 10)
In a further embodiment the n binding proteins or fragments specifically bind to BCMA and
inhibit the binding of BAFF and/or APRIL to BCMA wherein the antigen binding proteins or fragments
thereof have the ability to bind to chRlllA and mediate chRlllA mediated effector ons, or have
enhanced chRlllA mediated effector function. In one embodiment of the invention as herein provided
the antigen binding proteins are capable of internalisation.
In one aspect of the ion there is provided an antigen binding protein according to the invention
as herein described which binds to non-membrane bound BCMA, for example to serum BCMA.
In one aspect of the invention there is provided an antigen binding protein as herein described
wherein the antigen binding protein comprises CDRH3 of SEQ ID NO.3 or a variant of SEQ ID NO. 3.
In a further aspect of the invention there is provided an antigen binding protein as herein described
wherein the antigen binding protein further comprises one or more of: CDR H1 of SEQ. ID. NO: 1,
CDRH2: SEQ. ID. NO: 2: CDRL1: SEQ. ID. NO: 4, CDRL2: SEQ. ID. NO: 5 and/or CDRL3: SEQ. ID.
NO: 6 and or variants f.
In one aspect of the invention there is provided an antigen binding protein as herein described
wherein the antigen binding n comprises CDRH3 of SEQ ID NO.184 or a variant of SEQ ID NO.
184.
In a further aspect of the invention there is provided an antigen binding protein as herein described
wherein the antigen binding n further comprises one or more of: CDR H1 of SEQ. ID. NO: 182,
CDRH2: SEQ. ID. NO: 183: CDRL1: SEQ. ID. NO: 185, CDRL2: SEQ. ID. NO: 186 and/or CDRL3:
SEQ. ID. NO: 187 and or variants thereof.
In yet a further aspect the antigen binding protein comprises CDR H3 of SEQ. ID. NO: 3: CDRH2:
SEQ. ID. NO: 2: CDR H1 of SEQ. ID. NO:1: CDRL1: SEQ. ID. NO: 4: CDRL2: SEQ. ID. NO: 5 and
CDRL3: SEQ. ID. NO: 6.
In yet a further aspect the antigen binding protein comprises CDR H3 of SEQ. ID. NO: 184: CDRH2:
SEQ. ID. NO: 183: CDR H1 of SEQ. ID. NO:182: CDRL1: SEQ. ID. NO: 185: CDRL2: SEQ. ID. NO:
186 and CDRL3: SEQ. ID. NO: 187.
In one aspect of the invention the antgen binding protein has ed effector function. In r
aspect the antigen binding protein is conjugated to a cytotoxic agent. In yet a furher embodiment the
antigen binding protein has both enhanced effector on and is conjugated to a cytotoxic agent.
The antigen binding proteins of the present invention may comprise heavy chain variable regions and
40 light chain variable regions of the invention which may be formatted into the ure of a natural
antibody or functional fragment or equivalent thereof. An antigen binding n of the invention may
therefore comprise the VH s of the invention formatted into a full length antibody, a (Fab’)2
fragment, a Fab fragment, or equivalent thereof (such as scFV, bi- tri- or tetra-bodies, Tandabs etc.),
when paired with an appropriate light chain. The antibody may be an lgG1, IgG2, IgG3, or IgG4; or
IgM; IgA, IgE or IgD or a modified t thereof. The constant domain of the antibody heavy chain
may be selected accordingly. The light chain constant domain may be a kappa or lambda constant
domain. Furthermore, the antigen binding protein may comprise cations of all classes e.g. lgG
dimers, Fc mutants that no longer bind Fc receptors or mediate C1q binding. The antigen binding
protein may also be a chimeric antibody of the type described in W086/01533 which ses an
antigen binding region and a non-immunoglobulin .
The constant region is selected according to any functionality required e.g. an lgG1 may demonstrate
lytic ability through binding to complement and/or will mediate ADCC (antibody dependent cell
cytotoxicity).
The antigen binding proteins of the present invention are derived from the murine antibody having the
variable regions as described in SEQ ID NO:7 and SEQ ID NO:9 or non-murine equivalents thereof,
such as rat, human, ic or humanised variants thereof, for example they are d from the
antibody having the variable heavy chain sequences as described in SEQ ID NO:11, SEQ ID NO:13,
SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25, SEQ
ID N027 and SEQ ID NO:29 and/or the variable light chain ces as described in SEQ ID
NO:31, SEQ ID NO:33 and/or SEQ ID NO:35.
In another embodiment the antigen binding proteins of the present invention are derived from an
antibody having the variable heavy chain sequences as described in SEQ ID NO:116 or SEQ ID
NO:118 and/or the variable light chain ces as described in SEQ ID NO:120, or SEQ ID
NO:122.
In another embodiment the antigen binding proteins of the t invention are derived from an
antibody having the variable heavy chain sequences as described in SEQ ID NO:140 and/or the
variable light chain sequences as described in SEQ ID NO:144.
In one aspect of the invention there is provided an antigen binding protein comprising an isolated
heavy chain le domain selected from any one of the following: SEQ ID NO:11, SEQ ID NO:13,
SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25, SEQ
ID NO:27, SEQ ID NO:29, SEQ ID NO:116 or SEQ ID NO:118.
In another aspect of the invention there is ed an antigen g protein comprising an ed
light chain variable domain selected from any one of the following: SEQ ID NO:31, SEQ ID NO:33 or
40 SEQ ID NO:35, SEQ ID NO:120 or SEQ ID NO:122.
In a further aspect of the ion there is provided an antigen binding protein comprising an isolated
heavy chain variable domain selected from any one of the following: SEQ ID NO:11, SEQ ID NO:13,
SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25, SEQ
ID N027 and SEQ ID N029 and an isolated light chain variable domain selected from any one of the
following: SEQ ID NO:31, SEQ ID NO:33 and/or SEQ ID NO:35.
In one aspect the antigen binding protein of the present invention comprises a heavy chain le
region encoded by SEQ. ID. N023 and a light chain variable region encoded by SEQ. ID. NO:31
In one aspect the antigen binding protein of the present invention comprises a heavy chain le
region encoded by SEQ. ID. N027 and a light chain variable region encoded by SEQ. ID. NO:31.
In one aspect the antigen binding protein of the t invention comprises a heavy chain le
region encoded by SEQ. ID. N029 and a light chain variable region encoded by SEQ. ID. NO:31.
In one aspect the antigen binding protein of the present invention comprises a heavy chain variable
region encoded by SEQ. ID. NO:116 and a light chain variable region encoded by SEQ. ID. NO:120
In one aspect the antigen binding protein of the present invention comprises a heavy chain variable
region encoded by SEQ. ID. NO:118 and a light chain variable region d by SEQ. ID. NO:122
In one aspect there is provided a polynucleotide encoding an isolated variable heavy chain said
polynucleotide comprising SEQ. ID. NO. 12, or SEQ. ID. NO. 14, or SEQ. ID. NO. 16, or SEQ. ID. NO.
18, or SEQ. ID. NO. 20, or SEQ. ID. NO. 22, or SEQ. ID. NO. 24, or SEQ. ID. NO. 26, or SEQ. ID.
NO. 28, or SEQ. ID. NO. 30 or SEQ. ID. NO. 117 or SEQ. ID. NO. 119 or SEQ. ID. NO. 141..
In one aspect there is provided a cleotide ng an isolated variable light chain said
polynucleotide comprising SEQ. ID. NO. 32, or SEQ. ID. NO. 34, or SEQ. ID. NO. 36 or SEQ. ID. NO.
121 or SEQ. ID. NO.123 or SEQ. ID. NO. 145.
In a further aspect there is provided a polynucleotide encoding an isolated variable heavy chain said
polynucleotide comprising SEQ. ID. NO. 24, or SEQ. ID. NO. 28 or SEQ. ID. NO. 30 and a
polynucleotide encoding an isolated le light chain said polynucleotide comprising SEQ. ID. NO.
32, or SEQ. ID. NO. 34.
In yet a further aspect there is ed a polynucleotide encoding an isolated variable heavy chain
said polynucleotide comprising SEQ. ID. NO. 24 and a polynucleotide encoding an isolated variable
light chain said polynucleotide comprising SEQ. ID. NO.32.
In yet a further aspect there is provided a polynucleotide encoding an isolated variable heavy chain
said polynucleotide comprising SEQ. ID. NO. 117 and a polynucleotide encoding an isolated variable
light chain said polynucleotide comprising SEQ. ID. NO.121.
In yet a further aspect there is provided a polynucleotide encoding an ed variable heavy chain
said polynucleotide comprising SEQ. ID. NO. 119 and a polynucleotide encoding an isolated variable
light chain said polynucleotide comprising SEQ. ID. NO.123.
In yet a further aspect there is provided a polynucleotide encoding an isolated variable heavy chain
said polynucleotide sing SEQ. ID. NO. 141 and a polynucleotide encoding an isolated variable
light chain said polynucleotide sing SEQ. ID. NO.145.
In a further aspect the antigen binding protein may comprise any one of the variable heavy chains as
described herein in combination with any one of the light chains as described herein.
In one aspect the antigen binding protein is an antibody or antigen binding fragment thereof
comprising one or more CDR’s ing to the ion described , or one or both of the
heavy or light chain variable domains according to the invention described herein. In one embodiment
the antigen binding protein binds primate BCMA. In one such embodiment the antigen g protein
additionally binds non-human primate BCMA, for example lgus macaque monkey BCMA.
In another aspect the antigen g protein is selected from the group consisting of a dAb, Fab,
Fab’, F(ab’)2, Fv, diabody, triabody, tetrabody, miniantibody, and a dy,.
In one aspect of the present invention the antigen binding protein is a humanised or chimaeric
antibody, in a further aspect the antibody is humanised.
In one aspect the antibody is a monoclonal dy.
In one aspect of the present invention there is provided an antibody with the heavy chain sequence as
set forth in SEQ ID NO: 55 or SEQ ID NO: 59 or SEQ ID NO: 61.
In one aspect of the present invention there is provided an antibody with the light chain sequence as
set forth in SEQ ID NO: 63 or SEQ ID NO: 65.
In a further aspect of the invention there is ed an antibody with the heavy chain sequence of
SEQ ID NO: 55 and a light chain sequence as set forth in SEQ ID NO: 63.
In one embodiment there is provided an antigen binding protein which competes with an antigen
binding protein of the invention as herein described. In one such embodiment there is therefore
40 provided an antigen binding protein which es with an antigen binding protein which comprises
2012/059762
the heavy chain le ce of SEQ ID NO 23 and the light chain variable region of SEQ ID
NO 31.
In a further embodiment there is therefore provided an antigen g protein which competes with
an antigen binding protein which comprises a heavy chain variable sequence selected from one of
SEQ ID NO 27, SEQ ID NO 29, SEQ ID NO 116, SEQ ID NO 118 and SEQ ID NO 140 and alight
chain variable region selected from one of SEQ ID NO 31, SEQ ID NO 120, SEQ ID NO 122 and SEQ
ID NO 144.
In another aspect the antigen binding protein binds to human BCMA with high affinity for example
when measured by Biacore the antigen binding protein binds to human BCMA with an affinity of 20nM
or less or an affinity of 15nM or less or an affinity of 5nM or less or an affinity of 1000 pM or less or an
affinity of 500pM or less or an affinity of 400pM or less, or 300pM or less or for example about 120pM.
In a further embodiment the antigen binding protein binds to human BCMA when measured by
Biacore of between about 100pM and about 500pM or n about 100pM and about 400pM, or
between about 100pM and about 300pM. In one embodiment of the present invention the antigen
binding protein binds BCMA with an affinity of less than 150pm.
In one such embodiment, this is measured by Biacore, for example as set out in Example 4.
In another aspect the antigen binding protein binds to human BCMA and lises the binding of the
ligands BAFF and/or APRIL to the BCMA receptor in a cell neutralisation assay n the antigen
binding n has an IC50 of between about 1nM and about 500nM, or between about 1nM and
about 100nM, or between about 1nM and about 50nM, or between about 1nM and about 25nM, or
between about 5nM and about 15nM. In a further embodiment of the present invention the n
g protein binds BCMA and neutralises BCMA in a cell neutralisation assay wherein the antigen
binding protein has an IC50 of about 10nM.
In one such embodiment, this is measured by a cell neutralisation assay, for example as set out in
Example 4.6.
The antigen g proteins, for example antibodies of the present ion may be produced by
transfection of a host cell with an expression vector comprising the coding sequence for the antigen
binding n of the invention. An expression vector or recombinant plasmid is produced by placing
these coding sequences for the antigen g protein in operative association with conventional
regulatory control ces capable of controlling the replication and expression in, and/or secretion
from, a host cell. Regulatory sequences include promoter sequences, e.g., CMV promoter, and signal
sequences which can be derived from other known antibodies. Similarly, a second expression vector
can be produced having a DNA sequence which encodes a complementary antigen binding protein
light or heavy chain. In certain embodiments this second expression vector is identical to the first
except insofar as the coding sequences and selectable markers are concerned, so to ensure as far as
possible that each polypeptide chain is functionally sed. Alternatively, the heavy and light
chain coding sequences for the antigen binding protein may reside on a single vector.
A selected host cell is co-transfected by conventional techniques with both the first and second
vectors (or simply transfected by a single vector) to create the transfected host cell of the invention
comprising both the recombinant or synthetic light and heavy chains. The transfected cell is then
cultured by conventional techniques to produce the ered antigen binding protein of the
invention. The n binding protein which includes the association of both the inant heavy
chain and/or light chain is screened from culture by appropriate assay, such as ELISA or RIA. Similar
conventional techniques may be employed to construct other antigen binding proteins.
Suitable vectors for the g and subcloning steps employed in the methods and uction of
the compositions of this invention may be selected by one of skill in the art. For example, the
conventional pUC series of cloning vectors may be used. One vector, pUC19, is commercially
available from supply houses, such as am (Buckinghamshire, United m) or Pharmacia
la, Sweden). onally, any vector which is capable of replicating y, has an
abundance of cloning sites and selectable genes (e.g., antibiotic resistance), and is easily
manipulated may be used for cloning. Thus, the selection of the cloning vector is not a limiting factor
in this invention.
The expression vectors may also be characterized by genes suitable for amplifying expression of the
logous DNA sequences, e.g., the ian dihydrofolate reductase gene (DHFR). Other
vector sequences include a poly A signal sequence, such as from bovine growth hormone (BGH) and
the betaglobin promoter sequence (betaglopro). The expression vectors useful herein may be
synthesized by techniques well known to those skilled in this art.
The components of such vectors, e.g. replicons, ion genes, enhancers, promoters, signal
sequences and the like, may be obtained from commercial or natural sources or synthesized by
known procedures for use in directing the expression and/or secretion of the t of the
recombinant DNA in a selected host. Other appropriate expression vectors of which numerous types
are known in the art for mammalian, bacterial, insect, yeast, and fungal expression may also be
selected for this purpose.
The present invention also encompasses a cell line transfected with a recombinant d containing
the coding sequences of the antigen binding proteins of the present invention. Host cells useful for
the cloning and other lations of these cloning s are also conventional. However, cells
from various strains of E. Coli may be used for replication of the cloning vectors and other steps in the
construction of antigen binding proteins of this invention.
Suitable host cells or cell lines for the sion of the antigen binding proteins of the invention
include mammalian cells such as NSO, Sp2/0, CHO (e.g. DG44), COS, HEK, a fibroblast cell (e.g.,
3T3), and myeloma cells, for example it may be expressed in a CHO or a myeloma cell. Human cells
40 may be used, thus enabling the molecule to be modified with human glycosylation patterns.
Alternatively, other eukaryotic cell lines may be employed. The selection of suitable mammalian host
cells and methods for transformation, culture, amplification, screening and product production and
purification are known in the art. See, e.g., Sambrook et al., cited above.
Bacterial cells may prove useful as host cells le for the expression of the recombinant Fabs or
other embodiments of the present invention (see, e.g., Pliickthun, A., lmmunol. Rev., 130:151-188
(1992)). However, due to the tendency of ns expressed in bacterial cells to be in an ed or
improperly folded form or in a non-glycosylated form, any recombinant Fab produced in a bacterial
cell would have to be screened for retention of antigen binding ability. If the molecule expressed by
the bacterial cell was ed in a properly folded form, that bacterial cell would be a desirable host,
or in ative embodiments the molecule may express in the bacterial host and then be
subsequently re-folded. For example, various strains of E. Coli used for expression are well-known
as host cells in the field of biotechnology. Various strains of B. Subtilis, Streptomyces, other bacilli
and the like may also be employed in this method.
Where desired, strains of yeast cells known to those skilled in the art are also available as host cells,
as well as insect cells, e.g. Drosophila and Lepidoptera and viral expression systems. See, e.g. Miller
et al., Genetic Engineering, 8:277-298, Plenum Press (1986) and references cited therein.
The general methods by which the s may be ucted, the transfection methods required to
produce the host cells of the invention, and culture methods necessary to produce the n binding
protein of the invention from such host cell may all be conventional techniques. Typically, the e
method of the present invention is a serum-free culture method, usually by culturing cells serum-free
in suspension. Likewise, once produced, the n binding proteins of the invention may be purified
from the cell culture contents according to standard procedures of the art, including ammonium
16eroxidi precipitation, affinity columns, column chromatography, gel electrophoresis and the like.
Such techniques are within the skill of the art and do not limit this invention. For example,
preparations of altered antibodies are described in WO 99/58679 and WO 96/16990.
Yet another method of expression of the antigen binding proteins may utilize expression in a
transgenic animal, such as bed in U. 8. Patent No. 4,873,316. This relates to an sion
system using the animals casein promoter which when transgenically incorporated into a mammal
s the female to produce the desired recombinant n in its milk.
In a further ment of the invention there is provided a method of ing an antibody of the
invention which method comprises the step of culturing a host cell transformed or transfected with a
vector encoding the light and/or heavy chain of the antibody of the ion and recovering the
antibody thereby produced.
In accordance with the present invention there is provided a method of producing an anti-BCMA
antibody of the present invention which binds to and neutralises the activity of human BCMA which
method comprises the steps of;
40 ing a first vector encoding a heavy chain of the antibody;
providing a second vector encoding a light chain of the dy;
transforming a mammalian host cell (e.g. CHO) with said first and second vectors;
culturing the host cell of step (c) under conditions conducive to the secretion of the antibody from said
host cell into said culture media;
recovering the secreted antibody of step (d).
Once expressed by the desired method, the antibody is then examined for in vitro activity by use of an
appropriate assay. tly conventional ELISA assay formats are employed to assess qualitative
and quantitative binding of the antibody to BCMA. Additionally, other in vitro assays may also be
used to verify neutralizing efficacy prior to subsequent human clinical studies performed to evaluate
the persistence of the antibody in the body despite the usual clearance mechanisms.
The dose and duration of treatment s to the relative duration of the molecules of the present
invention in the human circulation, and can be adjusted by one of skill in the art ing upon the
condition being treated and the general health of the patient. It is envisaged that repeated dosing
(e.g. once a week or once every two weeks or once every 3 weeks) over an extended time period
(e.g. four to six months) maybe required to achieve maximal therapeutic efficacy..
In one embodiment of the present invention there is provided a recombinant transformed, transfected
or transduced host cell comprising at least one expression cassette, for example where the
sion cassette comprises a polynucleotide encoding a heavy chain of an antigen binding protein
according to the invention described herein and further comprises a polynucleotide encoding a light
chain of an antigen binding protein according to the invention bed herein or where there are two
sion cassettes and the 1St encodes the light chain and the second encodes the heavy chain.
For e in one embodiment the first expression cassette comprises a polynucleotide encoding a
heavy chain of an antigen binding protein sing a constant region or antigen binding fragment
thereof which is linked to a constant region according to the invention described herein and r
comprises a second cassette comprising a polynucleotide encoding a light chain of an antigen binding
protein comprising a constant region or antigen binding fragment thereof which is linked to a constant
region according to the invention described herein for example the first expression cassette
ses a polynucleotide encoding a heavy chain ed from SEQ. ID. NO:56, or SEQ. ID. NO:
60 or SEQ. ID. NO: 62 and a second expression cassette comprising a cleotide encoding a
light chain selected from SEQ. ID. NO: 64 or SEQ. ID. NO: 66.
In another ment of the ion there is provided a stably ormed host cell comprising a
vector comprising one or more expression cassettes ng a heavy chain and/or a light chain of
the antibody comprising a constant region or antigen binding fragment thereof which is linked to a
constant region as described herein. For example such host cells may comprise a first vector
encoding the light chain and a second vector encoding the heavy chain, for example the first vector
encodes a heavy chain selected from SEQ. ID. NO: 55, or SEQ. ID. NO: 59 or SEQ. ID. NO: 61 and a
40 second vector encoding a light chain for example the light chain of SEQ ID NO: 63 or SEQ. ID. NO:
WO 63805
65. In one such example the first vector encodes a heavy chain selected from SEQ. ID. NO: 55 and a
second vector encoding a light chain for example the light chain of SEQ ID NO: 63.
In another embodiment of the t invention there is provided a host cell according to the invention
described herein wherein the cell is eukaryotic, for example where the cell is mammalian. es
of such cell lines include CHO or NSO.
In another embodiment of the present invention there is provided a method for the production of an
antibody comprising a constant region or antigen g fragment thereof which is linked to a
nt region ing to the invention described herein which method comprises the step of
culturing a host cell in a culture media, for example serum- free e media.
In another ment of the present invention there is provided a method ing to the invention
described herein wherein said antibody is further purified to at least 95% or greater (e.g. 98% or
greater) with respect to said antibody containing serum- free culture media.
In yet r ment there is provided a pharmaceutical composition comprising an antigen
binding protein and a pharmaceutically acceptable carrier.
In r embodiment of the present invention there is provided a kit-of-parts comprising the
ition according to the invention described herein described together with instructions for use.
The mode of administration of the therapeutic agent of the invention may be any suitable route which
delivers the agent to the host. The antigen binding proteins, and pharmaceutical compositions of the
invention are particularly useful for parenteral administration, i.e., subcutaneously (s.c.), intrathecally,
intraperitoneally, uscularly (i.m.) or intravenously (i.v.). In one such embodiment the antigen
binding proteins of the present invention are administered intravenously or subcutaneously.
Therapeutic agents of the invention may be prepared as pharmaceutical compositions containing an
effective amount of the antigen binding protein of the invention as an active ingredient in a
pharmaceutically acceptable carrier. In one embodiment the prophylactic agent of the invention is an
aqueous suspension or solution containing the antigen g protein in a form ready for injection. In
one embodiment the suspension or solution is ed at physiological pH. In one embodiment the
compositions for parenteral administration will comprise a solution of the antigen binding protein of the
invention or a cocktail thereof dissolved in a pharmaceutically acceptable carrier. In one embodiment
the carrier is an aqueous r. A variety of aqueous carriers may be employed, e.g., 0.9% saline,
0.3% glycine, and the like. These solutions may be made sterile and generally free of particulate
matter. These solutions may be sterilized by conventional, well known sterilization techniques (e.g.,
filtration). The compositions may contain pharmaceutically able auxiliary substances as
40 required to approximate physiological conditions such as pH adjusting and buffering agents, etc. The
concentration of the antigen binding n of the invention in such pharmaceutical formulation can
vary widely, i.e., from less than about 0.5%, usually at or at least about 1% to as much as about 15 or
% by weight and will be selected primarily based on fluid volumes, viscosities, etc., according to the
particular mode of administration selected.
Thus, a pharmaceutical composition of the invention for intravenous infusion could be made up to
contain about 250 ml of sterile Ringer’s solution, and about 1 to about 30 or 5 mg to about 25 mg of
an antigen binding protein of the invention per ml of Ringer’s solution. Actual methods for preparing
parenterally administrable compositions are well known or will be apparent to those skilled in the art
and are described in more detail in, for example, Remington’s Pharmaceutical Science, 15th ed., Mack
Publishing Company, Easton, Pennsylvania. For the preparation of intravenously administrable
antigen binding protein ations of the invention see Lasmar U and Parkins D “The formulation of
Biopharmaceutical products”, . ch.today, page 7, Vol.3 (3rd April 2000); Wang, W
“Instability, stabilisation and formulation of liquid protein pharmaceuticals”, Int. J. Pharm 185 (1999)
129-188; Stability of Protein Pharmaceuticals Part A and B ed Ahern T.J., Manning M.C., New York,
NY: Plenum Press (1992); Akers,M.J. “Excipient—Drug ctions in Parenteral Formulations”,
J.Pharm Sci 91 (2002) 2283-2300; lmamura, K et al ts of types of sugar on stabilization of
Protein in the dried state”, J Pharm Sci 92 (2003) 266-274; lzutsu, Kkojima, S. “Excipient crystalinity
and its n-structure-stabilizing effect during freeze-drying”, J Pharm. Pharmacol, 54 (2002) 1033-
1039; Johnson, R, “Mannitol-sucrose mixtures-versatile formulations for protein peroxidise19g19n”, J.
Pharm. Sci, 91 (2002) 914-922; and Ha,E Wang W, Wang Y.j. “Peroxide formation in polysorbate 80
and protein stability”, J. Pharm Sci, 91, 2252-2264,(2002) the entire contents of which are
incorporated herein by reference and to which the reader is specifically referred.
In one ment the therapeutic agent of the ion, when in a pharmaceutical preparation, is
present in unit dose forms. The appropriate eutically effective dose will be determined readily
by those of skill in the art. Suitable doses may be ated for patients according to their weight, for
example suitable doses may be in the range of about 0.1 to about 20mg/kg, for example about 1 to
about 20mg/kg, for example about 10 to about 20mg/kg or for example about 1 to about 15mg/kg, for
example about 10 to about 15mg/kg or for example 1-5mg/kg. In one embodiment the dy is
given 1-5mg/kg every 3 weeks. To effectively treat conditions such as Multiple myeloma, SLE or lPT
in a human, le doses may be within the range of about 0.1 to about 1000 mg, for example about
0.1 to about 500mg, for example about 500mg, for example about 0.1 to about 100mg, or about 0.1
to about 80mg, or about 0.1 to about 60mg, or about 0.1 to about 40mg, or for example about 1 to
about 100mg, or about 1 to about 50mg, of an antigen g protein of this invention, which may be
administered parenterally, for example subcutaneously, enously or intramuscularly. Such dose
may, if ary, be repeated at appropriate time intervals selected as appropriate by a physician.
The antigen binding proteins described herein can be lized for e and reconstituted in a
suitable carrier prior to use. This technique has been shown to be effective with conventional
immunoglobulins and art-known peroxidise and reconstitution techniques can be employed.
In another aspect of the invention there is provided an antigen binding protein as herein described for
use in a medicament.
In one aspect of the present invention there is provided an antigen binding protein according to the
invention as herein described for use in the treatment of rheumatoid arthitis, Type 1 Diabetes Mellitus,
multiple sclerosis or psoriasis wherein said method comprises the step of administering to said patient
a therapeutically effective amount of the antigen binding protein as described herein.
In one embodiment of the present invention, methods are provided for treating cancer in a human
comprising administering to said human an antigen binding protein that specifically binds to BCMA. In
some instances the antigen g protein is part of an immunoconjugate.
In another aspect of the present invention there is provided an antigen binding protein according to
the invention as herein bed for use in the treatment of a B-cell mediated or plasma cell
mediated disease or antibody mediated disease or disorder selected from Multiple Myeloma (MM),
chronic lymphocytic leukemia (CLL), Non-secretory le myeloma, ring le myeloma,
Monoclonal gammopathy of undetermined significance , Solitary plasmacytoma (Bone,
Extramedullary), Lymphoplasmacytic lymphoma (LPL), strom’s Macroglobulinemia, Plasma
cell leukemia,, Primary Amyloidosis (AL), Heavy chain disease, Systemic lupus erythematosus (SLE),
POEMS syndrome / osteosclerotic myeloma, Type | and II obulinemia, Light chain deposition
disease, sture’s syndrome, Idiopathic thrombocytopenic purpura (ITP), Acute
glomerulonephritis, Pemphigus and Pemphigoid ers, and molysis bullosa acquisita; or
any Non-Hodgkin’s Lymphoma B-cell leukemia or Hodgkin’s lymphoma (HL) with BCMA expression
or any diseases in which patients develop neutralising dies to recombinant protein replacement
therapy n said method comprises the step of administering to said t a therapeutically
effective amount of the antigen binding protein as described herein.
B-cell disorders can be divided into defects of B-cell development/immunoglobulin production
(immunodeficiencies) and excessive/uncontrolled proliferation (lymphomas, ias). As used
herein, B-cell disorder refers to both types of diseases, and methods are provided for treating B-cell
disorders with an antigen binding protein.
In a particular aspect, the disease or disorder is selected from the group consisting of le
a (MM), Chronic cytic Leukaemia (CLL), Solitary Plasmacytoma (Bone,
Extramedullary), Waldenstrom’s Macroglobulinemia.
In one aspect of the present invention the disease is le Myeloma, Smoldering Multiple Myeloma
(SMM) or Solitary Plasmacytoma (Bone, Extramedullary).
In one aspect of the present invention the disease is Multiple Myeloma.
In one aspect of the present ion the disease is Systemic lupus erythematosus (SLE)
In one aspect of the present invention the disease is Idiopathic thrombocytopenic purpura (ITP)
Use of the antigen g protein as described herein in the manufacture of a medicament for the
treatment of diseases and disorders as described herein is also provided.
For example in one aspect of the invention there is provided the use of the n g protein as
described herein for use in the treatment or laxis of diseases and disorders responsive to
modulation (such as inhibiting or blocking) of the interaction between BCMA and the s BAFF
and APRIL.
In another aspect of the invention there is provided the use of the antigen binding protein as
described herein for use in the treatment or prophylaxis of an antibody mediated or plasma cell
mediated disease or disorder selected from rheumatoid arthitis, Type 1 Diabeted Mellitus, multiple
sis or psoriasis.
In another aspect of the invention there is provided the use of the antigen binding protein as
described herein for use in the treatment or prophylaxis of an antibody mediated or plasma cell
mediated disease or disorder selected from Multiple Myeloma (MM), chronic lymphocytic leukemia
(CLL), Monoclonal gammopathy of undetermined significance (MGUS), Smoldering multiple myeloma
(SMM), ry Plasmacytoma (Bone, Extramedullary), Waldenstrom’s Macroglobulinemia , y
Amyloidosis (AL), Heavy chain disease, Systemic lupus matosus (SLE), POEMS syndrome/
osteosclerotic myeloma, Type I and II cryoglobulinemia, Light chain deposition disease,
Goodpastures syndrome, Idiopathic thrombocytopenic purpura (ITP), Acute glomerulonephritis,
Pemphigus and Pemphigoid disorders and molysis bullosa acquisita, any Non-Hodgkin
Lymphoma and Leukemia with BCMA expression or any diseases in which patients develop
neutralising antibodies to recombinant n replacement therapy wherein said method comprises
the step of administering to said patient a therapeutically effective amount of the antigen binding
protein as described .
In one aspect, the invention provides a pharmaceutical composition sing an antigen binding
protein of the present invention or a functional nt thereof and a pharmaceutically acceptable
carrier for treatment or prophylaxis of rheumatoid arthitis, Type 1 Diabetes us, multiple sclerosis
or psoriasis or an antibody mediated or plasma cell mediated disease or disorder selected from
selected from le Myeloma (MM), chronic lymphocytic leukemia (CLL), Monoclonal gammopathy
of undetermined significance (MGUS), Smoldering multiple myeloma (SMM), Solitary Plasmacytoma
(Bone, Extramedullary), Waldenstrom’s Macroglobulinemia chain
, Primary dosis (AL), Heavy
disease, Systemic lupus erythematosus (SLE), POEMS syndrome / osteosclerotic myeloma, Type I
40 and II cryoglobulinemia, Light chain deposition disease, Goodpastures me, Idiopathic
thrombocytopenic purpura (ITP), Acute glomerulonephritis, Pemphigus and goid disorders and
Epidermolysis bullosa acquisita, any Non-Hodgkin Lymphoma and Leukemia with BCMA expression
or any diseases in which patients develop lising antibodies to recombinant protein replacement
y wherein said method comprises the step of administering to said t a eutically
effective amount of the antigen binding protein as described herein.
In another embodiment of the present invention there is provided a method of treating a human
patient afflicted with rheumatoid arthitis, Type 1 Diabetes Mellitus, multiple sclerosis or psoriasis or an
antibody ed or plasma cell mediated disorder or disease which method comprises the step of
administering a therapeutically effective amount of the antigen binding protein according to the
invention as described herein, for example there is provided a method of treating a human t
afflicted with an antibody mediated or plasma cell mediated disease or disorder selected from In
another aspect of the present invention there is provided an n binding protein according to the
invention as herein described for use in the treatment of an dy mediated or plasma cell
mediated disease or disorder selected from le Myeloma (MM), Chronic Lymphocytic Leukaemia
(CLL)Monoclonal gammopathy of undetermined significance (MGUS), Smoldering multiple a
(SMM), Solitary Plasmacytoma (Bone, edullary), Waldenstrom’s Macroglobulinemia , Primary
Amyloidosis (AL), Heavy chain disease, Systemic lupus erythematosus (SLE), POEMS syndrome/
osteosclerotic myeloma, Type | and II cryoglobulinemia, Light chain deposition disease,
Goodpastures syndrome, Idiopathic thrombocytopenic purpura (ITP), Acute glomerulonephritis,
Pemphigus and Pemphigoid disorders and Epidermolysis bullosa acquisita, any Non-Hodgkin
Lymphoma and Leukemia with BCMA expression or any diseases in which patients develop
neutralising antibodies to recombinant protein replacement therapy wherein said method comprises
the step of administering a pharmaceutical composition comprising an antigen binding protein
according to the ion herein in combination with a pharmaceutically acceptable carrier.
In a further ment there is provided a method of treating a human patient afflicted with Multiple
Myeloma (MM).
tions
As used , the terms "cancer, neoplasm," and "tumor" are used interchangeably and, in either
the singular or plural form, refer to cells that have undergone a malignant ormation that makes
them pathological to the host organism. Primary cancer cells can be readily distinguished from non-
cancerous cells by well-established ques, ularly histological examination. The definition of
a cancer cell, as used herein, includes not only a primary cancer cell, but any cell derived from a
cancer cell ancestor. This includes metastasized cancer cells, and in vitro cultures and cell lines
derived from cancer cells. When referring to a type of cancer that normally manifests as a solid
tumor, a "clinically detectable" tumor is one that is detectable on the basis of tumor mass; e.g., by
procedures such as computed tomography (CT) scan, magnetic resonance imaging (MRI), X-ray,
40 ound or palpation on physical examination, and/or which is detectable because of the
expression of one or more cancer-specific antigens in a sample obtainable from a patient. Tumors
may be a poietic (or hematologic or hematological or blood-related) cancer, for example,
cancers derived from blood cells or immune cells, which may be referred to as “liquid tumors.”
Specific examples of clinical conditions based on hematologic tumors include leukemias such as
chronic myelocytic leukemia, acute myelocytic leukemia, chronic lymphocytic leukemia and acute
lymphocytic leukemia; plasma cell malignancies such as multiple myeloma, MGUS and
Waldenstrom’s macroglobulinemia; lymphomas such as dgkin’s lymphoma, Hodgkin’s
lymphoma; and the like.
The cancer may be any cancer in which an abnormal number of blast cells or unwanted cell
proliferation is present or that is diagnosed as a logical cancer, including both lymphoid and
myeloid malignancies. Myeloid malignancies include, but are not limited to, acute d (or
myelocytic or myelogenous or lastic) leukemia (undifferentiated or differentiated), acute
promyeloid (or promyelocytic or promyelogenous or promyeloblastic) leukemia, acute onocytic
(or myelomonoblastic) ia, acute monocytic (or monoblastic) ia, erythroleukemia and
megakaryocytic (or megakaryoblastic) leukemia. These leukemias may be referred together as acute
d (or myelocytic or myelogenous) leukemia (AML). Myeloid malignancies also include
myeloproliferative disorders (MPD) which include, but are not d to, chronic myelogenous (or
myeloid) ia (CML), chronic myelomonocytic ia (CMML), essential thrombocythemia (or
thrombocytosis), and polcythemia vera (PCV). Myeloid ancies also include ysplasia (or
myelodysplastic syndrome or MDS), which may be referred to as refractory anemia (RA), refractory
anemia with excess blasts (RAEB), and refractory anemia with excess blasts in transformation
); as well as myelofibrosis (MFS) with or without agnogenic myeloid metaplasia.
Hematopoietic cancers also include lymphoid malignancies, which may affect the lymph nodes,
spleens, bone marrow, peripheral blood, and/or extranodal sites. Lymphoid cancers include B-cell
malignancies, which include, but are not limited to, B-cell non-Hodgkin’s lymphomas (B-NHLs). B-
NHLs may be nt (or low-grade), intermediate-grade (or aggressive) or high-grade (very
aggressive). lndolent Bcell lymphomas include follicular ma (FL); small lymphocytic
lymphoma (SLL); marginal zone lymphoma (MZL) including nodal MZL, extranodal MZL, splenic MZL
and splenic MZL with villous lymphocytes; lymphoplasmacytic lymphoma (LPL); and mucosa-
associated-lymphoid tissue (MALT or extranodal marginal zone) lymphoma. Intermediate-grade B-
NHLs include mantle cell lymphoma (MCL) with or without leukemic involvement, diffuse large cell
lymphoma (DLBCL), follicular large cell (or grade 3 or grade 38) lymphoma, and primary mediastinal
lymphoma (PML). High-grade B-NHLs include Burkitt’s lymphoma (BL), Burkitt-like lymphoma, small
non-cleaved cell lymphoma (SNCCL) and lymphoblastic lymphoma. Other B-NHLs include
immunoblastic lymphoma (or immunocytoma), primary effusion lymphoma, HIV associated (or AIDS
related) mas, and post-transplant lymphoproliferative disorder (PTLD) or lymphoma. B-cell
malignancies also include, but are not limited to, chronic lymphocytic leukemia (CLL), phocytic
40 leukemia (PLL), Waldenstrom’s macroglobulinemia (WM), hairy cell leukemia (HCL), large granular
lymphocyte (LGL) leukemia, acute lymphoid (or lymphocytic or lymphoblastic) leukemia, and
Castleman’s disease. NHL may also e T-cell non-Hodgkin’s lymphoma s(T-NHLs), which
include, but are not limited to T-cell non-Hodgkin’s lymphoma not otherwise specified (NOS),
peripheral T-cell lymphoma (PTCL), anaplastic large cell lymphoma (ALCL), angioimmunoblastic
lymphoid disorder , nasal natural killer (NK) cell / T-cell lymphoma, gamma/delta lymphoma,
cutaneous T cell lymphoma, mycosis fungoides, and Sezary syndrome.
Hematopoietic cancers also include n’s lymphoma (or disease) including classical Hodgkin’s
lymphoma, r sclerosing Hodgkin’s lymphoma, mixed cellularity Hodgkin’s lymphoma,
lymphocyte predominant (LP) Hodgkin’s lymphoma, nodular LP Hodgkin’s lymphoma,and cyte
ed Hodgkin’s lymphoma. Hematopoietic cancers also include plasma cell diseases or cancers
such as multiple myeloma (MM) ing smoldering MM, monoclonal gammopathy of rmined
(or unknown or unclear) significance (MGUS), plasmacytoma (bone, extramedullary),
lymphoplasmacytic lymphoma (LPL), Waldenstrom’s Macroglobulinemia, plasma cell leukemia, and
primary amyloidosis (AL). Hematopoietic cancers may also include other cancers of additional
hematopoietic cells, including rphonuclear leukocytes (or neutrophils), basophils, eosinophils,,
dendritic cells, ets, erythrocytes and natural killer cells. Tissues which e hematopoietic
cells referred herein to as opoietic cell tissues" include bone marrow; peripheral blood; thymus;
and peripheral lymphoid tissues, such as spleen, lymph nodes, lymphoid tissues associated with
mucosa (such as the gut-associated lymphoid tissues), tonsils, Peyer's patches and appendix, and
lymphoid tissues associated with other , for example, the ial linings.
The term “antigen binding protein” as used herein refers to antibodies, antibody fragments and other
protein constructs which are capable of binding to and neutralising human BCMA.
The terms Fv, Fc, Fd, Fab, or F(ab)2 are used with their rd meanings (see, e.g., Harlow et al.,
Antibodies A Laboratory Manual, Cold Spring Harbor Laboratory, (1988)).
The term “antibody” is used herein in the broadest sense and specifically covers monoclonal
antibodies (including full length monoclonal dies), polyclonal antibodies, multispecific antibodies
(e.g. bispecific antibodies)
The term lonal antibody” as used herein refers to an antibody obtained from a population of
substantially homogenous antibodies i.e. the individual antibodies sing the population are
identical except for possible naturally occurring mutations that may be t in minor amounts.
Monoclonal antibodies are highly ic being directed against a single antigenic binding site.
Furthermore, in contrast to polyclonal antibody preparations which typically include different
antibodies directed against different determinants (epitopes), each monoclonal antibody is directed
against a single determinant on the antigen.
A “chimeric antibody” refers to a type of engineered antibody in which a portion of the heavy and/ or
light chain is identical with or homologous to corresponding sequences in antibodies derived from a
particular donor dy class or subclass, while the remainder of the chain(s) is identical with or
homologous to corresponding sequences in antibodies derived from another species or belonging to
another antibody class or ss, as well as fragments of such antibodies, so long as they exhibit
the desired ical ty (US Patent No. 4, 816,567 and Morrison et al. Proc. Natl. Acad. Sci.
USA 81:6851-6855) (1984)).
A “humanised antibody” refers to a type of engineered antibody having its CDRs derived from a non-
human donor immunoglobulin, the remaining immunoglobulin-derived parts of the molecule being
derived from one (or more) human immunoglobulin(s). In addition, framework support residues may
be altered to preserve g affinity (see, e.g., Queen et al., Proc. Natl Acad Sci USA, 86:10029-
10032 (1989), Hodgson et al., Bio/Technology, 9:421 (1991)). A suitable human acceptor antibody
may be one selected from a conventional database, e.g., the KABAT® database, Los Alamos
database, and Swiss Protein database, by gy to the nucleotide and amino acid sequences of
the donor antibody. A human antibody characterized by a homology to the framework s of the
donor dy (on an amino acid basis) may be suitable to provide a heavy chain constant region
and/or a heavy chain variable framework region for insertion of the donor CDRs. A suitable acceptor
antibody capable of donating light chain constant or variable framework regions may be selected in a
similar . It should be noted that the acceptor antibody heavy and light chains are not required
to originate from the same acceptor antibody. The prior art describes several ways of producing such
humanised antibodies — see for example EP-A—0239400 and EP-A—054951.
For nucleic acids, the term “substantial identity” indicates that two nucleic acids, or designated
ces f, when lly aligned and compared, are identical, with appropriate nucleotide
insertions or deletions, in at least about 80% of the nucleotides, at least about 90% to about 95%, or
at least about 98% to about 99.5% of the nucleotides. atively, substantial identity exists when
the segments will hybridize under selective hybridization conditions, to the complement of the strand.
“Identity,” means, for polynucleotides and polypeptides, as the case may be, the comparison
calculated using an thm ed in (1) and (2) below:
(1) Identity for polynucleotides is calculated by multiplying the total number of nucleotides
in a given sequence by the integer defining the percent identity divided by 100 and then subtracting
that t from said total number of nucleotides in said sequence, or:
nn 3 xn — (xn o y),
wherein nn is the number of nucleotide alterations, xn is the total number of nucleotides in a given
sequence, y is 0.95 for 95%, 0.97 for 97% or 1.00 for 100%, and o is the symbol for the multiplication
operator, and wherein any non-integer product of xn and y is rounded down to the nearest integer
prior to subtracting it from xn. tions of a polynucleotide sequence encoding a polypeptide may
create nonsense, missense or frameshift mutations in this coding sequence and thereby alter the
40 polypeptide encoded by the cleotide following such alterations.
(2) Identity for polypeptides is ated by multiplying the total number of amino acids by
the integer defining the percent identity divided by 100 and then subtracting that product from said
total number of amino acids, or:
na 3 xa — (xa o y),
wherein na is the number of amino acid alterations, xa is the total number of amino acids in the
sequence, y is 0.95 for 95%, 0.97 for 97% or 1.00 for 100%, and o is the symbol for the lication
operator, and wherein any non-integer t of xa and y is rounded down to the t integer
prior to subtracting it from xa
For nucleotide and amino acid sequences, the term “identical” indicates the degree of identity
between two nucleic acid or amino acid sequences when optimally aligned and compared with
riate ions or deletions.
“lsolated” means altered “by the hand of man” from its natural state, has been changed or removed
from its original environment, or both. For example, a polynucleotide or a polypeptide lly
present in a living organism is not “isolated,” but the same polynucleotide or polypeptide ted
from the coexisting materials of its natural state is “isolated”, including but not limited to when such
polynucleotide or polypeptide is introduced back into a cell, even if the cell is of the same species or
type as that from which the polynucleotide or polypeptide was ted.
Throughout the present specification and the accompanying claims the term “comprising” and
“comprises” incorporates “consisting of” and “consists o That is, these words are intended to convey
the possible inclusion of other elements or rs not specifically recited, where the context allows.
The term fically binds” as used throughout the t specification in relation to antigen
binding proteins of the invention means that the antigen binding protein binds human BCMA
(hBCMA) with no or insignificant binding to other human proteins. The term however does not exclude
the fact that antigen binding proteins of the invention may also be cross-reactive with other forms of
BCMA, for example primate BCMA. For example in one embodiment the antigen binding protein
does not bind to TACI or BAFF-R.
The term “inhibits” as used throughout the present specification in relation to antigen binding proteins
of the invention means that the biological activity of BCMA is reduced in the presence of the antigen
binding proteins of the present invention in comparison to the activity of BCMA in the absence of such
antigen binding proteins. lnhibition may be due but not limited to one or more of ng ligand
binding, ting the ligand activating the receptor, and/ or down regulating the BCMA. lnhibits can
also refer to an antigen binding protein binding to BCMA and causing cell apoptosis or ADCC.The
antibodies of the invention may neutralise the activity of the BCMA ligands BAFF and/or APRIL
binding to BCMA. Levels of neutralisation can be measured in several ways, for example by use of
the assays as set out in the examples below, for e in 4.4 in an H929 cell NFkB signalling
40 assay. The BCMA ligands BAFF and APRIL are able to induce NFkB signalling and downstream
events following g to BCMA. The neutralisation of BCMA in this assay is measured by
assessing the ability of anti-BCMA monoclonal dies to inhibit BAFF or APRIL driven NFkB
inducfion.
If an antibody or antigen binding fragment thereof is capable of lisation then this is indicative of
inhibition of the interaction between human BAFF or APRIL and BCMA. Antibodies which are
considered to have lising activity against human BCMA would have an IC50 of less than 30
micrograms/ml, or less than 20 micrograms/ml, or less than 10 micrograms/ml, or less than 5
micrograms/ml or less than 1 micrograms/ml or less than 0.1 micrograms/ml in the H929 stimulation
assay as set out in Example 4.4
“CDRs” are defined as the complementarity determining region amino acid sequences of an antibody
which are the hypervariable domains of globulin heavy and light chains. There are three
heavy chain and three light chain CDRs (or CDR regions) in the variable portion of an
immunoglobulin. Thus, “CDRs” as used herein may refer to all three heavy chain CDRs, or all three
light chain CDRs (or both all heavy and all light chain CDRs, if appropriate).
CDRs e the majority of t residues for the binding of the antibody to the antigen or
epitope. CDRs of interest in this invention are derived from donor antibody variable heavy and light
chain sequences, and include analogs of the naturally occurring CDRs, which analogs also share or
retain the same antigen binding specificity and/or neutralizing ability as the donor antibody from which
they were derived.
The CDR sequences of antibodies can be determined by the Kabat numbering system (Kabat et al;
(Sequences of proteins of Immunological Interest NIH, 1987), alternatively they can be determined
using the Chothia numbering system (Al-Lazikani et al., (1997) JMB 273,927-948), the contact
definition method (MacCallum RM, and Martin A.C.R. and Thornton J.M, (1996), Journal of
Molecular Biology, 262 (5), 732-745) or any other established method for numbering the residues in
an antibody and determining CDRs known to the skilled man in the art
Other ing conventions for CDR sequences available to a skilled person include “AbM”
(University of Bath) and “contact” (University e London) s. The minimum overlapping
region using at least two of the Kabat, Chothia, AbM and contact methods can be ined to
provide the “minimum binding unit”. The minimum binding unit may be a sub-portion of a CDR.
Table A below ents one definition using each numbering convention for each CDR or g
unit. The Kabat numbering scheme is used in Table X to number the le domain amino acid
sequence. It should be noted that some of the CDR definitions may vary depending on the individual
publication used.
Table A
-Kabat CDR Chothia CDR AbM CDR ContactCDR
2012/059762
binding
unit
26-32/33/34 26-35/35A/35B 30-35/35A/35B 31-32
50-58 47-58 52-56
95-102 93-101 95-101
24-34 30-36 30-34
50-56 46-55 50-55
89-97 89-96 89-96
Throughout this specification, amino acid residues in antibody sequences are numbered according to
the Kabat scheme. Similarly, the terms “CDR”, “CDRL1”, “CDRL2”, “CDRL3”, “CDRH1”, “CDRH2”,
“CDRH3” follow the Kabat numbering system as set forth in Kabat et al; ces of proteins of
Immunological lnterest NIH, 1987.
The terms “Variant” refers to at least one, two or three amino acid changes in the sequence. These
amino acid changes may be deletion, substitution or addition but are preferably substitution. In one
such embodiment the substitutions are conservative substitutions.
In an alternative embodiment the variant sequence contains at least one substitution whilst retaining
the canonical of the antigen binding protein.
The complementarity determining regions (CDRs) L1, L2, L3, H1 and H2 tend to structurally exhibit
one of a finite number of main chain conformations. The ular canonical structure class of a CDR
is defined by both the length of the CDR and by the loop packing, determined by residues located at
key ons in both the CDRs and the ork regions (structurally determining es or
SDRs). Martin and Thornton (1996; J Mol Biol 263:800-815) have generated an automatic method to
define the “key residue” canonical templates. Cluster analysis is used to define the canonical classes
for sets of CDRs, and cal tes are then identified by analysing buried hydrophobics,
hydrogen-bonding residues, and e.g. conserved glycines. The CDRs of antibody sequences can be
assigned to cal classes by comparing the sequences to the key e templates and scoring
each template using identity or similarity matrices.
The terms “VH” and “VL” are used herein to refer to the heavy chain variable domain and light chain
variable domain respectively of an antibody.
As used herein the term “domain” refers to a folded protein structure which has tertiary structure
independent of the rest of the protein. Generally, domains are sible for discrete onal
properties of proteins and in many cases may be added, removed or transferred to other proteins
without loss of function of the remainder of the protein and/or of the domain. An “antibody single
variable domain” is a folded polypeptide domain comprising sequences characteristic of antibody
variable s. It therefore includes complete antibody le domains and modified variable
domains, for example, in which one or more loops have been replaced by sequences which are not
characteristic of antibody variable domains, or dy variable domains which have been truncated
or comprise N- or C-terminal ions, as well as folded fragments of variable domains which retain
at least the binding activity and specificity of the full-length domain.
The phrase “immunoglobulin single variable domain” refers to an antibody variable domain (VH, VHH,
VL) that specifically binds an antigen or epitope independently of a different V region or domain. An
immunoglobulin single le domain can be present in a format (e.g., homo- or hetero-multimer)
with other, different variable regions or variable domains where the other regions or domains are not
required for n binding by the single immunoglobulin variable domain (i.e., where the
immunoglobulin single variable domain binds n independently of the additional variable
domains). A “domain antibody” or “dAb” is the same as an “immunoglobulin single variable domain”
which is e of binding to an antigen as the term is used . An immunoglobulin single
variable domain may be a human antibody variable domain, but also includes single antibody variable
domains from other species such as rodent (for example, as disclosed in WO 00/29004), nurse shark
and Camelid VHH dAbs. Camelid VHH are immunoglobulin single variable domain polypeptides that
are derived from species including camel, llama, , dromedary, and guanaco, which produce
heavy chain antibodies naturally devoid of light chains. Such VHH domains may be sed
according to rd techniques available in the art, and such domains are still ered to be
“domain antibodies” according to the invention. As used herein “VH includes camelid VHH domains.
NARV are another type of immunoglobulin single variable domain which were identified in
cartilaginous fish including the nurse shark. These domains are also known as Novel Antigen
Receptor variable region (commonly abbreviated to V(NAR) or NARV). For further details see Mol.
Immunol. 44, 5 (2006) and U820050043519A.
The term “Epitope-binding ” refers to a domain that specifically binds an antigen or epitope
independently of a different V region or domain, this may be a domain antibody (dAb), for e a
human, camelid or shark immunoglobulin single variable domain or it may be a domain which is a
derivative of a scaffold selected from the group consisting of CTLA—4 (Evibody); lipocalin; Protein A
derived molecules such as Z-domain of Protein A (Affibody, SpA), A-domain (Avimer/Maxibody); Heat
shock proteins such as GroEl and GroES; 29eroxidise29g (trans-body); ankyrin repeat protein
(DARPin); peptide aptamer; C-type lectin domain (Tetranectin); human y-crystallin and human
ubiquitin (affilins); PDZ s; on toxinkunitz type domains of human protease inhibitors; and
fibronectin (adnectin); which has been subjected to protein engineering in order to obtain binding to a
ligand other than the natural ligand.
CTLA—4 (Cytotoxic T Lymphocyte-associated Antigen 4) is a CD28—family receptor expressed on
mainly CD4+ T-cells. lts extracellular domain has a variable domain-like lg fold. Loops corresponding
to CDRs of antibodies can be substituted with heterologous sequence to confer different binding
properties. CTLA—4 molecules engineered to have ent binding specificities are also known as
40 ies. For further s see Journal of Immunological Methods 248 (1-2), 31-45 (2001)
WO 63805
Lipocalins are a family of extracellular proteins which transport small hobic molecules such as
steroids, bilins, ids and lipids. They have a rigid t secondary structure with a numer of
loops at the open end of the conical structure which can be engineered to bind to different target
antigens. Anticalins are between 160-180 amino acids in size, and are derived from lins. For
further details see Biochim Biophys Acta 1482: 337-350 (2000), US7250297B1 and US20070224633
An affibody is a scaffold derived from n A of Staphylococcus aureus which can be engineered to
bind to antigen. The domain consists of a three-helical bundle of imately 58 amino acids.
Libraries have been generated by randomisation of surface residues. For further details see Protein
Eng. Des. Sel. 17, 455-462 (2004) and EP1641818A1
Avimers are multidomain proteins derived from the A-domain scaffold family. The native domains of
approximately 35 amino acids adopt a defined disulphide bonded structure. Diversity is generated by
shuffling of the natural variation exhibited by the family of A-domains. For further details see Nature
Biotechnology 23(12), 1556 — 1561 (2005) and Expert Opinion on lnvestigational Drugs 16(6), 909-
917 (June 2007)
A Transferrin is a monomeric serum transport glycoprotein. Transferrins can be engineered to bind
ent target antigens by insertion of peptide sequences in a permissive surface loop. es of
engineered transferrins scaffolds include the Trans-body. For further s see J. Biol. Chem 274,
24066-24073 (1999).
Designed Ankyrin Repeat Proteins (DARPins) are derived from Ankyrin which is a family of proteins
that mediate attachment of integral membrane proteins to the cytoskeleton. A single ankyrin repeat is
a 33 residue motif consisting of two or—helices and a B-turn. They can be engineered to bind different
target antigens by randomising es in the first or-helix and a B-turn of each repeat. Their binding
interface can be increased by increasing the number of s (a method of affinity maturation). For
further details see J. Mol. Biol. 332, 489-503 (2003), PNAS 100(4), 1700-1705 (2003) and J. Mol. Biol.
369, 1015-1028 (2007) and US20040132028A1.
ectin is a scaffold which can be engineered to bind to antigen. Adnectins consists of a
ne of the l amino acid sequence of the 10th domain of the 15 repeating units of human
fibronectin type III (FN3). Three loops at one end of the B-sandwich can be engineered to enable an
Adnectin to specifically recognize a therapeutic target of interest. For further details see Protein Eng.
Des. Sel. 18, 435-444 (2005), US20080139791, WO2005056764 and US6818418B1.
Peptide aptamers are combinatorial recognition molecules that t of a constant scaffold protein,
typically thioredoxin (TrxA) which contains a constrained variable peptide loop inserted at the active
40 site. For further details see Expert Opin. Biol. Ther.
, 783-797 (2005).
Microbodies are derived from naturally occurring microproteins of 25-50 amino acids in length which
contain 3-4 cysteine bridges — examples of microproteins include KalataB1 and conotoxin and
knottins. The roteins have a loop which can be engineered to include upto 25 amino acids
without affecting the overall fold of the microprotein. For further details of engineered knottin s,
see W02008098796.
Other epitope binding s include proteins which have been used as a scaffold to engineer
different target antigen binding properties include human y—crystallin and human ubiquitin (affilins),
kunitz type domains of human protease inhibitors, PDZ-domains of the Ras-binding protein AF-6,
scorpion toxins (charybdotoxin), C-type lectin domain (tetranectins) are reviewed in Chapter 7 — Non-
Antibody Scaffolds from Handbook of Therapeutic Antibodies (2007, edited by Stefan Dubel) and
Protein Science 15:14-27 (2006). Epitope binding domains of the present invention could be derived
from any of these alternative protein domains.
As used herein, the term “antigen-binding site” refers to a site on a protein which is capable of
specifically binding to antigen, this may be a single domain, for example an epitope-binding domain,
or it may be paired VH/VL domains as can be found on a standard antibody. In some embodiments of
the ion single-chain Fv (ScFv) s can provide antigen-binding sites.
The terms “mAbdAb” and ” are used herein to refer to antigen-binding proteins of the present
invention. The two terms can be used interchangeably, and are ed to have the same meaning
as used herein.
The term “antigen binding protein” as used herein refers to antibodies, antibody fragments for
example a domain antibody (dAb), ScFv, Fab, Fab2, and other protein constructs. Antigen binding
molecules may comprise at least one lg variable domain, for example antibodies, domain antibodies
(dAbs), Fab, Fab’, F(ab’)2, Fv, ScFv, ies, mAbdAbs, affibodies, heteroconjugate dies or
ific dies. In one embodiment the antigen binding molecule is an dy. In another
embodiment the antigen binding molecule is a dAb, i.e. an immunoglobulin single variable domain
such as a VH, VHH or VL that specifically binds an antigen or epitope independently of a different V
region or domain. Antigen binding molecules may be capable of g to two targets, i.e. they may
be dual targeting ns. Antigen binding molecules may be a combination of antibodies and antigen
g fragments such as for example, one or more domain antibodies and/or one or more ScFvs
linked to a monoclonal antibody. Antigen g molecules may also comprise a non-lg domain for
example a domain which is a derivative of a scaffold selected from the group consisting of CTLA—4
(Evibody); lipocalin; Protein A derived molecules such as Z-domain of Protein A (Affibody, SpA), A-
domain (Avimer/Maxibody); Heat shock ns such as GroEl and GroES; 31eroxidise31g (trans-
40 body); ankyrin repeat n (DARPin); peptide aptamer; C-type lectin domain (Tetranectin); human
tallin and human ubiquitin (affilins); PDZ domains; scorpion toxinkunitz type domains of human
protease inhibitors; and fibronectin (adnectin); which has been subjected to protein engineering in
order to obtain binding to OSM. As used herein “antigen binding protein” will be capable of
antagonising and/or neutralising human OSM. In addition, an antigen binding n may inhibit and
or block OSM activity by binding to 08M and preventing a natural ligand from binding and/or
activating the gp130 receptor.
The term “Effector Function” as used herein is meant to refer to one or more of Antibody dependant
cell mediated cytotoxic activity (ADCC) mediated
, Complement—dependant cytotoxic activity (CDC)
ses, Fc-mediated phagocytosis and antibody recycling via the FcRn or. For lgG
antibodies, effector functionalities including ADCC and ADCP are mediated by the interaction of the
heavy chain constant region with a family of ch receptors present on the surface of immune cells. In
humans these e chRl (CD64), chRll (CD32) and chRlll (CD16). Interaction between the
antigen binding protein bound to antigen and the formation of the Fc/ ch complex induces a range of
effects including cytotoxicity, immune cell activation, phagocytosis and release of inflammatory
cytokines.
The interaction between the constant region of an antigen binding protein and various Fc receptors
(FcR) is believed to e the effector functions of the antigen binding protein. Significant biological
effects can be a consequence of effector functionality, in particular, antibody-dependent cellular
cytotoxicity (ADCC), fixation of complement ement dependent cytotoxicity or CDC), and half-
learance of the antigen g protein. Usually, the y to mediate effector function requires
binding of the antigen binding protein to an antigen and not all antigen binding proteins will mediate
every effector function.
Effector function can be measured in a number of ways including for example via binding of the
chRlll to l Killer cells or via chRl to monocytes/macrophages to e for ADCC effector
on. For example an antigen g protein of the present ion can be assessed for ADCC
effector function in a Natural Killer cell assay. Examples of such assays can be found in Shields et al,
2001 The l of Biological try, Vol. 276, p6591-6604; Chappel et al, 1993 The Journal of
Biological Chemistry, Vol 268, p25124-25131; Lazar et al, 2006 PNAS, 103; 4005-4010.
Examples of assays to determine CDC function include that bed in 1995 J lmm Meth 184:29-38.
Some isotypes of human constant regions, in particular lgG4 and lgG2 isotypes, essentially lack the
functions of a) activation of complement by the classical pathway; and b) antibody-dependent cellular
cytotoxicity. Various modifications to the heavy chain constant region of n binding proteins may
be carried out depending on the desired effector property. lgG1 constant regions ning specific
ons have separately been described to reduce binding to Fc receptors and therefore reduce
ADCC and CDC (Duncan et al. Nature 1988, 332; 563-564; Lund et al. J. lmmunol. 1991, 147; 2657-
2662; Chappel et al. PNAS 1991, 88; 9036-9040; Burton and Woof, Adv. lmmunol. 1992, 51;1-84;
Morgan et al., Immunology 1995, 86; 319-324; Hezareh et al., J. Virol. 2001, 75 (24); 12161-12168).
In one embodiment of the present invention there is provided an antigen binding protein comprising a
constant region such that the antigen binding protein has reduced ADCC and/or ment
activation or effector functionality. In one such embodiment the heavy chain constant region may
comprise a naturally disabled constant region of lgG2 or lgG4 isotype or a mutated lgG1 constant
region. Examples of suitable modifications are bed in EP0307434. One example comprises the
substitutions of alanine residues at positions 235 and 237 (EU index numbering).
Human lgG1 constant regions containing specific mutations or altered glycosylation on residue
Asn297 have also been described to enhance binding to Fc receptors. In some cases these
ons have also been shown to enhance ADCC and CDC (Lazar et al. PNAS 2006, 103; 4005-
4010; Shields et al. J Biol Chem 2001, 276; 604; Nechansky et al. Mol Immunol, 2007, 44;
1815-1817).
In one embodiment of the present ion, such mutations are in one or more of positions ed
from 239, 332 and 330 (lgG1), or the equivalent positions in other lgG isotypes. Examples of suitable
mutations are S239D and l332E and A330L. In one embodiment the antigen binding n of the
invention herein described is mutated at positions 239 and 332, for example S239D and l332E or in a
further embodiment it is d at three or more positions selected from 239 and 332 and 330, for
example S239D and l332E and A330L. (EU index ing).
In an alternative embodiment of the present ion, there is provided an antigen binding protein
comprising a heavy chain constant region with an altered glycosylation profile such that the antigen
binding protein has enhanced or function. For example, n the antigen binding protein has
enhanced ADCC or enhanced CDC or wherein it has both enhanced ADCC and CDC effector
function. Examples of suitable methodologies to produce antigen binding proteins with an altered
glycosylation profile are described in WO2003011878, WO2006014679 and EP1229125, all of which
can be applied to the antigen g proteins of the present invention.
The present invention also provides a method for the production of an antigen binding protein
according to the invention comprising the steps of:
a) culturing a recombinant host cell comprising an expression vector sing the isolated nucleic
acid as described , wherein the FUT8 gene encoding alpha-1,6-fucosyltransferase has been
inactivated in the recombinant host cell; and
b) ring the antigen binding protein.
Such methods for the production of antigen binding proteins can be performed, for example, using the
POTELLIGENTTM technology system available from BioWa, lnc. (Princeton, NJ) in which CHOK1SV
cells g a functional copy of the FUT8 gene produce monoclonal antibodies having enhanced
antibody dependent cell mediated cytotoxicity (ADCC) activity that is increased ve to an identical
monoclonal antibody produced in a cell with a functional FUT8 gene. Aspects of the POTELLIGENTTM
technology system are described in US7214775, U86946292, W00061739 and WOO231240 all of
which are incorporated herein by reference. Those of ry skill in the art will also recognize other
appropriate systems.
In one embodiment of the present ion there is provided an antigen binding protein sing a
chimaeric heavy chain constant region for example an antigen binding protein comprising a chimaeric
heavy chain constant region with at least one CH2 domain from lgG3 such that the antigen binding
protein has enhanced effector on, for example wherein it has enhanced ADCC or enhanced
CDC, or enhanced ADCC and CDC functions,. In one such embodiment, the n binding protein
may se one CH2 domain from lgG3 or both CH2 domains may be from lgG3.
Also provided is a method of producing an antigen binding protein according to the invention
comprising the steps of:
a) culturing a recombinant host cell comprising an expression vector comprising an isolated nucleic
acid as described herein wherein the expression vector comprises a nucleic acid sequence encoding
an Fc domain having both lgG1 and lgG3 Fc domain amino acid residues; and
b) recovering the antigen binding protein.
Such methods for the production of antigen binding proteins can be performed, for example, using the
COMPLEGENTTM technology system available from BioWa, lnc. (Princeton, NJ) and Kyowa Hakko
Kogyo (now, Kyowa Hakko Kirin Co., Ltd.) Co., Ltd. In which a recombinant host cell comprising an
expression vector in which a nucleic acid sequence encoding a ic Fc domain having both lgG1
and lgG3 Fc domain amino acid residues is expressed to produce an antigen binding protein having
enhanced complement dependent cytotoxicity (CDC) activity that is increased relative to an otherwise
cal antigen binding protein lacking such a chimeric Fc domain. Aspects of the GENTTM
technology system are bed in W02007011041 and U820070148165 each of which are
incorporated herein by reference. In an alternative embodiment CDC activity may be increased by
ucing sequence specific ons into the Fc region of an lgG chain. Those of ordinary skill in
the art will also recognize other appropriate systems.
It will be apparent to those skilled in the art that such modifications may not only be used alone but
may be used in combination with each other in order to further enhance effector function.
In one such embodiment of the present invention there is provided an antigen binding protein
comprising a heavy chain constant region which comprises a mutated and chimaeric heavy chain
nt region for example n an n binding protein comprising at least one CH2 domain
from lgG3 and one CH2 domain from lgG1, wherein the lgG1 CH2 domain has one or more mutations
at ons selected from 239 and 332 and 330 (for example the mutations may be selected from
8239D and I332E and A330L) such that the antigen binding protein has enhanced effector function,
for e wherein it has one or more of the following functions, enhanced ADCC or enhanced
CDC, for example wherein it has enhanced ADCC and ed CDC. In one embodiment the lgG1
CH2 domain has the mutations 8239D and I332E.
In an alternative embodiment of the present ion there is provided an antigen binding protein
comprising a chimaeric heavy chain constant region and which has an altered glycosylation e. In
one such embodiment the heavy chain constant region comprises at least one CH2 domain from lgG3
and one CH2 domain from lgG1 and has an altered ylation profile such that the ratio of fucose
to mannose is 0.8:3 or less, for example wherein the antigen binding n is defucosylated so that
said antigen binding protein has an enhanced effector function in ison with an equivalent
antigen binding protein with an globulin heavy chain nt region lacking said mutations
and altered glycosylation profile, for example wherein it has one or more of the ing functions,
enhanced ADCC or enhanced CDC, for example n it has ed ADCC and enhanced CDC
In an alternative embodiment the antigen binding protein has at least one lgG3 CH2 domain and at
least one heavy chain constant domain from lgG1 wherein both lgG CH2 domains are mutated in
accordance with the limitations described herein.
In one aspect of the invention there is provided a method of producing an antigen binding protein
according to the invention described herein comprising the steps of:
a) culturing a recombinant host cell containing an expression vector containing an isolated nucleic
acid as described herein, said expression vector further comprising a Fc nucleic acid sequence
encoding a chimeric Fc domain having both lgG1 and lgG3 Fc domain amino acid residues, and
wherein the FUT8 gene encoding alpha-1,6-fucosyltransferase has been inactivated in the
recombinant host cell;and
b) recovering the antigen binding protein .
Such methods for the production of antigen binding proteins can be performed, for example, using the
ACCRETAMABTM technology system available from BioWa, lnc. (Princeton, NJ) which combines the
POTELLIGENTTM and COMPLEGENTTM technology systems to e an antigen binding protein
having both ADCC and CDC enhanced activity that is increased relative to an ise identical
monoclonal antibody lacking a chimeric Fc domain and which has fucose on the oligosaccharide
In yet another ment of the present ion there is provided an antigen binding protein
comprising a mutated and chimeric heavy chain constant region wherein said antigen g protein
has an altered glycosylation e such that the antigen binding protein has enhanced effector
function, for example wherein it has one or more of the following functions, enhanced ADCC or
enhanced CDC. In one embodiment the ons are selected from positions 239 and 332 and 330,
for example the mutations are selected from 8239D and I332E and A330L. In a further ment
the heavy chain constant region comprises at least one CH2 domain from lgG3 and one Ch2 domain
from lgG1. In one embodiment the heavy chain constant region has an altered glycosylation profile
such that the ratio of fucose to mannose is 0.8:3 or less for example the antigen g protein is
defucosylated, so that said antigen binding protein has an enhanced effector on in comparison
with an equivalent non-chimaeric antigen binding n or with an immunoglobulin heavy chain
constant region lacking said mutations and altered glycosylation profile.
40 lmmunoconjugates
2012/059762
Also provided is an immunoconjugate (interchangeably referred to as "antibody-drug conjugates," or
"ADCs")comprising an n binding protein according to the invention as herein described
including, but not limited to, an dy conjugated to one or more cytotoxic agents, such as a
chemotherapeutic agent, a drug, a growth tory agent, a toxin (e.g., a protein toxin, an
enzymatically active toxin of bacterial, fungal, plant, or animal origin, or fragments thereof), or a
ctive isotope (Le, a radioconjugate).
conjugates have been used for the local delivery of cytotoxic agents, i.e., drugs that kill or
inhibit the growth or proliferation of cells, in the treatment of cancer (Lambert, J. (2005) Curr. Opinion
in Pharmacology 5:543-549; Wu et al. (2005) Nature Biotechnology 23(9):1137-1146; Payne, G.
(2003) i 3:207-212; Syrigos and Epenetos (1999) Anticancer Research 19:605-614; scu-Duvaz
and Springer (1997) Adv. Drug Deliv. Rev. 26:151-172; US. Pat. No. 4,975,278). lmmunoconjugates
allow for the targeted ry of a drug moiety to a tumor, and intracellular accumulation therein,
where systemic administration of unconjugated drugs may result in unacceptable levels of toxicity to
normal cells as well as the tumor cells sought to be eliminated (Baldwin et al., Lancet (Mar. 15, 1986)
pp. 603-05; Thorpe (1985) "Antibody Carriers Of xic Agents In Cancer Therapy: A Review," in
Monoclonal dies '84: Biological And Clinical Applications (A. Pinchera et al., eds) pp. 6.
Both polyclonal antibodies and monoclonal antibodies have been reported as useful in these
strategies (Rowland et al., (1986) Cancer lmmunol. lmmunother. 21:183-87). Drugs used in these
methods include daunomycin, doxorubicin, methotrexate, and vindesine (Rowland et al., (1986)
supra). Toxins used in antibody-toxin conjugates include bacterial toxins such as diphtheria toxin,
plant toxins such as ricin, small molecule toxins such as geldanamycin (Mandler et al (2000) J. Nat.
Cancer Inst. 92(19):1573-1581; Mandler et al (2000) Bioorganic & Med. Chem. Letters 10:1025-1028;
Mandler et al (2002) Bioconjugate Chem. 13:786-791), maytansinoids (EP 1391213; Liu et al., (1996)
Proc. Natl. Acad. Sci. USA 93:8618-8623), and calicheamicin (Lode et al (1998) Cancer Res.
58:2928; Hinman et al (1993) Cancer Res. 53:3336-3342).
In one embodiment, the present invention includes immunoconjugates having the following l
structure:
ABP — ((Linker)n — Ctx)m
Wherein ABP is an antigen binding protein
Linker is either absent or any a cleavable or non-cleavable linker described herein
Ctx is any cytotoxic agent described herein
n is 0, 1, 2, or3and
m is 1, 2, 3, 4, 5, 6, 7, 8, 9or 10.
Examples of antibodies linked by an MC linker with auristatins such as MMAE and MMAF are
depicted in the following structures:
L- W? MMW
«UM r..WM ,3; l”
In certain ments, an immunoconjugate comprises an antigen binding n, including but not
limited to, an antibody and a chemotherapeutic agent or other toxin. Chemotherapeutic agents useful
in the generation of conjugates are described herein. Enzymatically active toxins and
fragments thereof that can be used include diphtheria A chain, nonbinding active fragments of
diphtheria toxin, exotoxin A chain (from Pseudomonas aeruginosa), ricin A chain, abrin A chain,
modeccin A chain, alpha-sarcin, Aleurites fordii proteins, dianthin proteins, aca americana
proteins (PAPI, PAP”, and PAP-S), momordica charantia inhibitor, , crotin, sapaonaria
officinalis inhibitor, gelonin, mitogellin, restrictocin, phenomycin, enomycin, and the tricothecenes.
See, e.g., WO 93/21232 published Oct. 28, 1993. A y of radionuclides are available for the
production of radioconjugated antibodies. Examples include 211At‘ 212Bi, 131|, 131In, 90Y, and 186Re.
Antigen binding proteins of the present ion may also be conjugated to one or more toxins,
including, but not limited to, a eamicin, maytansinoids, dolastatins, atins, a trichothecene,
and CC1065, and the derivatives of these toxins that have toxin activity. Suitable cytotoxic agents
include, but are not limited to, an auristatin including dovaline-valine-dolaisoleunine-dolaproine-
phenylalanine (MMAF) and monomethyl auristatin E (MMAE) as well as ester forms of MMAE, a DNA
minor groove binding agent, a DNA minor groove alkylating agent, an enediyne, a opsin, a
duocarmycin, a taxane, including paclitaxel and docetaxel, a puromycin, a dolastatin, a maytansinoid,
and a vinca alkaloid. Specific xic agents include topotecan, morpholino-doxorubicin, rhizoxin,
cyanomorpholino-doxorubicin, dolastatin-10, echinomycin, combretatstatin, chalicheamicin,
maytansine, DM-1, DM-4, netropsin. Other suitable cytotoxic agents include anti-tubulin agents, such
as an auristatin, a vinca alkaloid, a podophyllotoxin, a taxane, a baccatin tive, a physin, a
maytansinoid, a combretastatin, or a dolastatin. Antitubulin agent include ylvaline-valine-
dolaisoleuine-dolaproine-phenylalanine-p-phenylened- iamine (AFP), MMAF, MMAE, atin E,
vincristine, vinblastine, vindesine, vinorelbine, VP-16, camptothecin, paclitaxel, docetaxel, epothilone
A, epothilone B, nocodazole, colchicines, colcimid, estramustine, cemadotin, discodermolide,
maytansine, DM-1, DM-4 or erobin.
Antibody drug conjugates were produced by conjugating the small molecule anti-tubulin agent
monomethylauristatin E (MMAE) or monomethylauristatin F (MMAF) to the antibodies. In the case of
MMAE the linker consists of a thiol-reactive maleimide, a caproyl spacer, the dipeptide valine-
citrulline, and p-aminobenzyloxycarbonyl, a self-immolative nting group. In the case of MMAF a
protease-resistant maleimidocaproyl linker is used. The conjugation process leads to heterogeneity in
drug-antibody attachment, varying in both the number of drugs bound to each antibody molecule
(mole ratio [MR]), and the site of attachment. The most prevalent species is the al with an MR =
4; less ent are materials with MR of 0, 2, 6, and 8. The overall average drug-to-antibody MR is
approximately 4.
Production of lmmunoconjugates
The points of attachment are nes produced by mild reduction of the hain disulfides of the
antibody which is carried out whilst antibodies are immobilised on Protein G affinity resin (thus
enabling the use of large reagent es t intermediate purifications). While lized, a
large excess of TCEP will fully reduce the interchain disulfides but has no impact upon the binding of
the antibody to the resin.
The number of thiols per antibody generated by this procedure depends upon the source and
isotype of the antibodies. For example, human (and mouse-human chimeric) lgG1s have 4 reducible
disulfides, and thus te 8 thiols upon full reduction, whereas murine lgG1s have 5 reducible
disulfides and produce 10 thiols. lf ADCs with the maximal drug loading (e.g., 10 drugs per antibody
for the murine lgG1s) are desired, then the maleimido-drug-linker can simply be added to the
lized dies in sufficient excess to ensure complete ation. However, ADCs with
fewer drugs per antibody can also be prepared from fully reduced antibodies by including a
ically inert capping agent such as N-ethyl maleimide (NEM) which occupies some of the
available thiols on the antibody. When the maleimido-drug-linker and the capping agent are added
simultaneously to the fully reduced antibody and in large excess (at least 3-fold), the two maleimide
electrophiles compete for the limiting number of available thiols. In this fashion, the drug loading is
determined by the relative thiol reaction rates of the drug-linker and capping agent, and thus can be
considered to be under kinetic control. The relative reaction rates of maleimido-drug-linkers do vary
significantly, and thus the molar ratio of drug-linker to NEM present in a on mix must be
determined empirically to arrive at a panel of ADCs with a desired level of drug loading. The mole
fraction of the drug linkers SGD-1006 (chMAE) and SGD-1269 (mcMMAF) in NEM mixtures which
yield ADCs with approximately 4 drugs per antibody are summarized in Table 2 for common human
and murine lgG isotypes.
Auristatins and Dolastatins
WO 63805
In some embodiments, the immunoconjugate comprises an antigen binding protein or antibody
ated to dolastatins or dolostatin ic analogs and derivatives, the auristatins (US. Pat. Nos.
,635,483; 5,780,588). Dolastatins and auristatins have been shown to interfere with microtubule
cs, GTP hydrolysis, and r and cellular division (Woyke et al. (2001) Antimicrob. Agents
and Chemother. 45(12):3580-3584) and have anticancer (US. Pat. No. 5,663,149) and antifungal
activity (Pettit et al. (1998) Antimicrob. Agents Chemother. 42:2961-2965). The dolastatin or
auristatin (which are pentapeptide derivatives of dolastatins) drug moiety may be attached to the
antibody through the N (amino) terminus or the C (carboxyl) terminus of the peptidic drug moiety (WO
02/088172).
Exemplary auristatin embodiments include the N-terminus linked monomethylauristatin drug moieties
DE and DF, disclosed in "Monomethylvaline Compounds Capable of Conjugation to Ligands," US.
Patent No. 7,498,298, the disclosure of which is expressly incorporated by reference in its entirety.
As used herein, the abbreviation "MMAE" refers to monomethyl auristatin E. As used herein the
abbreviation "MMAF" refers to ne-valine-dolaisoleuine-dolaproine-phenylalanine.
Typically, peptide-based drug moieties can be prepared by forming a e bond between two or
more amino acids and/or peptide fragments. Such peptide bonds can be prepared, for example,
according to the liquid phase synthesis method (see E. er and K. Lubke, "The Peptides,"
volume 1, pp 76-136, 1965, Academic Press) that is well known in the field of peptide chemistry. The
auristatin/dolastatin drug es may be prepared according to the methods of: US. Pat. No.
,635,483; US. Pat. No. 5,780,588; Pettit et al. (1989) J. Am. Chem. Soc. 111:5463-5465; Pettit et al.
(1998) Anti-Cancer Drug Design 13:243-277; Pettit, G. R., et al. Synthesis, 1996, 719-725; and Pettit
et al. (1996) J. Chem. Soc. Perkin Trans. 15:859-863. See also Doronina (2003) Nat Biotechnol
21(7):778-784; "Monomethylvaline Compounds Capable of Conjugation to Ligands," US. Patent No.
7,498,298, filed Nov. 5, 2004, hereby incorporated by reference in its entirety (disclosing, e.g., linkers
and methods of preparing monomethylvaline compounds such as MMAE and MMAF ated to
linkers). Biologically active organic compounds which act as cytotoxic agents, specifically
pentapeptides, are disclosed in US Patent Nos. 869; 7,498,298; 7,098,308; 7,256,257; and
116. .
onal antibodies linked with MMAE adn MMAF as well as various derivatives of
auristatins and s of making them are described in US Patent NO. 7,964,566.
Examples of auristatins include MMAE and MMAF the structures of which are shown below:
WO 63805
Maytansine and Maytansinoids
sinoids are mitototic inhibitors which act by inhibiting tubulin polymerization. Maytansine was
first isolated from the east African shrub Maytenus serrata (US. Pat. No. 3,896,111). Subsequently, it
was discovered that certain microbes also produce maytansinoids, such as maytansinol and C-3
maytansinol esters (US. Pat. No. 4,151,042). Highly cytotoxic maytansinoid drugs drugs can be
prepared from ansamitocin sors produced by fermentation of microorganisms such as
synnema. Methods for isolating tocins are described in US Patent No. 6,573,074.
Synthetic maytansinol and derivatives and analogues thereof are disclosed, for example, in US. Pat.
Nos. 4,137,230; 4,248,870; 4,256,746; 608; 4,265,814; 4,294,757; 4,307,016; 4,308,268;
4,308,269; 4,309,428; 4,313,946; 4,315,929; 4,317,821; 4,322,348; 4,331,598; 4,361,650; 4,364,866;
4,424,219; 4,450,254; 4,362,663; and 4,371,533.
Antibody-maytansinoid conjugates are prepared by chemically linking an antibody to a maytansinoid
le without significantly diminishing the biological activity of either the antibody or the
maytansinoid molecule. See, e.g., US. Pat. No. 5,208,020. An average of 3-4 maytansinoid
molecules conjugated per antibody molecule has shown efficacy in enhancing cytotoxicity of target
cells without negatively affecting the function or solubility of the antibody, gh even one molecule
of toxin/antibody would be expected to enhance cytotoxicity over the use of naked antibody.
Maytansinoids are well known in the art and can be synthesized by known techniques or isolated from
l sources. Suitable maytansinoids are disclosed, for example, in US. Pat. No. 5,208,020 and in
the other patents and nonpatent publications referred to hereinabove. Maytansinoids are maytansinol
and maytansinol analogues modified in the aromatic ring or at other positions of the maytansinol
molecule, such as various maytansinol esters. Methods for preparing matansinoids for linkage with
antibodies are disclosed in US Patent Nos. 6,570,024 and 874.
Calicheamicin
The calicheamicin family of antibiotics is capable of producing double-stranded DNA breaks at sub-
lar trations. For the preparation of conjugates of the calicheamicin family, see US. Pat.
Nos. 5,712,374, 5,714,586, 116, 285, 5,770,701, 710, 5,773,001, 5,877,296 (all to
an Cyanamid Company). Structural analogues of calicheamicin which may be used include,
but are not limited to, .gamma.1|, .alpha.2|, .alpha.3l, N-acetyl-.gamma.1l, PSAG and .theta.l1
n et al., Cancer Research 53:3336-3342 (1993), Lode et al., Cancer Research 58:2925-2928
(1998) and the aforementioned US. patents to American Cyanamid). Another anti-tumor drug that the
antibody can be conjugated is QFA which is an antifolate. Both calicheamicin and QFA have
intracellular sites of action and do not readily cross the plasma membrane. Therefore, cellular uptake
of these agents through antibody mediated internalization greatly enhances their xic effects.
Other Cytotoxic Agents
Other mor agents that can be conjugated to the antibodies include BCNU, streptozoicin,
vincristine and 5-fluorouracil, the family of agents known collectively LL-E33288 complex described in
US. Pat. Nos. 5,053,394, 5,770,710, as well as esperamicins (US. Pat. No. 5,877,296).
Enzymatically active toxins and fragments thereof which can be used include diphtheria A chain,
nonbinding active fragments of diphtheria toxin, exotoxin A chain (from Pseudomonas aeruginosa),
ricin A chain, abrin A chain, modeccin A chain, alpha-sarcin, Aleurites fordii proteins, dianthin
proteins, Phytolaca americana proteins (PAPI, PAPII, and PAP-S), momordica charantia inhibitor,
curcin, crotin, sapaonaria officinalis inhibitor, n, mitogellin, ctocin, phenomycin, enomycin
and the tricothecenes. See, for example, WO 93/21232 published Oct. 28, 1993.
The present invention further contemplates an immunoconjugate formed between an antibody and a
compound with nucleolytic activity (e.g., a ribonuclease or a DNA endonuclease such as a
deoxyribonuclease; DNase).
For selective destruction of the tumor, the antibody may comprise a highly radioactive atom. A variety
of radioactive isotopes are available for the production of radioconjugated antibodies. Examples
include At211, I131, I125, Y90, Re186, Re188, Sm153, Bi212, P32, Pb212 and radioactive es
of Lu. When the ate is used for detection, it may comprise a radioactive atom for graphic
studies, for e tc99m or I123, or a spin label for nuclear magnetic nce (NMR) imaging
(also known as magnetic resonance g, mri), such as iodine-123 again, iodine-131, indium-111,
fluorine-19, carbon-13, nitrogen-15, oxygen-17, gadolinium, ese or iron.
The radio- or other labels may be incorporated in the conjugate in known ways. For example, the
peptide may be biosynthesized or may be synthesized by chemical amino acid sis using
suitable amino acid sors involving, for example, fluorine-19 in place of hydrogen. Labels such
as tc99m or I123, Re186, Re188 and |n111 can be attached via a cysteine residue in the peptide.
Yttrium-90 can be attached via a lysine residue. The IODOGEN method (Fraker et al. (1978)
Biochem. Biophys. Res. Commun. 80: 49-57) can be used to incorporate iodine-123. lonal
dies in Immunoscintigraphy" (Chatal, CRC Press 1989) describes other methods in detail.
Preparation of ADCs
In antibody drug conjugates, the antibody can be conjugated directly to the cytotoxic agent or via a
linker. Suitable linkers include, for example, cleavable and non-cleavable linkers. A cleavable linker
is lly susceptible to cleavage under intracellular conditions. Suitable cleavable s include,
for example, a peptide linker cleavable by an intracellular protease, such as lysosomal protease or an
endosomal protease. ln exemplary ments, the linker can be a dipeptide , such as a
valine-citrulline (val-cit) or a phenylalanine-lysine ys) linker. Other suitable linkers include linkers
yzable at a pH of less than 5.5, such as a one linker. onal suitable cleavable linkers
include disulfide linkers.
Bristol-Myers Squibb has described particular lysosomal enzyme-cleavable antitumor drug
conjugates. See, for example, US. Pat. No. 6,214,345. Seattle Genetics has published ations
US. Pat. Appl. 2003/0096743 and US. Pat. Appl. 130189, which describe p-
aminobenzylethers in drug delivery . The linkers described in these applications are d to
aminobenzyl ether compositions.
Conjugates of the antigen binding protein and cytotoxic agent may be made using a variety of
bifunctional protein coupling agents such as N-succinimidyl(2-pyridyldithio)propionate (SPDP),
succinimidyl(N-maleimidomethyl) cyclohexanecarboxylate (SMCC), iminothiolane (IT),
bifunctional derivatives of imidoesters (such as dimethyl idate HCI), active esters (such as
disuccinimidyl suberate), aldehydes (such as glutaraldehyde), bis-azido compounds (such as bis(pazidobenzoyl
) hexanediamine), bis-diazonium derivatives (such as bis-(p-diazoniumbenzoyl)-
ethylenediamine), diisocyanates (such as toluene 2,6-diisocyanate), and bis-active fluorine
compounds (such as fluoro-2,4-dinitrobenzene).
Additionally the linker may be composed of one or more linker components. Exemplary linker
components include 6-maleimidocaproyl ("MC"), maleimidopropanoyl ("MP"), -citrulline ("val-
cit"), alanine-phenylalanine ("ala-phe"), p-aminobenzyloxycarbonyl ("PAB"), N-Succinimidyl 4-(2-
pyridylthio)pentanoate ("SPP"), N-Succinimidyl 4-(N-maleimidomethyl)cyclohexane-1 carboxylate
("SMCC"), and N-Succinimidyl (4-iodo-acetyl)aminobenzoate ("SIAB"). Additional linker components
are known in the art and some are described herein. See also "Monomethylvaline Compounds
e of ation to Ligands," US. Patent No. US7,498,298, filed Nov. 5, 2004, the contents of
which are hereby incorporated by reference in its entirety.
Linkers may also ses amino acids and/or amino acid analogs. Amino acid linker components
e a dipeptide, a tripeptide, a tetrapeptide or a pentapeptide. Exemplary dipeptides include:
valine-citrulline (vc or val-cit), alanine-phenylalanine (af or ala-phe). Exemplary tripeptides include:
glycine-valine-citrulline (gly-val-cit) and glycine-glycine-glycine (gly-gly-gly). Amino acid residues
40 which comprise an amino acid linker component include those occurring naturally, as well as minor
amino acids and non-naturally occurring amino acid analogs, such as citrulline. Amino acid linker
components can be designed and optimized in their selectivity for tic cleavage by a particular
enzyme, for example, a tumor-associated protease, cathepsin B, C and D, or a plasmin protease.
Antigen g proteins and antibodies may be made reactive for conjugation with linker
reagents. philic groups on dies include, but are not limited to: (i) N-terminal amine
groups, (ii) side chain amine groups, e.g., lysine, (iii) side chain thiol groups, e.g. cysteine, and (iv)
sugar hydroxyl or amino groups where the antibody is glycosylated. Amine, thiol, and hydroxyl
groups are nucleophilic and capable of reacting to form covalent bonds with electrophilic groups on
linker moieties and linker reagents including: (i) active esters such as NHS esters, HOBt ,
haloformates, and acid halides; (ii) alkyl and benzyl halides such as haloacetamides; (iii) aldehydes,
ketones, carboxyl, and maleimide groups. n antibodies have reducible interchain ides, i.e.
cysteine bridges. Antibodies may be made reactive for conjugation with linker reagents by treatment
with a reducing agent such as DTT (dithiothreitol). Each cysteine bridge will thus form, theoretically,
two reactive thiol nucleophiles. Additional nucleophilic groups can be introduced into antibodies
h the reaction of lysines with 2-iminothiolane (Traut's reagent) resulting in conversion of an
amine into a thiol. Reactive thiol groups may be introduced into the antibody (or fragment thereof) by
introducing one, two, three, four, or more cysteine residues (e.g., preparing mutant antibodies
comprising one or more non-native cysteine amino acid es).
Antigen g proteins and antibodies may also be modified to introduce electrophilic moieties,
which can react with nucleophilic tuents on the linker reagent or drug. The sugars of
glycosylated antibodies may be oxidized, e.g. with ate oxidizing reagents, to form aldehyde or
ketone groups which may react with the amine group of linker reagents or drug moieties. The
resulting imine Schiff base groups may form a stable linkage, or may be reduced, e.g., by borohydride
reagents to form stable amine linkages. In one embodiment, reaction of the carbohydrate portion of a
glycosylated antibody with either glactose oxidase or sodium meta-periodate may yield carbonyl
(aldehyde and ketone) groups in the protein that can react with appropriate groups on the drug
(Hermanson, Bioconjugate Techniques). In r ment, proteins containing N-terminal
serine or threonine residues can react with sodium meta-periodate, resulting in production of an
aldehyde in place of the first amino acid (Geoghegan & Stroh, (1992) Bioconjugate Chem. 3:138—146;
US. Pat. No. 852). Such aldehydes can be reacted with a drug moiety or linker phile.
Nucleophilic groups on a drug moiety e, but are not limited to: amine, thiol, hydroxyl, hydrazide,
oxime, hydrazine, thiosemicarbazone, hydrazine carboxylate, and arylhydrazide groups capable of
reacting to form covalent bonds with electrophilic groups on linker moieties and linker reagents
including: (i) active esters such as NHS esters, HOBt esters, haloformates, and acid halides; (ii) alkyl
and benzyl halides such as haloacetamides; (iii) aldehydes, ketones, carboxyl, and maleimide groups.
In some embodiments, the linker is cleavable by a cleaving agent that is present in the ellular
environment (e.g., within a me or endosome or caveolea). The linker can be, e.g., a peptidyl
linker that is cleaved by an intracellular peptidase or se enzyme, including, but not limited to, a
mal or endosomal se. Typically, the peptidyl linker is at least two amino acids long or at
least three amino acids long. Cleaving agents can include cathepsins B and D and plasmin, all of
which are known to hydrolyze dipeptide drug derivatives resulting in the release of active drug inside
target cells (see, e.g., Dubowchik and Walker, 1999, Pharm. Therapeutics 123). Peptidyl
linkers may be cleavable by enzymes that are present cells. For example, a peptidyl linker that is
cleavable by the thiol-dependent protease cathepsin-B, which is highly expressed in cancerous
tissue, can be used (e.g., a Phe-Leu or a Gly—Phe-Leu-Gly (SEQ ID NO:50) linker). Other such linkers
are described, e.g., in US. Pat. No. 6,214,345. In specific embodiments, the peptidyl linker cleavable
by an intracellular protease is a Val-Cit linker or a Phe-Lys linker (see, e.g., US. Pat. No. 6,214,345,
which describes the synthesis of doxorubicin with the val-cit linker). One advantage of using
intracellular proteolytic release of the therapeutic agent is that the agent is typically attenuated when
conjugated and the serum ities of the conjugates are typically high.
In other embodiments, the cleavable linker is pH-sensitive, i.e., ive to hydrolysis at certain pH
values. Typically, the pH-sensitive linker yzable under acidic ions. For example, an acid-
labile linker that is hydrolyzable in the lysosome (e.g., a one, semicarbazone,
thiosemicarbazone, cis-aconitic amide, orthoester, acetal, ketal, or the like) can be used. (See, e.g.,
US. Pat. Nos. 368; 5,824,805; 5,622,929; Dubowchik and Walker, 1999, Pharm. Therapeutics
83:67-123; Neville et al., 1989, Biol. Chem. 264:14653-14661.) Such linkers are relatively stable
under l pH ions, such as those in the blood, but are unstable at below pH 5.5 or 5.0, the
approximate pH of the lysosome. In certain embodiments, the hydrolyzable linker is a thioether linker
(such as, e.g., a thioether ed to the therapeutic agent via an acylhydrazone bond (see, e.g.,
U.S. Pat. No. 5,622,929)).
In yet other embodiments, the linker is cleavable under reducing conditions (e.g., a disulfide linker). A
variety of disulfide linkers are known in the art, including, for example, those that can be formed using
SATA (N-succinimidylacetylthioacetate), SPDP (N-succinimidyl(2-pyridyldithio)propionate),
SPDB (N-succinimidyl(2-pyridyldithio)butyrate) and SMPT (N-succinimidyl-oxycarbonyl-alpha-
methyl-alpha-(2-pyridyl-dithio)toluene)- SPDB and SMPT (See, e.g., Thorpe et al., 1987, Cancer
Res. 47:5924-5931; nczak et al., In lmmunoconjugates: Antibody Conjugates in Radioimagery
and Therapy of Cancer (C. W. Vogel ed., Oxford U. Press, 1987. See also US. Pat. No. 4,880,935.)
In yet other specific embodiments, the linker is a malonate linker (Johnson et al., 1995, Anticancer
Res. 15:1387-93), a maleimidobenzoyl linker (Lau et al., 1995, Bioorg-Med-Chem. 3(10):1299-1304),
or a 3'-N-amide analog (Lau et al., 1995, Bioorg-Med-Chem. 3(10):1305-12).
Typically, the linker is not substantially sensitive to the ellular environment. As used herein, "not
substantially sensitive to the extracellular nment," in the context of a linker, means that no more
than about 20%, typically no more than about 15%, more typically no more than about 10%, and even
more typically no more than about 5%, no more than about 3%, or no more than about 1% of the
linkers, in a sample of ADC or ADC derivative, are cleaved when the ADC or ADC derivative present
in an extracellular environment (e.g., in plasma). Whether a linker is not substantially sensitive to the
ellular environment can be determined, for example, by incubating independently with plasma
both (a) the ADC or ADC derivative (the "ADC sample") and (b) an equal molar amount of
unconjugated dy or therapeutic agent (the "control sample") for a ermined time period
(e.g., 2, 4, 8, 16, or 24 hours) and then comparing the amount of unconjugated antibody or
therapeutic agent present in the ADC sample with that t in control sample, as ed, for
example, by high performance liquid chromatography.
In other, non-mutually exclusive embodiments, the linker promotes ar internalization. In n
embodiments, the linker promotes cellular internalization when conjugated to the therapeutic agent
(i.e., in the milieu of the linker-therapeutic agent moiety of the ADC or ADC derivate as described
herein). In yet other embodiments, the linker promotes cellular internalization when conjugated to both
the therapeutic agent and the antigen binding protein or antibody or derivative thereof (i.e., in the
milieu of the ADC or ADC tive as described herein).
A variety of linkers that can be used with the t compositions and methods are described in WO
2004010957 entitled "Drug Conjugates and Their Use for Treating Cancer, An Autoimmune e
or an Infectious Disease" filed Jul. 31, 2003, and US. Provisional Application No. 60/400,403, entitled
"Drug Conjugates and their use for treating cancer, an autoimmune disease or an infectious disease",
filed Jul. 31, 2002 (the disclosure of which is incorporated by nce herein).
Alternatively, a fusion protein comprising the antigen binding protein and cytotoxic agent may be
made, e.g., by recombinant techniques or peptide synthesis. The length of DNA may comprise
respective regions encoding the two portions of the conjugate either adjacent one another or
separated by a region encoding a linker peptide which does not destroy the desired properties of the
ate.
In yet another embodiment, the antibody may be conjugated to a "receptor" (such as streptavidin) for
utilization in tumor pre-targeting wherein the antibody-receptor conjugate is stered to the
patient, followed by removal of unbound conjugate from the circulation using a ng agent and
then administration of a "ligand" (e.g., avidin) which is conjugated to a cytotoxic agent (e.g., a
radionucleotide).
WO 63805 2012/059762
The term “Non Human dy or antibody fragment f” as used herein is meant to refer to
dies or fragments thereof which originate from any species other than human wherein human
includes chimeric antibodies.
The term “donor dy” refers to an antibody (monoclonal, and/or recombinant) which contributes
the amino acid sequences of its variable domains, CDRs, or other functional fragments or s
thereof to a first immunoglobulin partner, so as to provide the altered immunoglobulin coding region
and resulting expressed altered antibody with the antigenic specificity and neutralizing activity
characteristic of the donor antibody.
The term “acceptor antibody” refers to an antibody (monoclonal and/or recombinant) heterologous to
the donor antibody, which butes all (or any portion, but preferably all) of the amino acid
sequences encoding its heavy and/or light chain framework regions and/or its heavy and/or light chain
constant regions to the first immunoglobulin partner. The human dy is the acceptor antibody.
The term “Human acceptor sequence” as used herein is meant to refer to a framework of an antibody
or antibody fragment thereof comprising the amino acid sequence of a VH or VL ork derived
from a human antibody or antibody fragment f or a human consensus sequence framework into
which CDR’s from a non-human species may be incorporated.
The term “incorporation” of CDR’s or hypervariable regions as used herein encompasses any means
by which the man CDR’s are situated with the human acceptor framework. It will be
appreciated that this can be achieved in various ways, for example, c acids encoding the
desired amino acid sequence can be generated by mutating nucleic acids ng the non-human
variable domain sequence so that the framework residues thereof are changed to human acceptor
framework residues, or by mutating nucleic acid encoding the human variable domain sequence so
that the CDR’s are changed to non-human residues, or by synthesizing nucleic acids encoding the
desired sequence. In one embodiment the final sequence is generated in silico.
The present invention is now described by way of example only. The appended claims may include a
generalisation of one of more of the following examples.
Examples
Example 1 Monoclonal Antibody Generation and Selection
1.1 Immunisation strategies
The anti human BCMA mAb murine parental CA8 was identified from hybridomas d from mice
immunized with full length human BCMA. A BALB/c mouse was immunized i.p. with 25 ug of
recombinant (rBCMA) protein combined with CFA. The mouse was boosted three times at one-month
als with 25 ug of full length rBCMA protein + 10 ug monophosphoryl lipid A—stable emulsion
(MPL-SE) (Corixa Corporation, Seattle, WA) and given a pre-fusion boost of 30 ug rBCMA protein iv
3 days prior to fusion. Hybridomas were either generated and cloned using the ClonaCeII-HY
hybridoma cloning kit (StemCeII Technologies, Vancouver, BC) or using a conventional method. In
the conventional method, B cells from the spleens of the immunized animals were fused with Sp2/0
myeloma cells in the presence of PEG (Sigma-Aldrich, St. Louis, MO). After overnight recovery, fused
cells were plated at limiting dilution in 96-well plates and subjected to hypoxanthine-aminopterin-
thymidine selection. Hybridoma culture atants were examined for the ce of anti-BCMA
dies by ELISA and flow cytometry:
The anti human BCMA mAb murine parental 8G03 was identified from hybridomas derived
from SJL mice immunized with recombinant human BCMA/TNFRSF17-Fc chimera (R&D 193-Fc)
using the RIMMS method (Rapid immunisation multiple sites). At Day 0, 5ug protein per mouse was
fied in ASOZa adjuvant at 2 sites on back (over haunches and over shoulders) and subjacent to
the major lymph nodes at 4 sites on front. On day 6 and day 11 2.5ug n per mouse in RIBI
adjuvant was injected subjacent to the major lymph nodes at 4 sites on front. On day 14 the animals
were sacrificed. The lymph nodes and spleen were excised, disrupted and a PEG1500 d
somatic cell fusion performed using a 3:1 ratio with mouse a cells X63 AG8 653.GFP.BcI-2.11
t ; R17209/58). The fusion was plated out into 10 X 96 well plates and ed directly
from these.
The anti human BCMA mAb murine al S336105A07 was identified from hybridomas derived
from identical immunisations. The lymph nodes and spleen were excised at day 14, disrupted, and a
Cytopulse electrofusion was performed using a 1:1 ratio with mouse myeloma cells X63 AG8
653.GFP.BcI-2.11 (BioCat 112754; R17209/58). The fusion was plated out into omnitrays containing
semi solid medium prior to picking into 10 X 96 well plates and was screened directly from these 5
days later.
The anti human BCMA murine parental mAbs S332121F02 and S332126E04 were identified from
hybridomas derived from SJL mice immunized with recombinant Fc fusion of the extracellular domain
of human BCMA (4-53)BCMA using the RIMMS method (Rapid immunisation). At Day 0, 5ug protein
per mouse was emulsified in ASOZa adjuvant at 2 sites on back (over haunches and over ers)
and subjacent to the major lymph nodes at 4 sites on front. On day 6 5ug recombinant cyno BCMA-Fc
40 protein per mouse in RIBI adjuvant was injected subjacent to the major lymph nodes at 4 sites on
front. On day 11 2.5ug recombinant human BCMA-Fc and 2.5ug recombinant cyno BCMA-Fc per
mouse in RIBI adjuvant was injected subjacent to the major lymph nodes at 4 sites on front. On day
14 the animals were sacrificed and cells treated as for S307118G03.
The anti human BCMA murine parental mAb S322110D07 was identified from hybridomas derived
from SJL mice immunised with recombinant Fc fusion of the extracellular domain of human BCMA (4-
53) in complex with recombinant human April (R&D 5860-AP/CF) premixed at 1:1 molar ratio. The
mice were immunized i.p. with 5ug April/Cyno BCMA-Fc complex in PBS, suspended in RIBI
adjuvant, 100ul dose per mouse and boosted 3 times at 3-4 week intervals with 2.5ug April/Cyno
c complex in PBS, suspended in RIBI adjuvant, 100ul dose per mouse injected via
intraperitoneal route and given a pre-fusion boost of the same immunogen 1 day prior to fusion and
treated as for 8307118603.
The anti human BCMA mAb murine parental 5G01 and S335122F05 were identified from
hybridomas derived from SJL mice immunized with a mixture of recombinant Fc fusion of the
extracellular domain of human BCMA (4-53) and recombinant Fc fusion of the extracellular domain of
cyno BCMA (4-52) using the RIMMS method (Rapid immunisation multiple sites). At Day 0, 2, 5ug of
each protein per mouse was emulsified in ASO2a nt and injected at 2 sites on the back (over
es and over shoulders) and subjacent to the major lymph nodes at 4 sites on front. On day 6
and day 11 2.5ug of each protein per mouse in RIBI nt was injected subjacent to the major
lymph nodes at 4 sites on front. On day 14 the animals were sacrificed. The lymph nodes and spleen
were excised, disrupted and a Cytopulse electrofusion was med using a 1:1 ratio with mouse
myeloma cells X63 AG8 653.GFP.Bcl-2.11 (BioCat 112754; R17209/58). The fusion was plated out
into omnitrays containing semi solid medium prior to picking into 32 x 96 well plates and was
ed directly from these 5 days later.
Example 2 Humanisation.
2.1 Cloning of CA8 Hybridoma Variable Regions
Total RNA was extracted from CA8 hybridoma cells, heavy and light variable domain cDNA sequence
was then generated by reverse transcription and polymerase chain reaction (RT-PCR). The fonNard
primer for RT-PCR was a mixture of degenerate primers ic for murine immunoglobulin gene
leader-sequences and the reverse primer was specific for the antibody constant regions. Reverse
primers specific for lgG1, lgG2a and lgG2b were used in this case as the isotype was unknown. To
design the primers, DNA multiple sequence alignments of the leader ces of the mouse VH and
Vk genes were generated.
2.2 g of chimeric CA8
The DNA expression constructs encoding the chimeric antibody were prepared de novo by up of
overlapping oligonucleotides including restriction sites for cloning into mammalian expression vectors
as well as a human signal sequence. Hind/ll and Spel restriction sites were introduced to frame the
VH domain containing the signal sequence for cloning into mammalian expression vectors containing
the human v1 constant region. Hind/ll and Bsin restriction sites were introduced to frame the VL
domain containing the signal sequence for cloning into mammalian expression vector containing the
human kappa constant region.
2.3 Cloning of the humanised CA8 variants
The DNA expression constructs encoding the sed antibody variants were prepared de novo by
up of overlapping oligonucleotides including restriction sites for cloning into mammalian
expression vectors as well as a human signal sequence. Hind/ll and Spel restriction sites were
introduced to frame the VH domain containing the signal sequence for cloning into ian
sion vectors containing the human v1 constant region. Hind/ll and Bsin restriction sites were
introduced to frame the VL domain containing the signal sequence for cloning into mammalian
expression vector containing the human kappa constant region.
2.4 Expression of the recombinant CA8 antibodies (including antibody quantification)
Expression ds encoding the heavy and light chains respectively were transiently co-transfected
into HEK 293 BE cells and expressed at small scale to produce dy. Antibodies were quantified
by ELISA. ELISA plates were coated with anti human lgG (Sigma l3382) at 1mg/ml and blocked with
blocking solution (4% BSA in Tris buffered saline). Various dilutions of the tissue culture supernatants
were added and the plate was incubated for 1 hour at room temperature. Dilutions of a known
standard antibody were also added to the plate. The plate was washed in TBST and binding was
detected by the addition of a dise labelled anti human kappa light chain antibody (Sigma
A7164) at a dilution of 1/1000 in ng on. The plate was incubated for 1 hour at room temp
before g in TBST. The plate was developed by addition of CPD substrate (Sigma P9187) and
colour development stopped by addition of 2M H2SO4. Absorbance was measured at 490nm and a
standard curve plotted using data for the known standard dilutions. The standard curve was used to
estimate the concentration of antibody in the tissue culture atants. Larger scale antibody
preparations were purified using n A and trations were measured using a Nanodrop
(Thermo Scientific).
Table 1. Design of CA8 variable heavy and light humanised variants
WO 63805
Humanised Template Backmutations
VH Kabat#
ht graft of CA8 VH CDRs onto |GHV1_69 + JH1 minigene None
. 27v T
1 A T
I_ A .
_ w: . A
_ _...
l_. N°°D
1 N"D
_2 N"D
_. _...
Straight graft of CA8 VL CDRs onto |GKV1_39 + JK2 minigene None
M F71Y
M1 M4L K4 E
2.5 Defucosylated antibody production
To generate defucosylated antibodies the heavy and light chains respectively were co-transfected
into CHO DG44 M8705 BioWa cells and expressed at scale to produce antibody. Briefly, 30ug DNA
was linearised overnight with Not1, the DNA was ethanol precipitated and solved in TE buffer.
From culture, 2.4X107 BioWa DG44 cells were obtained and washed in 14ml of warmed PBS-
sucrose. The cells were spun and the pellet resuspended in 1.6ml of PBS-sucrose. Half (0.8ml) of
aforementioned cells, suspended in PBS-sucrose, were added to a BioRad cuvette with the 30ug of
ised DNA (in 50u| TE buffer). A BioRad GenePuIser was programmed to 380V with a
capacitance of 25uF and the cuvette was entered for electroporation. The resulting 850u| of
electroporated cells and DNA were added to (80ml) warmed SFM512 medium (including phenol red,
2XHT (nucleosides), glutamax and Gibco supplement4). Finally, the resulting 80ml of cell suspension
was transferred (150pl/well) to each well of one of 4 X 96-well plates. After 48 hours, the medium
was changed to nucleoside free by removing approximately 130u| of conditioned and replacing with
150u| of fresh ion medium SFM512 medium ding phenol red and ax). Every 3-4
days, 130-150ul of conditioned medium was removed and replaced with fresh, selection medium.
Wells were monitored for colour change and assayed for IgG concentration as discussed previously.
2.6 Additional antibodies — Cloning of Hybridoma Variable Regions
Total RNA was extracted from S307118G03, S332121F02, S332126E04, S322110D07,
S336105A07, S335115G01 and S335122F05 hybridoma cells. Heavy and light le domain
cDNA sequence was then generated by reverse transcription and polymerase chain on (RT-
PCR). The d primer for RT-PCR was a mixture of degenerate primers specific for murine
immunoglobulin gene leader-sequences and the reverse primer was specific for the antibody constant
regions, in this case isotype lgG2a. Primers were designed based on a strategy described by Jones
and Bendig (Bio/Technology 9:88, 1991). RT-PCR was carried out for both V-region ces to
enable subsequent verification of the t V-region sequences. DNA sequence data was obtained
for the V-region products generated by the RT-PCR.
2.7 Additional antibodies — Cloning of the chimeras
The DNA expression constructs encoding the chimeric antibodies were prepared de novo by on
advantage PCR cloning (Clonetech) of the V-gene PCR ts into mammalian expression
vectors. This cloning method enabled fusion the murine variable regions to human lgG1 H chain and
kappa L chain nt regions.
2.8 S307118G03 — Cloning of the humanized variants
Cloning was carried out as for paragraph 2.3.
2.9 S307118G03 sion of the recombinant antibodies
Expression plasmids encoding the relevant heavy and light chains (listed in Table 8 below) were
transiently co-transfected into HEK 293 BE cells and expressed at small scale to produce antibody.
The antibodies were Protein A ed from the supernatants and quantified using the Nanodrop
spectrophotometer.
8 below) were transiently co-transfected into HEK 293 BE cells and sed at small scale to
produce antibody. The antibodies were Protein A purified from the supernatants and quantified using
the Nanodrop spectrophotometer.
Example 3 Conjugation of antibodies to chMAE and mcMMAF to form antibody drug
ates (ADC)
Table B Chemical structures of drug-linkers
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Gammabind Plus Protein G Sepharose (GE Healthcare) resin slurry (75 uL) was added to a each well
of a deep well (2 mL capacity) filter plate. The antibodies to be conjugated were d by species
and isotype and up to 0.5 mg of each antibody transferred to each well of the plate. Each antibody
was transferred to two separate wells to facilitate the ation of two conjugates, with the drug-
linkers SGD-1006 and SGD-1269. The filter plate was then shaken at 1200 RPM for 2 hours at 5 °C
to bind the antibodies to the resin. The filter plate was then centrifuged at 500x g for 3 minutes to
ensure complete pulldown of all fluids and resin to the bottom of the each well.
The bound dies were then reduced by adding 500 uL of 10 mM TCEP in 100 mM KPO4, 150
mM NaCl, pH 7, 1mM EDTA and shaking for 30 minutes at 22 °C. Following reduction, the plate was
again fuged to remove the TCEP solution and subsequently washed with PBS + 1mM EDTA, 1
mL per well. The wash solution was removed by centrifugation and the process repeated 3 times for
a total of 4 washes. The bound and reduced antibodies were then conjugated using a mixture of NEM
and drug linker prepared in accordance with the mole fractions indicated in Table 2.
Table 2.
Antibody Reducible SGD-1006 mole SGD-1269 mole
(species / isotype) Disulfides fraction fraction
Human lgG1* 0.675 0.688
WO 63805
Murine lgG2b “ 0.463
* also for murine/ human lgG1 chimerics
Separate mixtures of NEM and drug linker were thus prepared for each antibody species/ isotype
using 10 mM DMSO stock ons of SGD-1006, SGD-1269 (See Table B) and NEM. When mixed
at the appropriate ratio the total maleimide concentration was ore still 10 mM, and this value
was used to calculate the volume of maleimide solution to be added to each well. For example for a
murine lgG1 with 5 reducible disulfides (10 available thiols when reduced) 0.5 mg of dy at 150
kD is 3.33 nmol corresponding to 33.3 nmol of thiol. A 3-fold excess is therefore 100 nmol of total
maleimide or 10 uL of the 10 mM drug linker/ NEM mix. For the SGD-1269 conjugate this mix would
then be prepared with 5.86 uL of SGD-1269 and 4.14 uL of NEM. The maleimide mix would then be
diluted into 500 uL of PBS prior to addition to the immobilized reduced antibody. In practice, since
multiple antibodies of each isotype were ated simultaneously a single SGD-1269 / NEM mixed
solution for each isotype was prepared by lying the number of wells containing that isotype by
uL per well then diluting into a volume of PBS equal to 500 uL times the number of wells. In like
n a total of eight drug-linker/ NEM mixes were prepared—four with SGD-1006 and four with
SGD-1269—and d into PBS. These mixes were then added to the reduced antibodies (500 uL
per well) and the plate was shaken for 30 minutes at 22 °C. The plate was then centrifuged as above
to remove the excess reaction solution, and subsequently washed 4 times with PBS as before.
The bound ADCs were then eluted by adding 200 uL of 50 mM glycine pH 2.5 to each well and
shaking the plate for 3 minutes at 1200 RPM. While shaking 20 uL of neutralization buffer (1M
potassium phosphate, pH 7.4, 500 mM NaCl, 0.2% Tween-20) was added to each well ofa 1 mL
collection plate. The ADCs were then eluted into the collection plate by spinning at 1500x g for 6
minutes. The collection plate was then shaken briefly to ensure complete mixing of the neutralization
buffer.
The concentration of each ADC was then determined with an absorbance plate reader by erring
the solutions into a UV assay plate (Costar model 3635, Corning) and measuring the optical density at
280 nm. An average lgG extinction coefficient of 1.45 mL mg-1 cm-1 was used to provide an
adequate estimation of ADC concentration across the panel. To confirm successful conjugation, a
ed phase protein HPLC method (described below) was used to estimate the drug loading of the
isotype controls. For the plate containing the humanization ts of CA8 this method was used to
te the loading of all ADCs directly.
The reversed phase protein chromatography method for determining drug loading employs the PLRP-
S polymeric stationary phase (Agilent Technologies). Since the antibodies were fully reduced during
the conjugation s all of the antibody subunits elute from the column as single polypeptide
chains allowing the subpopulations of light and heavy chain species with varying levels of drug
g to be evaluated separately. Thus, the is of these data allow for the calculation of the
average light chain drug loading and the average heavy chain drug loading as independent factors
which can then be ed to determine average antibody drug loading with the basic knowledge
that each antibody is comprised of two light and two heavy chains. The chromatographic conditions
were as follows: A PRLP-S column, 1000 A, 50 X 2.1 mm, 8 um particle size (Agilent Technologies)
with water + 0.05% TFA as mobile phase A and itrile + 0.01% TFA as mobile phase B; elution
with a linear gradient of 27% B to 42% B in 12.5 minutes.
Anti-BCMA dies were conjugated with SGD-1006 and SGD-1269 in three separate batches
over a period of seven months. In the first batch a total of 29 antibodies were ated (resulting in
58 ADCs). The drug g of each isotype l determined by PLRP chromatography and the
data are summarized in Table 3.
Table 3.
lsotype SGD-1006 loading SGD-1269 loading
For the second batch an additional 25 antibodies were conjugated (resulting in 50 ADCs). The drug
loading of each isotype control was again determined by PLRP chromatography and the data are
summarized in Table 4.
Table 4.
SGD-1269
lsotype SGD-1006 loading loading
In the third batch 30 antibodies were conjugated (resulting in 60 ADCs), including 13 humanized
variants of CA8. In this final batch, the drug loading of all ADCs were determined and are
summarized in the following two plate maps. (Table 5 & 6)
2012/059762
SGD-lUUES (VB-m) ADCS SGD-l269 AF) ADCS
3.? 3.8 3.6 3.8 3.4 3.7 3.8 3.7"
4.1% 5.1% 3.4% 4.8% 2.8% 8. 5% 24.1% 3.4%
Table 6.
control l
EB S336106D07 83361051407
F (3.48 J7M2 GRITS28T85
S341106G02 -§
_§CA8 J8M1
SGD-lOOG (vs—MMAE) ADCs SGD-1269 (mu-ME) ADCs
Mean drug loading and %CV are indicated for each isotype series at the bottom. An
uncharacteristically large variability in drug loading was observed for the SGD-‘I269 ADCs prepared
with mlgG2b antibodies; the reason for this is unclear. Also, the Fc-enhanced CA8 antibodies yielded
somewhat lower drug loading levels than the other CA8 human variants; to s this, additional
Fc-enhanced CA8 was conjugated in a solution-phase reaction to better match the drug loading
achieved for the other antibodies.
Example 4 — Binding Data
4.1 FMAT binding assay to show binding of Chimeric CA8 to cells expressing human or cyno
BCMA .
Cryopreserved transfected human, cyno BCMA and mock transfected HEK293 cells were red
from LN2 storage. Assay wells were prepared with human chimeric CA8 antibody, at a range of
different concentrations, mixed with human BCMA HEK293, cyno BCMA HEK293 and mock
transfected cells respectively. Anti-human IgG FMAT Blue secondary conjugate was added for
detection of human chimeric CA8. The assay plates were left for a minimum of 90 minutes before the
result was read on the ABI8200 (FMAT) plate reader.
This showed that the CA8 antibody in chimeric form binds well to both human and cyno BCMA
proteins sed on HEK293 cells.
Results are shown in Figure 1.
4.2 ELISA experiment showing binding of chimeric CA8 to recombinant BCMA protein
Chimeric CA8 antibodies were tested for binding to human BCMA and cyno BCMA expressed as Fc
s. Human BCMA-Fc and cyno BCMA-Fc were coated to ELISA plates and the plates were
blocked using BSA to reduce non specific binding. CA8 chimeric antibodies were added in a
tration range from 5ug/ml to 0.1ug/ml to the human and cyno BCMA coated ELISA plates. Any
bound human chimeric CA8 antibody was detected using anti-human IgG HRP conjugated secondary
antibody as appropriate. HRP substrate (TMB) was added to develop the ELISA. This showed that
CA8 antibody binds to recombinant human and cyno BCMA in an ELISA assay.
Results are shown in Figure 2.
4.3 Biacore experiment to show CA8 antibody binding to BCMA and TACI proteins to determine
cross reactivity with TACI n.
CA8 chimera antibody was injected and captured on protein A. (A protein A derivitised sensorchip
was used). Residual protein A binding was blocked with an ion of a high concentration of human
IgG on. BCMA-Fc, TACI-Fc or BAFF-R-Fc solutions were then tested for binding to the antibody.
The 3 ns were injected in sequence and binding events were measured. The surface was
regenerated between injection of each n.
Sensorgrams were ed in the Biaevaluation program. Double nce ction was done to
remove instrument noise and any non-specific binding from the sensorgram curves.
This showed that CA8 was specific for binding to BCMA binding and not to TACI and BAFFR.
Binding of the CA8 antibody to BCMA-Fc, TACI-Fc and BAFF-R-Fc was plotted out as shown in
Figure 3.
4.4 Cell binding and neutralisation data
4.4.1 Binding of murine anti BCMA antibodies to le myeloma cells and BCMA expressing
cells
Multiple a cell line H929 and ARH77-hBCMA 10B5 BCMA expressing ectant cells were
stained with murine S332211D07, S3332121F02 or S332126E04 or murine isotype control at 5
pg/mL. Multiple myeloma cell line H929 was stained with murine S307118G03. Cells were incubated
for 20 mins at room temperature (RT) and then washed with FACS buffer (PBS + 0.5% BSA + 0.1%
sodium azide) to remove unbound antibody. Cells were incubated with a secondary PE labelled anti-
mouse lgG antibody for 15 minutes at RT and then washed with FACS buffer to remove unbound
dy. Cells were analysed by FACS to detect antibody bound to the cells.
The results (Figure 4) showed that all 4 murine antibodies bound to the H929 multiple myeloma cell
line and the three antibodies tested on ARH77 BCMA transfected cells bound to these.
4.4.2 Binding curve of chimeric CA8 to multiple myeloma cells as ined by FACS
A panel of multiple a cell lines were used to determine the binding of chimeric CA8. Cell lines
H929, OPM-2, JJN-3 and U266 were stained with either chimeric CA8 or irrelevant antibody (Synagis)
at varying concentrations for 20 minutes at RT. Cells were then washed with FACS buffer (PBS +
0.5% BSA + 0.1% sodium azide) to remove d antibody. Cells were incubated with a
secondary PE labelled uman lgG antibody for 15 minutes at RT and then washed with FACS
buffer to remove unbound antibody. Cells were analysed by FACS and mean fluorescence intensity
(MFI) values measured to determine binding.
Results showed that chimeric CA8 bound to multiple myeloma cell lines H929, OPM-2, JJN-3 & U266
in a dose ent manner (Figure 5).
4.4.3 Binding of humanised CA8 to BCMA transfected cells as determined by FACS
ARH77-hBCMA 10B5 BCMA expressing transfectant cells or H929 cells were stained with either
chimeric CA8 or humanised variants of CA8 designated J6M0, J6M1, J6M2, J9M0, J9M1, J9M2 at
varying concentrations for 20 minutes at RT. Cells were then washed with FACS buffer (PBS + 0.5%
BSA + 0.1% sodium azide) to remove unbound antibody. Cells were incubated with a secondary PE
labelled anti-human lgG antibody for 15 minutes at RT and then washed with FACS buffer to remove
unbound dy. Cells were analysed by FACS and mean fluorescence intensity (MFI) values
measured to determine binding.
Results showed that chimeric CA8 and all antibodies tested apart from J9M2 bound to ARH77-
hBCMA 10B5 BCMA expressing ectant cells and H929 cells in a dose dependent manner
(Figure 6).
4.5 Demonstration of ability of CA8 and the humanised version J6M0 to neutralise binding of
BAFF or APRIL to recombinant BCMA.
The aim of this assay was to assess the ability of antibody CA8, and humanised version J6M0 in both
wild type and ylated (Potelligent) form, at s concentrations, to neutralise the binding
ability of either BCMA ligand, BAFF or APRIL.
96 well flat bottomed plates were coated overnight with 1pg/mL solution of recombinant human BCMA
Fc 4-53 in PBS. Following a wash step using 0.05% TWEEN20, plates were blocked with 2% Bovine
Serum Albumin solution in PBS for 1 hour at room temperature. Plates were washed as before and
40pL of each antibody e IgG, murine CA8, and chimeric CA8), starting at 10pg/mL, titrated at 1
in 2 in duplicate was added to the relevant wells and incubated for 1hour at room temperature. 40pL
of 2% BSA was added to the relevant control wells. 10pL of either recombinant human BAFF (2149-
BF/CF, R&D Systems) or recombinant human APRIL AP/CF, R&D Systems) was added at
30ng/mL and 750ng/mL respectively, giving a final concentration of 6ng/mL and 150ng/mL
respectively in each well. Equivalent volume of 2% BSA was added to the relevant control wells.
Plates were allowed to incubate for 2 hours at room ature, after which they were washed as
before. Biotinylated uman ligand (BAFF BAF124 or APRIL BAF884, R&D Systems) was added
to the relevant wells at 50ng/mL and incubated for 1 hour. Following a wash step, 50pL of a 1:4000
on of Streptavidin-HRP (Amersham RPN4401) was added to each well and incubated for 30
minutes at room temperature. The wash process was repeated again followed by the addition of
100pL of Tetramethylbenzidine substrate solution (T8665, Sigma) into each well. Plates were
incubated for 20-25 minutes at room temperature, wrapped in foil. The reaction was stopped with the
addition of 100pL of 1M H2804. Optical density was determined at 450nm using omax reader.
See Figure 7A and B.
In a plate based assay for neutralisation of binding of BAFF or APRIL to BCMA, the EC50 values
calculated for ic CA8 were 0.695pg/mL and 0.773pg/mL tively. The values for the
humanised J6M0 were 0.776ng/ml and g/ml. The vaues for the J6M0 potelligent version were
0.748 and 0.616ng/ml respectively. .
4.6 Effect of chimerised CA8 and humanised J6M0 BCMA antibody on BAFF or APRIL induced
phosphorylation of NFkB in H929 cells.
In one set of experiments, H-929 cells were plated at 75,000cells/well in a 96 well plate in serum free
. The chimeric CA8 antibody was added 24 hours later to give final well concentrations up to
200ug/ml. Ten minutes later, BAFF or APRIL ligand were added to the cells to give final well
concentrations of 0.6 or 0.3ug/ml respectively. After 30 minutes the cells were Iysed and
phosphorylated kaappaB levels ed using a MSD pNFkappaB assay.
The chimeric BCMA antibody CA8 neutralised both BAFF and APRIL induced kaappaB cell
ling in H-929 cells. It was particularly potent at neutralising BAFF d kaappaB cell
signalling in this cell type with a mean IC50 of 10nM, compared to 257nM for APRIL induced
kaappaB cell signalling.
Meaned data for 2 experiments
IC50s were 10nM for BAFF induced kaappaB neutralisation and 257nM for APRIL induced
kaappaB lisation (mean of 2 independent experiments) are shown in Table 7.
Table 7
A further set of experiments were carried out to aim to understand why there was such a discrepancy
between the potency in neutralisation of APRIL and BAFF in the cell based system. Following the
discovery of the soluble form of BCMA the experimental design was changed to include a step where
the H929 cells were washed prior to the assay to reduce the interference from the antibody binding to
e BCMA. H-929 cells were washed 3 times to remove any sBCMA and resuspended in serum
free medium. J6M0 potelligent antibody was added to a 96 well plate to give a final well
concentrations up to 100ug/ml along with BAFF or APRIL ligand to give a final well concentration of
0.6 or 0.2 ug/ml respectively. H-929 cells were then plated at 7.5x104cells/well in serum free medium.
s later the cells were Iysed and phosphorylated NFkappaB levels ed using a MSD
paB assay.This is data from one experiment. Each data point is the d of two
replicates.The data from this experiment is shown in Figure 7c. The IC50s for inhibition of BAFF and
APRIL signalling were determined as 0.91ug/ml and 2.43ug/ml respectively.
4.7 ProteOn analysis of anti-BCMA CA8 chimeric and humanised constructs
The initial screen of CA8 chimeric and sed ts was carried out on the ProteOn XPR36
(Biorad). The method was as follows; Protein A was immobilised on a GLC chip (Biorad, Cat No: 176-
5011) by primary amine coupling, CA8 variants were then captured on this surface and recombinant
human BCMA (in house or commercial US Biological, BO410) materials (run 2 only)) passed over at
256, 64, 16, 4, 1nM with a OnM injection (i.e. buffer alone) used to double reference the binding
curves, the buffer used is the HBS—EP buffer. 50mM NaOH was used to regenerate the capture
surface. The data was fitted to the 1:1 model using the analysis software inherent to the ProteOn
XPR36.. Run 1 corresponds to the first screen of humanised CA8 variants (J0 to J5 series) and run 2
to the second screen of humanised CA8 variants (J5 to J9 series). Both runs were carried out at
°C.
The data ed from run1 are set out in Table 8 and data from run 2 are set in Table 9 Several
les in the Run 2 (Table 09) failed to give affinity values measurable by ProteOn, this was due
to the off-rate being beyond the sensitivity of the machine in this assay, this does however indicate
that all these molecules bind tightly to recombinant human BCMA. From Run 1 the data tes that
some constructs did not show any binding to recombinant cyno BCMA,.
Table 8: Run 1-Kinetics analyses of anti-BCMA molecules against Recombinant Human BCMA
Human in house BCMA Cyno in house BCMA
—nnmnnm
Table 9. Run 2-Kinetics analyses of anti-BCMA molecules against Recombinant Human BCMA
For antibodies J8MO, J9MO, J8M1, J9M2, J7M2, J5M0, J7M1, J7M0, J8M2, J9M1, J5M2, J5M1 the off
rate was beyond the sensitivity of the assay hence no data shown.
commercial human
Human in house BCMA BCMA Cyno in house BCMA
KD KD
Sample Name (nM) (nM) KD (nM)
2.51E+0 1.03E 0.41 7.05E+0 9.79E 0.13 5.89E+0
2.17E+0 2.70E 0.12 0 3.75E 0.06 4.88E+0
2.40E+0 7.40E 0.30 6.23E+0 5.37E 0.08 5.64E+0
0 4.06E 0.20 0 3.97E 0.07 4.41E+0
8307118G03 H5L0 No Analysable Binding v weak signal Analysable
S307118G03 H5L1 No Analysable Binding_ _ _ Analysable Binding
V weak Signal
0 1.65E 1.55E+0 1.48E 0.95
No Analysable Binding
S307118G03Chimera 5 -03 3.44 6 -03 6
4.8 BIAcore analysis of anti-BCMA CA8 chimeric and humanised constructs (J7 to J9 series)
Protein A was immobilised on a CM5 chip (GE Healthcare, Cat No: BR30) by primary amine
coupling and this surface was then used to capture the antibody les. Recombinant human
BCMA (US Biological, BO410) was used as e at 256nM, 64nM, 16nM, 4nM and 1nM.
Regeneration of the capture surface was carried out using 50mM NaOH. All binding curves were
double referenced with a buffer injection (i.e. OnM) and the data was fitted to the using the 1:1 model
inherent to T100 evaluation re. The run was carried out at 37°C, using HBS—EP as the running
buffer.
The results showed the molecules tested with the exception of J9M2 bind to recombinant human
BCMA, with similar affinity as the chimeric molecule. Data generated from this experiment are
presented in table 10.
Table 10: cs analysis of anti-BCMA humanised les against Recombinant Human BCMA
Human commercial BCMA Cyno in house BCMA
-nnmn“liimname
humanised 6.77E+05 04 0.442
J9M1 1.96E+07 3.50E-04 0.018
humanised 7.03E+05 3.24E-04 0.46
J9M0 4.95E+06 1.74E-04 0.035
“5306 349504 0'305
3.27E+07 1.18E-03 0.036
humanised 2.82E+05 3.62E-04 1.284
J8M1 2.66E+06 1.34E-04 0.05
3.89E+05 4.18E-04 1.076
244306 126E“ 0052
J8M0
humanised 3.70E+05 3.91E-04 1.057
J7M1 2.35E+06 04 0.056
humanised 05 5.06E-04 1.324
J8M2 2.63E+06 1.50E-04 0.057
humanised 3.46E+05 4.47E-04 1.293
J7M2 2.37E+06 1.35E-04 0.057
humanised 3.21E+05 3.67E-04 1.143
J7M0 2.36E+06 1.51 E-04 0.064
humanised 4.88E+05 2.52E-04 0.515
J9M2 No Anal sable Bindino
4.9 BIAcore analysis of anti-BCMA CA8 chimeric and humanised constructs J6M0 and J9M0
Protein A was immobilised on a CM5 chip (GE Healthcare, Cat No: BR30) by y amine
coupling and this surface was then used to capture the antibody les. Recombinant human
BCMA (US ical, B0410) was used as analyte at 256nM, 64nM, 16nM, 4nM and 1nM.
Regeneration of the capture surface was carried out using 50mM NaOH. All g curves were
double referenced with a buffer injection (i.e. OnM) the data was fitted to the using the 1:1 model
inherent to T100 evaluation software. The run was carried out at 25°C and 37°C for experiment 1 and
only 37°C for experiment 2 using HBS—EP as the running buffer.
The both runs identified J9M0 as the best molecule in term of l affinity to human BCMA. Data
generated from this experiment are presented in table 11.
Table 11 cs analyses of anti-BCMA humanised molecules against Human BCMA
Human commercial BCMA
37°C
1.59E+ 3. 38E- 3. 75E+ 1.58E- 3. 62E+
J9MO 0.02106 0.042
1.01E+ 1.22E- 2.12E+ 1 .48E- 3. 78E+
J6MO 0.12106 0.698
Chimera 1.88E+ 2. 63E- 1. 72E+ 8.72E- 1. 88E+
0. 140 0.051
4.10. ProteOn analysis of new anti-BCMA chimeric constructs
The initial screen of the new chimeric variants from the second batch of hybridomas was carried out
on the ProteOn XPR36 d). The method was as follows; Protein A was immobilised on a GLM
chip (Biorad, Cat No: 176-5012) by y amine coupling, anti-BCMA variants were then captured
on this surface and recombinant human BCMA (in house material) passed over at 256, 64, 16, 4, 1nM
with a OnM injection (i.e. buffer alone) used to double nce the binding curves, the buffer used is
the HBS—EP buffer. Regeneration of the e surface was carried out using 50mM NaOH. The data
was fitted to the 1:1 model using the analysis software inherent to the n XPR36. The run was
carried out at 25°C.
Data generated from this experiment are presented in table 12.
Table 12: Kinetics analyses of anti-BCMA humanised les against Human BCMA
_In house human BCMA
—n“KD (“w
S332110D07 3.11E+05 3.77E-03 12.100
S332121F02 3.73E+05 6.45E-03 17.300
Example 5 Cell Killing Assays.
5.1 ADCC potencies of chimeric CA8 and defucosylated chimeric CA8 n in ARH77 cells
expressing BCMA
Human natural killer (NK) cells were incubated with europium labelled ARH77 BCMA transfected
target cells (1085) in the presence of varying concentrations of antibody at an E:T ratio of 5:1 for 2
hours. Europium release from the target cells was measured and specific lysis calculated.
Result: Chimeric CA8 and defucosylated chimeric CA8 killed BCMA sing target cells via
ADCC. The defucosylated chimeric antibody showed more potent ADCC activity, as ed by a
higher percent lysis achieved with all the target cells tested and a ld lower EC50 on the high
BCMA expressing target cell line 1085, compared to the parent chimeric antibody. See Figure 8A and
BB.
.2 ADCC activity of CA8 humanised antibodies using ARH77 BCMA expressing target cells and
PBMC as effectors
Human PBMC were incubated with europium labelled ARH77 BCMA transfected target cells (1085)
in the presence of varying concentrations of humanised versions of CA8 antibody (5ug/ml to
0.005ug/ml) at an E:T ratio of 5:1 for 2 hours. Europium e from the target cells was measured
and specific lysis calculated.
Result:
Result: All the J5, J6, J7 J8 & J9 series of sed variants of CA8 showed ADCC activity against
the ARH77 high BCMA expressing cell line 10B5 in a dose dependent manner. ADCC was at a
similar level as that found in the experiments using chimeric CA8 molecule. See Figure 9.
.3 ADCC potencies of chimeric S322110F02, S322110D07 and S307118G03 and humanised
S307118G03 H3L0 against ARH77 10B5 cells expressing BCMA with purified NK cells as effector
cells
Human natural killer (NK) target cells were incubated with europium labelled ARH77 BCMA
transfected target cells (10B5) in the presence of varying concentrations of antibody at an E:T ratio of
:1 for 2 hours. um release from the target cells was measured and ic lysis calculated.
: all 4 antibodies tested showed ADCC activity against ARH77 10B5 cells. See Figure 10.
.4 Antibody-Drug Conjugate (ADC) activity of Chimeric CA8 ADCs.
ing ADC activity of chimeric CA8 antibody, chimeric CA8-mcMMAF antibody drug conjugates
and chimeric CA8-chMAE antibody drug conjugates against human multiple myeloma cell lines.
Muliple Myeloma cell lines were treated with chimeric CA8 antibody-drug conjugates to determine the
ADC concentrations required for growth inhibition and death.
The dy drug conjugates tested were added to wells containing multiple myeloma cells at
concentrations ranging from 1ug/ml to . The plates were incubated at 370C for 96 hours at
which point viable cells were tated using Cell titre Glo. The unconjugated chimeric CA8
antibody showed no significant growth inhibitory activity at the antibody concentrations that were
. The chimeric CA8-mcMMAF antibody-drug conjugate showed greater growth inhibitory
activity than the chimeric CA8-chMAE dy-drug conjugate in all 4 of the multiple myeloma cell
lines that were tested. See Figure 11 and Table 13
Table 13 |C50 values represented in ng/mL for the ic CA8-chMAE and the chimeric CA8-
mcMMAF antibody-drug conjugates in 4 different le myeloma cell lines
Multiple Myeloma IC50 (ng/mL)
cell lines
CA8 chimera- CA8 chimerachMAE
mcMMAF
NCl-H929 29.5 8.8
U266-Bl 18.9 9.7
JJN3 21.8 12.4
OPMZ 92.7 58.1
.5 Measuring cell cycle arrest activity of chimeric CA8 antibody, chimeric CA8-mcMMAF
antibody drug conjugates and chimeric CA8-chMAE antibody drug conjugates against human
le a cell line H929.
To determine the mechanism that chimeric CA8 Antibody Drug Conjugates ) cause growth
inhibition in multiple myeloma cells, the cell cycle of NCl-H929 cells was monitored by measuring
cellular DNA content through fixed cell propidium iodide staining at multiple timepoints following
ic CA8 antibody and chimeric CA8 ADC ent.
At the chimeric CA8 ADC concentration tested mL), the chimeric CA8-mcMMAF ADC caused
icant GZ/M cell cycle arrest (4N DNA content) which peaked at 48 hours. At the later timepoints
48, 72 and 96 hours, treatment with the chimeric CA8-mcMMAF ADC resulted in accumulation of a
cell tion with sub-2N DNA content, which is representative of cell death. At the 50ng/mL
concentration tested the chimeric CA8-chMAE ADC had no significant effect on GZ/M cell cycle
arrest or sub-G1 accumulation. See Figure 12.
.6 Phospho-Histone-H3 (Thr11) ng as a marker for chimeric CA8-mcMMAF antibody drug
conjugate and chimeric CA8-chMAE antibody drug conjugate induced mitotic arrest.
To determine if the accumulation of cells with 4N DNA content is a specific result of mitotic arrest
induced by the chimeric CA8 ADCs NCl-H929 cells were stained with an anti-phospho-
Histone H3 antibody following treatment with increasing concentrations of unconjugated chimeric
CA8, chimeric CA8-chMAE or chimeric CA8-mcMMAFfor 48 hours.
Treatment with chimeric CA8 ADCs resulted in a dose-dependent accumulation of 29 cells
that stained positive for 65eroxidi-Histone H3 (Thr11), a specific marker of mitotic cells. The chimeric
CA8-mcMMAF ADC caused accumulation of idi-Histone H3 positive cells at lower
concentrations than the chimeric CA8-chMAE ADC. See Figure 13.
.7 Measuring apoptosis in NCl-H929 cells in response to chimeric CA8 ADCs by staining for
AnneXin V.
To determine if the accumulation of cells with sub-2N DNA content is a specific result of apoptosis
induced by the chimeric CA8 ADCs, NCl-H929 cells were stained with an anti-Annexin-V antibody
following treatment with increasing trations of unconjugated chimeric CA8, chimeric CA8-
chMAE or chimeric CA8-mcMMAFfor 48 hours. Treatment with chimeric CA8 ADCs resulted in a
dose-dependent accumulation of NCl-H929 cells that d positive for Annexin-V, a specific marker
of apoptosis. The chimeric CA8-mcMMAF ADC caused accumulation of Annexin-V ve cells at
lower concentrations than the ic CA8- chMAE ADC. See Figure 14.
.8 Antibody-Drug Conjugate (ADC) activity of humanised variants of CA8 CMA antibody-drug
40 conjugates.
Cells were plated in 96-well plates (4,000 cells per well in 100uL of RPMI + 10% FBS)
Naked antibody or ADC was added 6 hours after cell seeding and plates were incubated for 144
hours. Growth inhibition in the presence of the antibodies or ADCs was measured at 144 hours using
Cell Titre glo. Data points represent the mean of triplicate CellTiterGlo measurements. Error bars
represent standard error.
le Myeloma cell lines 29 and OPM2 were treated with zed CA8 anti-BCMA
antibody-drug conjugates to determine the ADC concentrations required for growth inhibition and
death. The mcMMAF and chMAE antibody-drug conjugate forms of these antibodies showed
significant growth inhibitory activity comparable to that found with the CA8 chimera. Variant J6M0
showed higher potency than the a and data is shown in figure 15 in H929 cells and OPM2
cells.. The mcMMAF antibody-drug conjugate showed greater growth inhibitory activity than the
chMAE antibody-drug conjugate for all antibodies in both cell lines tested. s for all humanized
variants are shown in Table 14.
Table 14. |C50 values represented in ng/mL for the anti BCMA antibody-drug conjugates in NCl-H929
and U266-B1 cells
NCI-H929 OPM2
mcMMAF chMAE mcMMAF chMAE
e Average Average
|C50 |C50 Average |C50 |C50
(ng/mL) (ng/mL) (ng/mL) (ng/mL)
chimera 11.64 37.96 57.04 80.01
CA8 J6M0 5.97 27.67 87.22 121.2
CA8 J6M1 14.6 51.89 205.6 239.9
CA8 J6M2 9.5 39.71 112.9 144.7
CA8 J7M0 18.97 52.25 93.27 127.1
CA8 J7M1 17.87 43.97 95.35 107.5
CA8 J7M2 31.63 55.13 102.6 115.9
CA8 J8M0 15.67 59.94 89.95 132
CA8 J8M1 17.04 46.55 82.96 115.8
CA8 J8M2 15.08 55.98 72.63 124.5
CA8 J9M0 14.95 48.5 58.6 109.8
CA8 J9M1 15.19 55.1 55.88 115
CA8 J9M2 20.87 55.77 80.35 111.7
.9 Antibody-Drug Conjugate (ADC) activity of other murine anti-BCMA dy-drug conjugates.
Cells were plated in 96-well plates (4,000 cells per well in 100uL of RPMI + 10% FBS)
Antibody or ADC was added 6 hours after cell seeding and plates were incubated for 144 hours.
Growth inhibition in the presence of the ADCs was measured at 144 hours using Cell Titre glo. The
mean of triplicate CellTiterGlo measurements are shown. Table 15a and 15b are from experiments
carried out at different times on different series of antibodies. Multiple a cell lines NCl-H929
and U266-B1 were used for dies in Table 15a.
The mcMMAF and chMAE antibody-drug conjugate forms of murine antibodies 0D07,
S332121F02 and S332136E04 showed significant growth inhibitory activity. The mcMMAF antibody-
drug conjugate showed greater growth inhibitory activity than the chMAE dy-drug conjugate in
all of the murine anti-BCMA antibodies tested where activity was seen. |C50 figures are shown in
40 Table 15a. See Figure 16 for dose response curves for these three antibodies and also
8107118G03. Error bars represent standard error. 29, U266-Bl, JJN3 and OPM2 cells for
dies in Table 15b were treated with a ent series of murine anti—BCMA antibody—drug
conjugates to determine the ADC concentrations required for growth inhibition and death. ICSO s
are shown in Table 15b. All 5 antibodies shown on the table had significant ADC activity.
Table 15a. l050 values represented in ng/mL for the anti BCMA antibody—drug conjugates in NCI—
H929 and U266-B1 cells
Antibody NCl-H929 U226-B1
-chMAE -mcMMAF -chMAE -mcMMAF
S322110D07 mlgG1 M Q _m m
$332121 F02 mlgG1 24_.5 Z _ L 2+5
S332126E04 mlgG1 m y _m m
Table 15b IC50 values represented in ng/mL for the anti BCMA antibody-drug conjugates in NCI-
H929, U266-B1, JJN3 and OPM2 cells
NCI-H929 U26631 JJN3 OPM2
Average IC50 chMAE mcMMAF chMAE mcMMAF chMAE mcMMAF chMAE
n lmL
8335115601 _.914 4 2 .
_ m 1_.5 m. m %
S336105A07 M g m E fl & 95_.5
S335122F05 m E m u 29_.5 M M
S335106E08 m L9 m m M M &
S335128A12 w M m M >500 >500 >500
.10 ADCC potency of conjugated, afucosylated JGMO (Potelligent)
Afucosylated J6M0 conjugated to MMAE or MMAF was tested in ADCC assays using BCMA
transfectants to ensure that its ADCC activity was not compromised by the conjugation. Europium
labelled ARH77-1OB5 cells were incubated with various J6M0 WT and Potelligent BCMA antibodies at
trations up to g/ml for 30 minutes prior to the on of PBMCs (PBMC: target cell
ratio 50:1). Two hours later an aliquot of cell media was d and mixed with enhancement
solution. After 30 minutes on a plate shaker, europium release was monitored on the Victor 2 1420
multi-label reader. Datapoints represent means of triplicate values. This data is representative of 2
experiments.
There were no significant ences in ADCC potency between the ugated and ADC forms of
J6M0 Potelligent. In the same experiment a wild type n of J6M0 was included to show how the
potency compares to the afucosylated version. As expected, defucosylation resulted in a lower EC50
and higher l lysis. No lysis was observed with the Fc disabled form of J6M0. (Figure 17)
.11 ADCC potency of afucosylated J6M0 on MM cell lines
Human PBMC were incubated with multiple myeloma target cells at an E:T ratio of 50:1 in presence
of g concentrations of afucosylated (Potelligent) J6M0 The percentage of target cells remaining
in the effector + target cell mixture after 18 hours was measured by FACS using a fluorescently
labelled anti-CD138 antibody to detect the target cells and the percent lysis calculated. This is
representative of several experiments.
J6M0 Potelligent antibody showed ADCC activity against all five multiple myeloma target cell lines
tested. This was important to test since earlier s were carried out using transfected cells.
Results are shown in Figure 18. Full dataset with multiple donors is shown in Table 16 The potencies
were all in a similar range as those found with the transfectants. The ADCC activity was not directly
related to BCMA surface expression on these cell lines.
Table 16 EC50 values generated on 13 independent assays using 11 donors (designated A-K) across
the five multiple myeloma cell lines.
A: AA
Example 6. Xenograft data
6.1 Murine xenografts of human MM cell lines were tested to ensure that antibody potency
detected in vitro can also be demonstrated in vivo. The cell line selected for xenograft studies was
NCI-H929 which is sensitive to ADC and ADCC killing in vitro,. Studies were carried out in
immunocompromised CB.17 SCID mice which lack T and B cells but maintain NK cells to allow for
ADCC ty. However it should be noted that gh human IgG1 can engage murine Fc
receptors, the Potelligent enhancement does not improve the affinity as it does with human Fc
receptors.
6.2 Impact of unconjugated and MMAE or MMAF ated J6M0 on NCI-H929 tumour growth.
In order to independently analyze both the ADCC and ADC activities of J6M0 we tested J6M0
antibody in the presence and e of MMAF or MMAE conjugation. By testing the unconjugated
J6M0, any anti-tumour s could be attributed to some combination of ADDC and functional
inhibitory ty.
Mice with NCI-H929 tumours that had reached a volume of 200 mm3 on average were treated with a
human IgG1 control or the J6M0 antibody (unconjugated, MMAE or MMAF) twice weekly at a dose of
50 ug or 100ug, for 2 weeks. Results from this study show that a 100 ug dose of the MAF
conjugate ed in elimination of tumours in those mice which have completed the dosing. The
J6M0-MMAF mice were maintained for 40 days after the last dose with no recurrence of tumour
ing. These results from this experiment demonstrate that MMAF conjugation had increased
anti-tumour activity over both unconjugated J6M0 antibody and J6M0-MMAE conjugate See Figure
Example 7 Evaluation of e BCMA Levels from MM Patient Serum
7.1 It is currently unknown whether BCMA is present extracellularly and can be detected in the
blood. In this work, we determined the serum level of human BCMA from MM patients. Serum
samples from 54 MM and plasma cell dyscrasia patients and 20 normal control samples were
analyzed by ELISA. Human Subject Approval was obtained from Western Institutional Review Board.
7.2 Assessment of Serum Human BCMA Levels
Blood, from patients and normal controls in the clinic, were ted in serum collection tubes. MM
patient samples were from a variety of stages (progressive disease, remission, relapsed, newly
diagnosed, and others). The Blood samples were spun at 10,000 rpm for 10 s and serum
transferred into sterile micro-centrifuge plastic tubes.
A Human BCMA/TNFRSF17 ELISA kit from R& D Systems (catalog # DY193E) which measures
e human BCMA levels was used to detect BCMA ing the standard protocol supplied with
the kit.
Briefly, 96 well micro-plates were coated with 100u| per well capture dy and incubated overnight
at 40C. The plates were washed three times with wash buffer (0.05% Tween 20 in PBS, pH 7.2) and
blocked with 300ul of 1% BSA in PBS at room ature for 2 hours. The plates were washed three
times with washing buffer. 100ul of serum sample or rd was added into each well and
incubated for 2 hours at room temperature. The plates were washed three times with washing buffer
and then 100ul of the detection antibody was added to each well and incubated 2 hours at room
temperature. 100ul of Streptavidin-HRP was added in each well after washing plates three times and
incubated in dark room for 20 minutes. The plates were washed three times and added 50ul stop
solution and then determined by micro-plate reader with 570nM wavelength.
A series of assays were carried out in order to determine the serum dilution factor appropriate for the
levels of BCMA which were present. A dilution factor of 1:500 was found to be suitable for the majority
of samples and is the dilution factor used in the data shown in Figure 20. The full data set is shown in
Table 17.
Patient and normal control serum samples diluted and run in triplicates had BCMA levels ined.
The serum levels of BCMA were significantly elevated in the sera from MM patients ed with
normal controls in this study.When the disease subset was divided further there was a trend towards
elevated serum levels of BCMA in the sera from progressing MM patients compared with those in
remission.. This is the first report identifying serum BCMA in any human disease and suggests that
these levels may be a novel ker for monitoring disease status and therapeutic se of MM
patients and for other patients with plasma cell mediated diseases.
Table 17. Figures represent serum concentration of soluble BCMA in ng/ml calculated from samples
diluted at 1/50, 1/500 and 1/5000. P values were calculated using the one tailed T-Test and 95%
significance values are below the table.
Myeloma: Myeloma: Myeloma: Myeloma: Other Plasma Cell
1-5000 Normal Progressive Stable Remission Other MGUS sias
14.130 500.804 154.762 151.201 94.457 84.912 22.838
1-500 a: Myeloma: Myeloma: Myeloma: Other Plasma Cell
Triplicate Normal Progressive Stable Remission Other MGUS Dyscrasias
.901 215.877 81.135 43.294 97.584 53.894 22.838
1-500 a: Myeloma: Myeloma: Myeloma: Other Plasma Cell
Single Normal Progressive Stable Remission Other MGUS Dyscrasias
16.620 207.028 61.576 42.796 71.372 40.623 14.099
Myeloma: Myeloma: Myeloma: Myeloma: Other Plasma Cell
1-50 Trial 1 Normal Progressive Stable Remission Other MGUS Dyscrasias
.568 129.544 41.983 40.507 65.120 42.067 51.650
1-50 Myeloma: Myeloma: Myeloma: Myeloma: Other Plasma Cell
Trial 2 Normal ssive Stable Remission Other MGUS Dyscrasias
17.160 119.220 34.567 34.264 54.780 26.333 51.650
P-Values (One Tailed T-Test, 95% Significance)
~1-500 Single
Normal vs Progressive: p=.0010*
ssive vs ion:p=.0146*
~1-500 Triplicate
Normal vs Progressive: p=.0004*
Progressive vs Remission: p=.0091*
~1-50 Trial 1
Normal vs Progressive: p=.0171*
Progressive vs Remission: p=.0777
~1-50 Trial 2
Normal vs Progressive: p=.0184*
Progressive vs Remission: p=.0876
* shows significance
Sequence Summary (Table C)
Description Amino acid ce Polynucleotide
CA8 VH domain (murine) SEQ.I.D.NO:7 SEQ.I.D.NO:8
CA8 VL domain (murine) SEQ.I.D.NO:9 SEQ.I.D.NO:10
CA8 Humanised VH J0 SEQ.I.D.NO:11 SEQ.I.D.NO:12
CA8 Humanised VH J1 SEQ.I.D.NO:13 SEQ.I.D.NO:14
CA8 Humanised VH J2 SEQ.I.D.NO:15 SEQ.I.D.NO:16
CA8 Humanised VH J3 SEQ.I.D.NO:17 SEQ.I.D.NO:18
CA8 Humanised VH J4 SEQ.I.D.NO:19 SEQ.I.D.NO:20
CA8 Humanised VH J5 SEQ.I.D.NO:21 SEQ.I.D.NO:22
CA8 Humanised VH J6 SEQ.I.D.NO:23 SEQ.I.D.NO:24
CA8 Humanised VH J7 SEQ.I.D.NO:25 SEQ.I.D.NO:26
CA8 Humanised VH J8 SEQ.I.D.NO:27 SEQ.I.D.NO:28
CA8 Humanised VH J9 SEQ.I.D.NO:29 SEQ.I.D.NO:3O
CA8 Humanised VL M0 SEQ.I.D. NO:31 D.NO:32
CA8 sed VL M1 SEQ.I.D. NO:33 SEQ.I.D.NO:34
CA8 Humanised VL M2 SEQ.I.D. NO:35 SEQ.I.D.NO:36
Human BCMA SEQ.I.D.NO:37 D.NO:38
CD33-hBCMA ECD (1-53) TEV-Fc
Human BCMA SEQ.I.D.NO:39 SEQ.I.D.NO:4O
CD33-hBCMA ECD (4-53) TEV-Fc
Cyno BCMA SEQ.I.D.NO:41 SEQ.I.D.NO:42
CD33 cyno BCMA ECD (4-52) TEV-Fc
CA8 J5 sed heavy chain D.NO:53 SEQ.|.D.NO:54
CA8 J6 Humanised heavy chain SEQ.|.D.NO:55 D.NO:56
CA8 J7 Humanised heavy chain SEQ.|.D.NO:57 SEQ.|.D.NO:58
CA8 J8 Humanised heavy chain SEQ.|.D.NO:59 SEQ.|.D.NO:60
CA8 J9 Humanised heavy chain SEQ.|.D.NO:61 SEQ.|.D.NO:62
CA8 M0 Humanised light chain D.NO:63 SEQ.|.D.NO:64
CA8 M1 Humanised light chain SEQ.|.D.NO:65 SEQ.|.D.NO:66
CA8 M2 Humanised light chain SEQ.|.D.NO:67 SEQ.|.D.NO:68
S307118GO3 VH domain (murine) SEQ.|.D.NO:69 SEQ.|.D.NO:7O
S307118GO3 VL domain (murine) SEQ.|.D.NO:71 SEQ.|.D.NO:72
S307118G03 heavy chain (chimeric) SEQ.|.D.NO:73 SEQ.|.D.NO:74
S307118G03 light chain(chimeric) SEQ.|.D.NO:75 SEQ.|.D.NO:76
S307118G03 Humanised VH H0 SEQ.|.D.NO:77 SEQ.|.D.NO:78
S307118G03 Humanised VH H1 SEQ.|.D.NO:79 SEQ.|.D.NO:8O
S307118G03 humanised VH H2 SEQ.|.D.NO:81 SEQ.|.D.NO:82
S307118G03 humanised VH H3 SEQ.|.D.NO:83 SEQ.|.D.NO:84
S307118G03 humanised VH H4 SEQ.|.D.NO:85 SEQ.|.D.NO:86
S307118G03 humanised VH H5 SEQ.|.D.NO:87 SEQ.|.D.NO:88
S307118G03 humanised VL L0 SEQ.|.D.NO:89 SEQ.|.D.NO:90
8G03 humanised VL L1 D.NO:91 SEQ.|.D.NO:92
8307118603 CDRH1 SEQ.|.D.NO:93
8307118603 CDRH2 SEQ.|.D.NO:94
8307118603 CDRH3 SEQ.|.D.NO:95
8307118603 CDRL1 SEQ.|.D.NO:96
8307118603 CDRL2 SEQ.|.D.NO:97
8307118603 CDRL3 SEQ.|.D.NO:98
S307118G03 humanised H5 CDRH3 SEQ.|.D.NO:99
S307118GO3 H0 Humanised heavy SEQ.|.D.NO:1OO SEQ.|.D.NO:101
chain
S307118G03 H1 humanised heavy SEQ.|.D.NO:102 SEQ.|.D.NO:103
chain
S307118G03 H2 humanised heavy SEQ.|.D.NO:104 SEQ.|.D.NO:105
chain
8GO3 H3 humanised heavy D.NO:106 SEQ.|.D.NO:107
chain
S307118G03 H4 sed heavy SEQ.|.D.NO:108 SEQ.|.D.NO:109
chain
S307118GO3 H5 sed heavy SEQ.|.D.NO:11O SEQ.|.D.NO:111
chain
2012/059762
8307118603 L0 humanised light chain SEQ.I.D.NO:112 D.NO:113
8307118603 L1 humanised light chain SEQ.I.D.NO:114 SEQ.I.D.NO:115
1F02 murine variable heavy SEQ.I.D.NO:116 SEQ.I.D.NO:117
chain
S332121F02 chimeric variable heavy SEQ.I.D.NO:118 SEQ.I.D.NO:119
chain
S332121F02 murine variable light chain SEQ.I.D.NO:120 D.NO:121
S332121F02 chimeric variable light SEQ.I.D.NO:122 SEQ.I.D.NO:123
chain
S322110D07 murine variable heavy SEQ.I.D.NO:124 SEQ.I.D.NO:125
chain
S322110D07 murine variable light SEQ.I.D.NO:128 SEQ.I.D.NO:129
chain
S332126E04 murine variable heavy SEQ.I.D.NO:132 SEQ.I.D.NO:133
chain
SEQJDNQMO SEQ'I'D'NO:141
S336105A07 murine variable heavy
chain
S335115G01 murine le heavy SEQ.I.D.NO:148 SEQ.I.D.NO:149
833-5122F05 murine variable heavy SEQ.I.D.NO:156 SEQ.I.D.NO:158
c an
S335122F05 Chimeric heavy chain SEQ.I.D.NO:158 SEQ.I.D.NO:159
S335122F05 murine le light chain SEQ.I.D.NO:160 SEQ.I.D.NO:161
S335122F05 Chimeric light chain SEQ.I.D.NO:162 SEQ.I.D.NO:163
S332121F02 CDRH1
SEQ.I.D.NO: 164
S332121F02 CDRH2
SEQ.I.D.NO: 165
\] U‘I
S332121F02 CDRH3
SEQ.|.D.NO: 166
S332121F02 CDRL1
SEQ.|.D.NO: 167
S332121F02 CDRL2
SEQ.|.D.NO: 168
S332121F02 CDRL3
SEQ.|.D.NO: 169
S322110D07 CDRH1
SEQ.|.D.NO: 170
0D07 CDRH2
SEQ.|.D.NO: 171
S322110D07 CDRH3
SEQ.|.D.NO: 172
S322110D07CDRL1
SEQ.|.D.NO: 173
S322110D07 CDRL2
SEQ.|.D.NO: 174
S322110D07 CDRL3
SEQ.|.D.NO: 175
S332126E04CDRH1
SEQ.|.D.NO: 176
S332126E04 CDRH2
SEQ.|.D.NO: 177
S332126E04 CDRH3
SEQ.|.D.NO: 178
S332126E04 CDRL1
SEQ.|.D.NO: 179
S332126E04 CDRL2
SEQ.|.D.NO: 180
S332126E04 CDRL3
SEQ.|.D.NO: 181
S336105A07 CDRH1
SEQ.|.D.NO: 182
S336105A07 CDRH2
SEQ.|.D.NO: 183
S336105A07 CDRH3
SEQ.|.D.NO: 184
5A07 CDRL1
SEQ.|.D.NO: 185
S336105A07 CDRL2
SEQ.|.D.NO: 186
S336105A07 CDRL3
SEQ.|.D.NO: 187
S335115G01 CDRH1
SEQ.|.D.NO: 188
S335115G01 CDRH2
SEQ.|.D.NO: 189
5G01 CDRH3
SEQ.|.D.NO: 190
S335115G01 CDRL1
SEQ.|.D.NO: 191
S335115G01 CDRL2
SEQ.|.D.NO: 192
S335115G01 CDRL3
SEQ.|.D.NO: 193
S335122F05 CDRH1
SEQ.|.D.NO: 194
S335122F05 CDRH2
D.NO: 195
2F05 CDRH3
SEQ.|.D.NO: 196
S335122F05 CDRL1
SEQ.|.D.NO: 197
S335122F05 CDRL2
D.NO: 198
S335122F05 CDRL3
SEQ.|.D.NO: 199
CE LISTING
SEQ ID 1 — CA8 CDRH1
NYWMH
SEQ ID 2 — CA8 CDRH2
ATYRGHSDTYYNQKFKG
SEQ ID 3 — CA8 CDRH3
GAIYNGYDVLDN
SEQ ID 4 — CA8 CDRL1
SASQDISNYLN
SEQ ID 5 — CA8 CDRL2
YTSNLHS
SEQ ID 6 — CA8 CDRL3
QQYRKLPWT
SEQ ID 7 — CA8 VH domain (murine)
EVQLQQSGAVLARPGASVKMSCKGSGYTFTNYWMHWVKQRPGQGLEWIGATYRGHSDTYYNQKF
TAVTSTSTAYMELSSLTNEDSAVYYCTRGAIYNGYDVLDNWGQGTLVTVSS
SEQ ID 8 — CA8 VH domain (murine) (Polynucleotide)
GAGGTGCAGCTGCAGCAGAGCGGCGCCGTGCTGGCCAGGCCCGGAGCTAGCGTGAAGATGAG
CTGCAAGGGCAGCGGCTACACCTTCACCAACTACTGGATGCACTGGGTGAAACAGAGGCCCGG
CCAGGGACTGGAGTGGATCGGCGCCACCTACAGGGGCCACAGCGACACCTACTACAACCAGAA
GTTCAAGGGCAAGGCCAAGCTGACCGCCGTGACCTCAACCAGCACCGCCTACATGGAACTGAG
CAGCCTGACCAACGAGGACAGCGCCGTCTATTACTGCACCAGGGGCGCCATCTACAACGGCTA
CGACGTGCTGGACAATTGGGGCCAGGGAACACTAGTGACCGTGTCCAGC
SEQ ID 9 — CA8 VL domain (murine)
DIQLTQTTSSLSASLGDRVTISCSASQDISNYLNWYQQKPDGTVELVIYYTSNLHSGVPSRFSGSGSG
TDYSLTIGYLEPEDVATYYCQQYRKLPWTFGGGSKLE|KR
SEQ ID 10 — CA8 VL domain (murine) (Polynucleotide)
GATATCCAGCTGACCCAGACCACAAGCAGCCTGAGCGCCTCCCTGGGCGACAGGGTGACCATT
AGCTGCAGCGCCAGCCAGGACATCAGCAACTACCTGAACTGGTACCAGCAGAAGCCCGACGGC
40 ACCGTGGAGCTCGTGATCTACTACACCTCCAACCTGCACAGCGGCGTGCCCAGCAGGTTCTCTG
WO 63805
GCAGCGGCAGCGGCACCGACTACAGCCTGACCATCGGCTATCTGGAGCCCGAGGACGTCGCCA
CCTACTACTGCCAGCAGTACAGGAAGCTGCCCTGGACCTTCGGCGGAGGCTCTAAGCTGGAGA
TTAAGCGT
SEQ ID 11 — CA8 Humanised VH J0
QVQLVQSGAEVKKPGSSVKVSCKASGGTFSNYWMHWVRQAPGQGLEWMGATYRGHSDTYYNQK
FKGRVTITADKSTSTAYMELSSLRSEDTAVYYCARGAIYNGYDVLDNWGQGTLVTVSS
SEQ ID 12 — CA8 Humanised VH J0 (Polynucleotide)
CAGGTGCAGCTGGTCCAGAGCGGCGCCGAAGTGAAGAAGCCCGGCAGCTCCGTGAAAGTGAG
CTGCAAGGCCAGCGGCGGCACCTTCAGCAACTACTGGATGCACTGGGTGAGGCAGGCCCCCG
GACAGGGCCTGGAGTGGATGGGCGCCACCTACAGGGGCCACAGCGACACCTACTACAACCAGA
AGTTCAAGGGCCGGGTGACCATCACCGCCGACAAGAGCACCAGCACCGCCTACATGGAACTGA
GCAGCCTCAGGAGCGAGGACACCGCTGTGTATTACTGCGCCAGGGGCGCCATCTACAACGGCT
ACGACGTGCTGGACAACTGGGGCCAGGGCACACTAGTGACCGTGTCCAGC
SEQ ID 13 — CA8 Humanised VH J1
QVQLVQSGAEVKKPGSSVKVSCKASGYTFTNYWMHWVRQAPGQGLEWMGATYRGHSDTYYNQK
FKGRVTITADKSTSTAYMELSSLRSEDTAVYYCARGAIYNGYDVLDNWGQGTLVTVSS
SEQ ID 14 — CA8 Humanised VH J1 (Polynucleotide)
CAGCTGGTCCAGAGCGGCGCCGAAGTGAAGAAGCCCGGCAGCTCCGTGAAAGTGAG
CTGCAAGGCCAGCGGCTACACCTTCACCAACTACTGGATGCACTGGGTGAGGCAGGCCCCCGG
ACAGGGCCTGGAGTGGATGGGCGCCACCTACAGGGGCCACAGCGACACCTACTACAACCAGAA
GTTCAAGGGCCGGGTGACCATCACCGCCGACAAGAGCACCAGCACCGCCTACATGGAACTGAG
CAGCCTCAGGAGCGAGGACACCGCTGTGTATTACTGCGCCAGGGGCGCCATCTACAACGGCTA
CGACGTGCTGGACAACTGGGGCCAGGGCACACTAGTGACCGTGTCCAGC
SEQ ID 15 — CA8 Humanised VH J2
QVQLVQSGAEVKKPGSSVKVSCKASGYTFTNYWMHWVRQAPGQGLEWMGATYRGHSDTYYNQK
FKGRVTITADKSTSTAYMELSSLRSEDTAVYYCTRGAIYNGYDVLDNWGQGTLVTVSS
SEQ ID 16 — CA8 Humanised VH J2 (Polynucleotide)
CAGGTGCAGCTGGTCCAGAGCGGCGCCGAAGTGAAGAAGCCCGGCAGCTCCGTGAAAGTGAG
CTGCAAGGCCAGCGGCTACACCTTCACCAACTACTGGATGCACTGGGTGAGGCAGGCCCCCGG
ACAGGGCCTGGAGTGGATGGGCGCCACCTACAGGGGCCACAGCGACACCTACTACAACCAGAA
GTTCAAGGGCCGGGTGACCATCACCGCCGACAAGAGCACCAGCACCGCCTACATGGAACTGAG
CAGCCTCAGGAGCGAGGACACCGCTGTGTATTACTGCACCAGGGGCGCCATCTACAACGGCTA
CGACGTGCTGGACAACTGGGGCCAGGGCACACTAGTGACCGTGTCCAGC
SEQ ID 17 — CA8 Humanised VH J3
QVQLVQSGAEVKKPGSSVKVSCKGSGYTFTNYWMHWVRQAPGQGLEWMGATYRGHSDTYYNQK
FKGRVTITADTSTSTAYMELSSLRSEDTAVYYCTRGAIYNGYDVLDNWGQGTLVTVSS
SEQ ID 18 — CA8 Humanised VH J3 (Polynucleotide)
CAGGTGCAGCTGGTCCAGAGCGGCGCCGAAGTGAAGAAGCCCGGCAGCTCCGTGAAAGTGAG
CTGCAAGGGCAGCGGCTACACCTTCACCAACTACTGGATGCACTGGGTGAGGCAGGCCCCCGG
CCTGGAGTGGATGGGCGCCACCTACAGGGGCCACAGCGACACCTACTACAACCAGAA
GTTCAAGGGCCGGGTGACCATCACCGCCGACACGAGCACCAGCACCGCCTACATGGAACTGAG
CAGGAGCGAGGACACCGCTGTGTATTACTGCACCAGGGGCGCCATCTACAACGGCTA
CGACGTGCTGGACAACTGGGGCCAGGGCACACTAGTGACCGTGTCCAGC
SEQ ID 19 — CA8 Humanised VH J4
QVQLVQSGAEVKKPGSSVKVSCKGSGYTFTNYWMHWVRQAPGQGLEWIGATYRGHSDTYYNQKF
KGRATLTADTSTSTAYMELSSLRSEDTAVYYCTRGAIYNGYDVLDNWGQGTLVTVSS
SEQ ID 20 — CA8 Humanised VH J4 (Polynucleotide)
CAGGTGCAGCTGGTCCAGAGCGGCGCCGAAGTGAAGAAGCCCGGCAGCTCCGTGAAAGTGAG
CTGCAAGGGCAGCGGCTACACCTTCACCAACTACTGGATGCACTGGGTGAGGCAGGCCCCCGG
ACAGGGCCTGGAGTGGATCGGCGCCACCTACAGGGGCCACAGCGACACCTACTACAACCAGAA
GTTCAAGGGCCGGGCGACCCTCACCGCCGACACGAGCACCAGCACCGCCTACATGGAACTGAG
CAGGAGCGAGGACACCGCTGTGTATTACTGCACCAGGGGCGCCATCTACAACGGCTA
CGACGTGCTGGACAACTGGGGCCAGGGCACACTAGTGACCGTGTCCAGC
SEQ ID 21 — CA8 Humanised VH J5
QVQLVQSGAEVKKPGSSVKVSCKGSGYTFTNYWMHWVRQAPGQGLEWMGATYRGHSDTYYNQK
FKGRVTITADTSTSTAYMELSSLRSEDTAVYYCTRGAIYDGYDVLDNWGQGTLVTVSS
SEQ ID 22 — CA8 Humanised VH J5 (Polynucleotide)
CAGGTGCAGCTGGTCCAGAGCGGCGCCGAAGTGAAGAAGCCCGGCAGCTCCGTGAAAGTGAG
CTGCAAGGGCAGCGGCTACACCTTCACCAACTACTGGATGCACTGGGTGAGGCAGGCCCCCGG
ACAGGGCCTGGAGTGGATGGGCGCCACCTACAGGGGCCACAGCGACACCTACTACAACCAGAA
GTTCAAGGGCCGGGTGACCATCACCGCCGACACGAGCACCAGCACCGCCTACATGGAACTGAG
CAGCCTCAGGAGCGAGGACACCGCTGTGTATTACTGCACCAGGGGCGCCATCTACGACGGCTA
CGACGTGCTGGACAACTGGGGCCAGGGCACACTAGTGACCGTGTCCAGC
SEQ ID 23 — CA8 Humanised VH J6
QVQLVQSGAEVKKPGSSVKVSCKASGGTFSNYWMHWVRQAPGQGLEWMGATYRGHSDTYYNQK
FKGRVTITADKSTSTAYMELSSLRSEDTAVYYCARGAIYDGYDVLDNWGQGTLVTVSS
SEQ ID 24 — CA8 Humanised VH J6 (Polynucleotide)
CAGGTGCAGCTGGTCCAGAGCGGCGCCGAAGTGAAGAAGCCCGGCAGCTCCGTGAAAGTGAG
CTGCAAGGCCAGCGGCGGCACCTTCAGCAACTACTGGATGCACTGGGTGAGGCAGGCCCCCG
GACAGGGCCTGGAGTGGATGGGCGCCACCTACAGGGGCCACAGCGACACCTACTACAACCAGA
AGTTCAAGGGCCGGGTGACCATCACCGCCGACAAGAGCACCAGCACCGCCTACATGGAACTGA
GCAGCCTCAGGAGCGAGGACACCGCTGTGTATTACTGCGCCAGGGGCGCCATCTACGACGGCT
ACGACGTGCTGGACAACTGGGGCCAGGGCACACTAGTGACCGTGTCCAGC
SEQ ID 25 — CA8 Humanised VH J7
QVQLVQSGAEVKKPGSSVKVSCKASGYTFTNYWMHWVRQAPGQGLEWMGATYRGHSDTYYNQK
FKGRVTITADKSTSTAYMELSSLRSEDTAVYYCARGAIYDGYDVLDNWGQGTLVTVSS
SEQ ID 26 — CA8 Humanised VH J7 (Polynucleotide)
CAGGTGCAGCTGGTCCAGAGCGGCGCCGAAGTGAAGAAGCCCGGCAGCTCCGTGAAAGTGAG
CTGCAAGGCCAGCGGCTACACCTTCACCAACTACTGGATGCACTGGGTGAGGCAGGCCCCCGG
ACAGGGCCTGGAGTGGATGGGCGCCACCTACAGGGGCCACAGCGACACCTACTACAACCAGAA
GTTCAAGGGCCGGGTGACCATCACCGCCGACAAGAGCACCAGCACCGCCTACATGGAACTGAG
CAGCCTCAGGAGCGAGGACACCGCTGTGTATTACTGCGCCAGGGGCGCCATCTACGACGGCTA
CGACGTGCTGGACAACTGGGGCCAGGGCACACTAGTGACCGTGTCCAGC
SEQ ID 27 — CA8 Humanised VH J8
QVQLVQSGAEVKKPGSSVKVSCKASGYTFTNYWMHWVRQAPGQGLEWMGATYRGHSDTYYNQK
FKGRVTITADKSTSTAYMELSSLRSEDTAVYYCTRGAIYDGYDVLDNWGQGTLVTVSS
SEQ ID 28 — CA8 sed VH J8 (Polynucleotide)
CAGGTGCAGCTGGTCCAGAGCGGCGCCGAAGTGAAGAAGCCCGGCAGCTCCGTGAAAGTGAG
CTGCAAGGCCAGCGGCTACACCTTCACCAACTACTGGATGCACTGGGTGAGGCAGGCCCCCGG
ACAGGGCCTGGAGTGGATGGGCGCCACCTACAGGGGCCACAGCGACACCTACTACAACCAGAA
GTTCAAGGGCCGGGTGACCATCACCGCCGACAAGAGCACCAGCACCGCCTACATGGAACTGAG
CAGCCTCAGGAGCGAGGACACCGCTGTGTATTACTGCACCAGGGGCGCCATCTACGACGGCTA
CGACGTGCTGGACAACTGGGGCCAGGGCACACTAGTGACCGTGTCCAGC
SEQ ID 29 — CA8 Humanised VH J9
SGAEVKKPGSSVKVSCKGSGYTFTNYWMHWVRQAPGQGLEWIGATYRGHSDTYYNQKF
KGRATLTADTSTSTAYMELSSLRSEDTAVYYCTRGAIYDGYDVLDNWGQGTLVTVSS
SEQ ID 30 — CA8 Humanised VH J9 ucleotide)
CAGGTGCAGCTGGTCCAGAGCGGCGCCGAAGTGAAGAAGCCCGGCAGCTCCGTGAAAGTGAG
40 CTGCAAGGGCAGCGGCTACACCTTCACCAACTACTGGATGCACTGGGTGAGGCAGGCCCCCGG
ACAGGGCCTGGAGTGGATCGGCGCCACCTACAGGGGCCACAGCGACACCTACTACAACCAGAA
GTTCAAGGGCCGGGCGACCCTCACCGCCGACACGAGCACCAGCACCGCCTACATGGAACTGAG
CAGCCTCAGGAGCGAGGACACCGCTGTGTATTACTGCACCAGGGGCGCCATCTACGACGGCTA
CGACGTGCTGGACAACTGGGGCCAGGGCACACTAGTGACCGTGTCCAGC
SEQ ID 31 — CA8 Humanised VL M0
D|QMTQSPSSLSASVGDRVTITCSASQDISNYLNWYQQKPGKAPKLLIYYTSNLHSGVPSRFSGSGS
GTDFTLTISSLQPEDFATYYCQQYRKLPWTFGQGTKLEIKR
SEQ ID 32 — CA8 Humanised VL M0 (Polynucleotide)
GACATCCAGATGACCCAGAGCCCTAGCTCACTGAGCGCCAGCGTGGGCGACAGGGTGACCATT
ACCTGCTCCGCCAGCCAGGACATCAGCAACTACCTGAACTGGTACCAGCAGAAGCCCGGCAAG
GCCCCCAAGCTGCTGATCTACTACACCTCCAACCTGCACTCCGGCGTGCCCAGCAGGTTCAGCG
GAAGCGGCAGCGGCACCGATTTCACCCTGACCATCTCCAGCCTGCAGCCCGAGGACTTCGCCA
CCTACTACTGCCAGCAGTACAGGAAGCTCCCCTGGACTTTCGGCCAGGGCACCAAACTGGAGAT
CAAGCGT
SEQ ID 33 — CA8 Humanised VL M1
SPSSLSASVGDRVTITCSASQDISNYLNWYQQKPGKAPKLLIYYTSNLHSGVPSRFSGSGS
GTDYTLTISSLQPEDFATYYCQQYRKLPWTFGQGTKLE|KR
SEQ ID 34 — CA8 Humanised VL M1 (Polynucleotide)
GACATCCAGATGACCCAGAGCCCTAGCTCACTGAGCGCCAGCGTGGGCGACAGGGTGACCATT
ACCTGCTCCGCCAGCCAGGACATCAGCAACTACCTGAACTGGTACCAGCAGAAGCCCGGCAAG
GCCCCCAAGCTGCTGATCTACTACACCTCCAACCTGCACTCCGGCGTGCCCAGCAGGTTCAGCG
GAAGCGGCAGCGGCACCGATTACACCCTGACCATCTCCAGCCTGCAGCCCGAGGACTTCGCCA
CCTACTACTGCCAGCAGTACAGGAAGCTCCCCTGGACTTTCGGCCAGGGCACCAAACTGGAGAT
CAAGCGT
SEQ ID 35 — CA8 Humanised VL M2
D|QLTQSPSSLSASVGDRVTITCSASQDISNYLNWYQQKPGKAPELVIYYTSNLHSGVPSRFSGSGSG
ISSLQPEDFATYYCQQYRKLPWTFGQGTKLEIKR
SEQ ID 36 — CA8 Humanised VL M2 (Polynucleotide)
GACATCCAGCTGACCCAGAGCCCTAGCTCACTGAGCGCCAGCGTGGGCGACAGGGTGACCATT
ACCTGCTCCGCCAGCCAGGACATCAGCAACTACCTGAACTGGTACCAGCAGAAGCCCGGCAAG
GCCCCCGAGCTGGTGATCTACTACACCTCCAACCTGCACTCCGGCGTGCCCAGCAGGTTCAGC
GGAAGCGGCAGCGGCACCGATTACACCCTGACCATCTCCAGCCTGCAGCCCGAGGACTTCGCC
TACTGCCAGCAGTACAGGAAGCTCCCCTGGACTTTCGGCCAGGGCACCAAACTGGAGA
40 TCAAGCGT
SEQ ID 37 — Human BCMA CD33-hBCMA ECD (1-53) TEV-FC
MPLLLLLPLLWAGALAMLQMAGQCSQNEYFDSLLHACIPCQLRCSSNTPPLTCQRYCNASVTNSVKG
TNSGENLYFQGDPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVWDVSHEDP
EVKFNWWDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISK
AKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFF
LYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
SEQID38—HumanBCMACD33hBCMAECD($531EVJ%(PdwmdemMe)
ATGCCGCTGCTGCTACTGCTGCCCCTGCTGTGGGCAGGGGCGCTAGCTATGCTGCAGATGGCC
GGCCAGTGCAGCCAGAACGAGTACTTCGACAGCCTGCTGCACGCCTGCATCCCCTGCCAGCTG
AGATGCAGCAGCAACACACCTCCTCTGACCTGCCAGAGATACTGCAACGCCAGCGTGACCAACA
AGGGCACCAACTCCGGAGAGAACCTGTACTTCCAAGGGGATCCCAAATCTTGTGACAA
AACTCACACATGCCCACCGTGCCCAGCACCTGAACTCCTGGGGGGACCGTCAGTCTTCCTCTTC
AAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTG
GACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCAT
AATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTC
ACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCC
TCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAGCCACAGGTGT
ACACCCTGCCCCCATCCCGGGATGAGCTGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCA
AAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACT
ACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGT
GGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCA
CAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAA
SEQ ID 39— Human BCMA CD33-hBCMA ECD (4-53) TEV-FC
MPLLLLLPLLWAGALAMAGQCSQNEYFDSLLHACIPCQLRCSSNTPPLTCQRYCNASVTNSVKGTNS
GENLYFQGDPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVK
FNWWDGVEVHNAKTKPREEQYNSTYRWSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKG
QPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYS
KLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
SEQ ID 40 — Human BCMA CD33-hBCMA ECD (4-53) TEV-FC ucleotide)
ATGCCGCTGCTGCTACTGCTGCCCCTGCTGTGGGCAGGGGCGCTAGCTATGGCCGGCCAGTGC
AGCCAGAACGAGTACTTCGACAGCCTGCTGCACGCCTGCATCCCCTGCCAGCTGAGATGCAGC
AGCAACACACCTCCTCTGACCTGCCAGAGATACTGCAACGCCAGCGTGACCAACAGCGTGAAGG
GCACCAACTCCGGAGAGAACCTGTACTTCCAAGGGGATCCCAAATCTTGTGACAAAACTCACAC
40 ATGCCCACCGTGCCCAGCACCTGAACTCCTGGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAA
CCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGC
CACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAG
ACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTG
CACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCC
CCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAGCCACAGGTGTACACCCTG
CCCCCATCCCGGGATGAGCTGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCT
ATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCA
CGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAG
CAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTAC
ACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAA
SEQ ID 41— Cynomolgous BCMA CD33 cyno BCMA ECD (4-52) TEV-Fc
MPLUJLPLUNAGALAMARQCSQNEYFDSLLHDCKPCQLRCSSTPPLTCQRYCNASMTNSVKGMNS
QGDPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTUWSRTPEVTCVVVDVSHEDPEVK
FNWYVDGVEVHNAKTKPREEQYNSTYRWSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKG
VYTLPPSRDELTKNQVSLTCLVKGFYPSDVfiflflNESNGQPENNYKTTPPVLDSDGSFFLYS
KLTVDKSRWKMQGNVFSCSVMHEALHNHYTQKSLSLSPGK
SEQ ID 42 — Cynomolgous BCMA CD33 cyno BCMA ECD (4-52) TEV-Fc (Polynucleotide)
ATGCCGCTGCTGCTACTGCTGCCCCTGCTGTGGGCAGGGGCGCTAGCTATGGCCAGACAGTGC
AGCCAGAACGAGTACTTCGACAGCCTGCTGCACGACTGCAAGCCCTGCCAGCTGAGATGCAGC
AGCACACCTCCTCTGACCTGCCAGAGATACTGCAACGCCAGCATGACCAACAGCGTGAAGGGCA
TGAACTCCGGAGAGAACCTGTACTTCCAAGGGGATCCCAAATCTTGTGACAAAACTCACACATGC
CCACCGTGCCCAGCACCTGAACTCCTGGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCA
AGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACG
AAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAA
GCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCA
GGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATC
ACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAGCCACAGGTGTACACCCTGCCCCCA
TCCCGGGATGAGCTGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCA
GCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTC
CCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTG
GCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAG
AAGAGCCTCTCCCTGTCTCCGGGTAAA
SEQ ID 43— CA8 J0 Humanised heavy chain
QVQLVQSGAEVKKPGSSVKVSCKASGGTFSNYMMMfiNVRQAPGQGLEMHWGATYRGHSDTYYNQK
FKGRVHTADKSTSTAYMELSSLRSEDTAVYYCARGAWNGYDVUNMNGQGTLVTVSSASTKGPSVF
40 PLAPSSKSTSGGTAALGCLVKDYFPEPVTVSNNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSL
GTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTC
WVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNK
ALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTT
PPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
SEQ ID 44 — CA8 J0 Humanised heavy chain (Polynucleotide)
CAGGTGCAGCTGGTCCAGAGCGGCGCCGAAGTGAAGAAGCCCGGCAGCTCCGTGAAAGTGAG
CTGCAAGGCCAGCGGCGGCACCTTCAGCAACTACTGGATGCACTGGGTGAGGCAGGCCCCCG
GACAGGGCCTGGAGTGGATGGGCGCCACCTACAGGGGCCACAGCGACACCTACTACAACCAGA
AGTTCAAGGGCCGGGTGACCATCACCGCCGACAAGAGCACCAGCACCGCCTACATGGAACTGA
TCAGGAGCGAGGACACCGCTGTGTATTACTGCGCCAGGGGCGCCATCTACAACGGCT
ACGACGTGCTGGACAACTGGGGCCAGGGCACACTAGTGACCGTGTCCAGCGCCAGCACCAAGG
GCGTGTTCCCCCTGGCCCCCAGCAGCAAGAGCACCAGCGGCGGCACAGCCGCCCTG
GGCTGCCTGGTGAAGGACTACTTCCCCGAACCGGTGACCGTGTCCTGGAACAGCGGAGCCCTG
ACCAGCGGCGTGCACACCTTCCCCGCCGTGCTGCAGAGCAGCGGCCTGTACAGCCTGAGCAGC
GTGGTGACCGTGCCCAGCAGCAGCCTGGGCACCCAGACCTACATCTGTAACGTGAACCACAAG
AACACCAAGGTGGACAAGAAGGTGGAGCCCAAGAGCTGTGACAAGACCCACACCTGC
CCCCCCTGCCCTGCCCCCGAGCTGCTGGGAGGCCCCAGCGTGTTCCTGTTCCCCCCCAAGCCT
AAGGACACCCTGATGATCAGCAGAACCCCCGAGGTGACCTGTGTGGTGGTGGATGTGAGCCAC
GAGGACCCTGAGGTGAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCACAATGCCAAGACC
AAGCCCAGGGAGGAGCAGTACAACAGCACCTACCGGGTGGTGTCCGTGCTGACCGTGCTGCAC
CAGGATTGGCTGAACGGCAAGGAGTACAAGTGTAAGGTGTCCAACAAGGCCCTGCCTGCCCCTA
TCGAGAAAACCATCAGCAAGGCCAAGGGCCAGCCCAGAGAGCCCCAGGTGTACACCCTGCCCC
CTAGCAGAGATGAGCTGACCAAGAACCAGGTGTCCCTGACCTGCCTGGTGAAGGGCTTCTACCC
CAGCGACATCGCCGTGGAGTGGGAGAGCAACGGCCAGCCCGAGAACAACTACAAGACCACCCC
CCCTGTGCTGGACAGCGATGGCAGCTTCTTCCTGTACAGCAAGCTGACCGTGGACAAGAGCAGA
TGGCAGCAGGGCAACGTGTTCAGCTGCTCCGTGATGCACGAGGCCCTGCACAATCACTACACC
CAGAAGAGCCTGAGCCTGTCCCCTGGCAAG
SEQ ID 45— CA8 J1 Humanised heavy chain
QVQLVQSGAEVKKPGSSVKVSCKASGYTFTNYWMHWVRQAPGQGLEWMGATYRGHSDTYYNQK
FKGRVHTADKSTSTAYMELSSLRSEDTAVYYCARGAWNGYDVUNMNGQGTLVTVSSASTKGPSVF
PLAPSSKSTSGGTAALGCLVKDYFPEPVTVSNNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSL
GTQTWCNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTUWSRTPEVTC
HEDPEVKHmNYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWlNGKEYKCKVSNK
ALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTT
PPVLDSDGSFFLYSKLTVDKSRWKMDGNVFSCSVMHEALHNHYTQKSLSLSPGK
SEQ ID 46 — CA8 J1 Humanised heavy chain (Polynucleotide)
CAGCTGGTCCAGAGCGGCGCCGAAGTGAAGAAGCCCGGCAGCTCCGTGAAAGTGAG
CTGCAAGGCCAGCGGCTACACCTTCACCAACTACTGGATGCACTGGGTGAGGCAGGCCCCCGG
ACAGGGCCTGGAGTGGATGGGCGCCACCTACAGGGGCCACAGCGACACCTACTACAACCAGAA
GTTCAAGGGCCGGGTGACCATCACCGCCGACAAGAGCACCAGCACCGCCTACATGGAACTGAG
CAGCCTCAGGAGCGAGGACACCGCTGTGTATTACTGCGCCAGGGGCGCCATCTACAACGGCTA
CGACGTGCTGGACAACTGGGGCCAGGGCACACTAGTGACCGTGTCCAGCGCCAGCACCAAGG
GCCCCAGCGTGTTCCCCCTGGCCCCCAGCAGCAAGAGCACCAGCGGCGGCACAGCCGCCCTG
GGCTGCCTGGTGAAGGACTACTTCCCCGAACCGGTGACCGTGTCCTGGAACAGCGGAGCCCTG
ACCAGCGGCGTGCACACCTTCCCCGCCGTGCTGCAGAGCAGCGGCCTGTACAGCCTGAGCAGC
ACCGTGCCCAGCAGCAGCCTGGGCACCCAGACCTACATCTGTAACGTGAACCACAAG
CCCAGCAACACCAAGGTGGACAAGAAGGTGGAGCCCAAGAGCTGTGACAAGACCCACACCTGC
CCCCCCTGCCCTGCCCCCGAGCTGCTGGGAGGCCCCAGCGTGTTCCTGTTCCCCCCCAAGCCT
AAGGACACCCTGATGATCAGCAGAACCCCCGAGGTGACCTGTGTGGTGGTGGATGTGAGCCAC
GAGGACCCTGAGGTGAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCACAATGCCAAGACC
AAGCCCAGGGAGGAGCAGTACAACAGCACCTACCGGGTGGTGTCCGTGCTGACCGTGCTGCAC
CAGGATTGGCTGAACGGCAAGGAGTACAAGTGTAAGGTGTCCAACAAGGCCCTGCCTGCCCCTA
TCGAGAAAACCATCAGCAAGGCCAAGGGCCAGCCCAGAGAGCCCCAGGTGTACACCCTGCCCC
GAGATGAGCTGACCAAGAACCAGGTGTCCCTGACCTGCCTGGTGAAGGGCTTCTACCC
CAGCGACATCGCCGTGGAGTGGGAGAGCAACGGCCAGCCCGAGAACAACTACAAGACCACCCC
CCCTGTGCTGGACAGCGATGGCAGCTTCTTCCTGTACAGCAAGCTGACCGTGGACAAGAGCAGA
TGGCAGCAGGGCAACGTGTTCAGCTGCTCCGTGATGCACGAGGCCCTGCACAATCACTACACC
CAGAAGAGCCTGAGCCTGTCCCCTGGCAAG
SEQ ID 47 — CA8 J2 Humanised heavy chain
QVQLVQSGAEVKKPGSSVKVSCKASGYTFTNYWMHWVRQAPGQGLEWMGATYRGHSDTYYNQK
FKGRVWTADKSTSTAYMELSSLRSEDTAVYYCTRGAWNGYDVUNMNGQGTLVTVSSASTKGPSVF
PLAPSSKSTSGGTAALGCLVKDYFPEPVTVSNNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSL
GTQTWCNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTUWSRTPEVTC
VVVDVSHEDPEVKHmNYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWlNGKEYKCKVSNK
ALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTT
PPVLDSDGSFFLYSKLTVDKSRWKMDGNVFSCSVMHEALHNHYTQKSLSLSPGK
SEQ ID 48 — CA8 J2 Humanised heavy chain (Polynucleotide)
CAGGTGCAGCTGGTCCAGAGCGGCGCCGAAGTGAAGAAGCCCGGCAGCTCCGTGAAAGTGAG
CTGCAAGGCCAGCGGCTACACCTTCACCAACTACTGGATGCACTGGGTGAGGCAGGCCCCCGG
ACAGGGCCTGGAGTGGATGGGCGCCACCTACAGGGGCCACAGCGACACCTACTACAACCAGAA
GGGCCGGGTGACCATCACCGCCGACAAGAGCACCAGCACCGCCTACATGGAACTGAG
CAGCCTCAGGAGCGAGGACACCGCTGTGTATTACTGCACCAGGGGCGCCATCTACAACGGCTA
CGACGTGCTGGACAACTGGGGCCAGGGCACACTAGTGACCGTGTCCAGCGCCAGCACCAAGG
40 GCCCCAGCGTGTTCCCCCTGGCCCCCAGCAGCAAGAGCACCAGCGGCGGCACAGCCGCCCTG
GGCTGCCTGGTGAAGGACTACTTCCCCGAACCGGTGACCGTGTCCTGGAACAGCGGAGCCCTG
GGCGTGCACACCTTCCCCGCCGTGCTGCAGAGCAGCGGCCTGTACAGCCTGAGCAGC
GTGGTGACCGTGCCCAGCAGCAGCCTGGGCACCCAGACCTACATCTGTAACGTGAACCACAAG
CCCAGCAACACCAAGGTGGACAAGAAGGTGGAGCCCAAGAGCTGTGACAAGACCCACACCTGC
CCCCCCTGCCCTGCCCCCGAGCTGCTGGGAGGCCCCAGCGTGTTCCTGTTCCCCCCCAAGCCT
AAGGACACCCTGATGATCAGCAGAACCCCCGAGGTGACCTGTGTGGTGGTGGATGTGAGCCAC
CCTGAGGTGAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCACAATGCCAAGACC
AAGCCCAGGGAGGAGCAGTACAACAGCACCTACCGGGTGGTGTCCGTGCTGACCGTGCTGCAC
CAGGATTGGCTGAACGGCAAGGAGTACAAGTGTAAGGTGTCCAACAAGGCCCTGCCTGCCCCTA
TCGAGAAAACCATCAGCAAGGCCAAGGGCCAGCCCAGAGAGCCCCAGGTGTACACCCTGCCCC
CTAGCAGAGATGAGCTGACCAAGAACCAGGTGTCCCTGACCTGCCTGGTGAAGGGCTTCTACCC
CAGCGACATCGCCGTGGAGTGGGAGAGCAACGGCCAGCCCGAGAACAACTACAAGACCACCCC
CCCTGTGCTGGACAGCGATGGCAGCTTCTTCCTGTACAGCAAGCTGACCGTGGACAAGAGCAGA
TGGCAGCAGGGCAACGTGTTCAGCTGCTCCGTGATGCACGAGGCCCTGCACAATCACTACACC
CAGAAGAGCCTGAGCCTGTCCCCTGGCAAG
SEQ ID 49— CA8 J3 Humanised heavy chain
QVQLVQSGAEVKKPGSSVKVSCKGSGYTFTNYMMMflNVRQAPGQGUENMGATYRGHSDTYYNQK
FKGRVTITADTSTSTAYMELSSLRSEDTAVYYCTRGAIYNGYDVLDNWGQGTLVTVSSASTKGPSVF
PLAPSSKSTSGGTAALGCLVKDYFPEPVTVSNNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSL
GTQTWCNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTUWSRTPEVTC
VVVDVSHEDPEVKHmNYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWlNGKEYKCKVSNK
ALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTT
PPVLDSDGSFFLYSKLTVDKSRWKMDGNVFSCSVMHEALHNHYTQKSLSLSPGK
SEQ ID 50 — CA8 J3 Humanised heavy chain ucleotide)
CAGGTGCAGCTGGTCCAGAGCGGCGCCGAAGTGAAGAAGCCCGGCAGCTCCGTGAAAGTGAG
CTGCAAGGGCAGCGGCTACACCTTCACCAACTACTGGATGCACTGGGTGAGGCAGGCCCCCGG
ACAGGGCCTGGAGTGGATGGGCGCCACCTACAGGGGCCACAGCGACACCTACTACAACCAGAA
GTTCAAGGGCCGGGTGACCATCACCGCCGACACGAGCACCAGCACCGCCTACATGGAACTGAG
CAGCCTCAGGAGCGAGGACACCGCTGTGTATTACTGCACCAGGGGCGCCATCTACAACGGCTA
CGACGTGCTGGACAACTGGGGCCAGGGCACACTAGTGACCGTGTCCAGCGCCAGCACCAAGG
GCCCCAGCGTGTTCCCCCTGGCCCCCAGCAGCAAGAGCACCAGCGGCGGCACAGCCGCCCTG
GGCTGCCTGGTGAAGGACTACTTCCCCGAACCGGTGACCGTGTCCTGGAACAGCGGAGCCCTG
ACCAGCGGCGTGCACACCTTCCCCGCCGTGCTGCAGAGCAGCGGCCTGTACAGCCTGAGCAGC
ACCGTGCCCAGCAGCAGCCTGGGCACCCAGACCTACATCTGTAACGTGAACCACAAG
CCCAGCAACACCAAGGTGGACAAGAAGGTGGAGCCCAAGAGCTGTGACAAGACCCACACCTGC
CCCCCCTGCCCTGCCCCCGAGCTGCTGGGAGGCCCCAGCGTGTTCCTGTTCCCCCCCAAGCCT
AAGGACACCCTGATGATCAGCAGAACCCCCGAGGTGACCTGTGTGGTGGTGGATGTGAGCCAC
40 GAGGACCCTGAGGTGAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCACAATGCCAAGACC
AAGCCCAGGGAGGAGCAGTACAACAGCACCTACCGGGTGGTGTCCGTGCTGACCGTGCTGCAC
CAGGATTGGCTGAACGGCAAGGAGTACAAGTGTAAGGTGTCCAACAAGGCCCTGCCTGCCCCTA
TCGAGAAAACCATCAGCAAGGCCAAGGGCCAGCCCAGAGAGCCCCAGGTGTACACCCTGCCCC
CTAGCAGAGATGAGCTGACCAAGAACCAGGTGTCCCTGACCTGCCTGGTGAAGGGCTTCTACCC
CAGCGACATCGCCGTGGAGTGGGAGAGCAACGGCCAGCCCGAGAACAACTACAAGACCACCCC
CCCTGTGCTGGACAGCGATGGCAGCTTCTTCCTGTACAGCAAGCTGACCGTGGACAAGAGCAGA
TGGCAGCAGGGCAACGTGTTCAGCTGCTCCGTGATGCACGAGGCCCTGCACAATCACTACACC
CAGAAGAGCCTGAGCCTGTCCCCTGGCAAG
SEQ ID 51 — CA8 J4 Humanised heavy chain
QVQLVQSGAEVKKPGSSVKVSCKGSGYTFTNYMMMflNVRQAPGQGLEMHGATYRGHSDTYYNQKF
KGRATLTADTSTSTAYMELSSLRSEDTAVYYCTRGAIYNGYDVLDNWGQGTLVTVSSASTKGPSVFP
LAPSSKSTSGGTAALGCLVKDYFPEPVTVSVWVSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLG
TQTWCNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTUWSRTPEVTCV
VVDVSHEDPEVKHMNYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDMANGKEYKCKVSNKA
LPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTP
PVLDSDGSFFLYSKLTVDKSRWKDQGNVFSCSVMHEALHNHYTQKSLSLSPGK
SEQ ID 52 — CA8 J4 Humanised heavy chain (Polynucleotide)
CAGGTGCAGCTGGTCCAGAGCGGCGCCGAAGTGAAGAAGCCCGGCAGCTCCGTGAAAGTGAG
CTGCAAGGGCAGCGGCTACACCTTCACCAACTACTGGATGCACTGGGTGAGGCAGGCCCCCGG
ACAGGGCCTGGAGTGGATCGGCGCCACCTACAGGGGCCACAGCGACACCTACTACAACCAGAA
GTTCAAGGGCCGGGCGACCCTCACCGCCGACACGAGCACCAGCACCGCCTACATGGAACTGAG
CAGCCTCAGGAGCGAGGACACCGCTGTGTATTACTGCACCAGGGGCGCCATCTACAACGGCTA
CGACGTGCTGGACAACTGGGGCCAGGGCACACTAGTGACCGTGTCCAGCGCCAGCACCAAGG
GCGTGTTCCCCCTGGCCCCCAGCAGCAAGAGCACCAGCGGCGGCACAGCCGCCCTG
GGCTGCCTGGTGAAGGACTACTTCCCCGAACCGGTGACCGTGTCCTGGAACAGCGGAGCCCTG
GGCGTGCACACCTTCCCCGCCGTGCTGCAGAGCAGCGGCCTGTACAGCCTGAGCAGC
ACCGTGCCCAGCAGCAGCCTGGGCACCCAGACCTACATCTGTAACGTGAACCACAAG
CCCAGCAACACCAAGGTGGACAAGAAGGTGGAGCCCAAGAGCTGTGACAAGACCCACACCTGC
CCCCCCTGCCCTGCCCCCGAGCTGCTGGGAGGCCCCAGCGTGTTCCTGTTCCCCCCCAAGCCT
AAGGACACCCTGATGATCAGCAGAACCCCCGAGGTGACCTGTGTGGTGGTGGATGTGAGCCAC
GAGGACCCTGAGGTGAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCACAATGCCAAGACC
AAGCCCAGGGAGGAGCAGTACAACAGCACCTACCGGGTGGTGTCCGTGCTGACCGTGCTGCAC
CAGGATTGGCTGAACGGCAAGGAGTACAAGTGTAAGGTGTCCAACAAGGCCCTGCCTGCCCCTA
TCGAGAAAACCATCAGCAAGGCCAAGGGCCAGCCCAGAGAGCCCCAGGTGTACACCCTGCCCC
GAGATGAGCTGACCAAGAACCAGGTGTCCCTGACCTGCCTGGTGAAGGGCTTCTACCC
CAGCGACATCGCCGTGGAGTGGGAGAGCAACGGCCAGCCCGAGAACAACTACAAGACCACCCC
CCCTGTGCTGGACAGCGATGGCAGCTTCTTCCTGTACAGCAAGCTGACCGTGGACAAGAGCAGA
TGGCAGCAGGGCAACGTGTTCAGCTGCTCCGTGATGCACGAGGCCCTGCACAATCACTACACC
CAGAAGAGCCTGAGCCTGTCCCCTGGCAAG
SEQ ID 53 — CA8 J5 Humanised heavy chain
QVQLVQSGAEVKKPGSSVKVSCKGSGYTFTNYMMMflNVRQAPGQGUENMGATYRGHSDTYYNQK
FKGRVTITADTSTSTAYMELSSLRSEDTAVYYCTRGAIYDGYDVLDNWGQGTLVTVSSASTKGPSVF
KSTSGGTAALGCLVKDYFPEPVTVSNNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSL
NVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTUWSRTPEVTC
VVVDVSHEDPEVKHmNYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWlNGKEYKCKVSNK
ALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTT
PPVLDSDGSFFLYSKLTVDKSRWKMDGNVFSCSVMHEALHNHYTQKSLSLSPGK
SEQ ID 54 — CA8 J5 Humanised heavy chain (Polynucleotide)
CAGGTGCAGCTGGTCCAGAGCGGCGCCGAAGTGAAGAAGCCCGGCAGCTCCGTGAAAGTGAG
CTGCAAGGGCAGCGGCTACACCTTCACCAACTACTGGATGCACTGGGTGAGGCAGGCCCCCGG
CCTGGAGTGGATGGGCGCCACCTACAGGGGCCACAGCGACACCTACTACAACCAGAA
GTTCAAGGGCCGGGTGACCATCACCGCCGACACGAGCACCAGCACCGCCTACATGGAACTGAG
CAGCCTCAGGAGCGAGGACACCGCTGTGTATTACTGCACCAGGGGCGCCATCTACGACGGCTA
CGACGTGCTGGACAACTGGGGCCAGGGCACACTAGTGACCGTGTCCAGCGCCAGCACCAAGG
GCCCCAGCGTGTTCCCCCTGGCCCCCAGCAGCAAGAGCACCAGCGGCGGCACAGCCGCCCTG
GGCTGCCTGGTGAAGGACTACTTCCCCGAACCGGTGACCGTGTCCTGGAACAGCGGAGCCCTG
GGCGTGCACACCTTCCCCGCCGTGCTGCAGAGCAGCGGCCTGTACAGCCTGAGCAGC
GTGGTGACCGTGCCCAGCAGCAGCCTGGGCACCCAGACCTACATCTGTAACGTGAACCACAAG
CCCAGCAACACCAAGGTGGACAAGAAGGTGGAGCCCAAGAGCTGTGACAAGACCCACACCTGC
CCCCCCTGCCCTGCCCCCGAGCTGCTGGGAGGCCCCAGCGTGTTCCTGTTCCCCCCCAAGCCT
AAGGACACCCTGATGATCAGCAGAACCCCCGAGGTGACCTGTGTGGTGGTGGATGTGAGCCAC
GAGGACCCTGAGGTGAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCACAATGCCAAGACC
AAGCCCAGGGAGGAGCAGTACAACAGCACCTACCGGGTGGTGTCCGTGCTGACCGTGCTGCAC
CAGGATTGGCTGAACGGCAAGGAGTACAAGTGTAAGGTGTCCAACAAGGCCCTGCCTGCCCCTA
TCGAGAAAACCATCAGCAAGGCCAAGGGCCAGCCCAGAGAGCCCCAGGTGTACACCCTGCCCC
CTAGCAGAGATGAGCTGACCAAGAACCAGGTGTCCCTGACCTGCCTGGTGAAGGGCTTCTACCC
CAGCGACATCGCCGTGGAGTGGGAGAGCAACGGCCAGCCCGAGAACAACTACAAGACCACCCC
CCCTGTGCTGGACAGCGATGGCAGCTTCTTCCTGTACAGCAAGCTGACCGTGGACAAGAGCAGA
TGGCAGCAGGGCAACGTGTTCAGCTGCTCCGTGATGCACGAGGCCCTGCACAATCACTACACC
CAGAAGAGCCTGAGCCTGTCCCCTGGCAAG
SEQ ID 55 — CA8 J6 Humanised heavy chain
QVQLVQSGAEVKKPGSSVKVSCKASGGTFSNYMMMhNVRQAPGQGLEMHWGATYRGHSDTYYNQK
FKGRVHTADKSTSTAYMELSSLRSEDTAVYYCARGAWDGYDVUNMNGQGTLVTVSSASTKGPSVF
40 PLAPSSKSTSGGTAALGCLVKDYFPEPVTVSNNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSL
CNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTC
WVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNK
ALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTT
PPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
SEQ ID 56 — CA8 J6 Humanised heavy chain (Polynucleotide)
CAGGTGCAGCTGGTCCAGAGCGGCGCCGAAGTGAAGAAGCCCGGCAGCTCCGTGAAAGTGAG
CTGCAAGGCCAGCGGCGGCACCTTCAGCAACTACTGGATGCACTGGGTGAGGCAGGCCCCCG
GACAGGGCCTGGAGTGGATGGGCGCCACCTACAGGGGCCACAGCGACACCTACTACAACCAGA
AGTTCAAGGGCCGGGTGACCATCACCGCCGACAAGAGCACCAGCACCGCCTACATGGAACTGA
GCAGCCTCAGGAGCGAGGACACCGCTGTGTATTACTGCGCCAGGGGCGCCATCTACGACGGCT
ACGACGTGCTGGACAACTGGGGCCAGGGCACACTAGTGACCGTGTCCAGCGCCAGCACCAAGG
GCCCCAGCGTGTTCCCCCTGGCCCCCAGCAGCAAGAGCACCAGCGGCGGCACAGCCGCCCTG
GGCTGCCTGGTGAAGGACTACTTCCCCGAACCGGTGACCGTGTCCTGGAACAGCGGAGCCCTG
GGCGTGCACACCTTCCCCGCCGTGCTGCAGAGCAGCGGCCTGTACAGCCTGAGCAGC
GTGGTGACCGTGCCCAGCAGCAGCCTGGGCACCCAGACCTACATCTGTAACGTGAACCACAAG
CCCAGCAACACCAAGGTGGACAAGAAGGTGGAGCCCAAGAGCTGTGACAAGACCCACACCTGC
CCCCCCTGCCCTGCCCCCGAGCTGCTGGGAGGCCCCAGCGTGTTCCTGTTCCCCCCCAAGCCT
AAGGACACCCTGATGATCAGCAGAACCCCCGAGGTGACCTGTGTGGTGGTGGATGTGAGCCAC
GAGGACCCTGAGGTGAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCACAATGCCAAGACC
AAGCCCAGGGAGGAGCAGTACAACAGCACCTACCGGGTGGTGTCCGTGCTGACCGTGCTGCAC
CAGGATTGGCTGAACGGCAAGGAGTACAAGTGTAAGGTGTCCAACAAGGCCCTGCCTGCCCCTA
TCGAGAAAACCATCAGCAAGGCCAAGGGCCAGCCCAGAGAGCCCCAGGTGTACACCCTGCCCC
CTAGCAGAGATGAGCTGACCAAGAACCAGGTGTCCCTGACCTGCCTGGTGAAGGGCTTCTACCC
CAGCGACATCGCCGTGGAGTGGGAGAGCAACGGCCAGCCCGAGAACAACTACAAGACCACCCC
GCTGGACAGCGATGGCAGCTTCTTCCTGTACAGCAAGCTGACCGTGGACAAGAGCAGA
TGGCAGCAGGGCAACGTGTTCAGCTGCTCCGTGATGCACGAGGCCCTGCACAATCACTACACC
CAGAAGAGCCTGAGCCTGTCCCCTGGCAAG
SEQ ID 57 — CA8 J7 Humanised heavy chain
QVQLVQSGAEVKKPGSSVKVSCKASGYTFTNYWMHWVRQAPGQGLEWMGATYRGHSDTYYNQK
FKGRVHTADKSTSTAYMELSSLRSEDTAVYYCARGAWDGYDVUNMNGQGTLVTVSSASTKGPSVF
PLAPSSKSTSGGTAALGCLVKDYFPEPVTVSNNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSL
GTQTWCNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTUWSRTPEVTC
VVVDVSHEDPEVKHmNYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWlNGKEYKCKVSNK
ALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTT
DGSFFLYSKLTVDKSRWKMDGNVFSCSVMHEALHNHYTQKSLSLSPGK
SEQ ID 58 — CA8 J7 Humanised heavy chain (Polynucleotide)
WO 63805
CAGGTGCAGCTGGTCCAGAGCGGCGCCGAAGTGAAGAAGCCCGGCAGCTCCGTGAAAGTGAG
CTGCAAGGCCAGCGGCTACACCTTCACCAACTACTGGATGCACTGGGTGAGGCAGGCCCCCGG
ACAGGGCCTGGAGTGGATGGGCGCCACCTACAGGGGCCACAGCGACACCTACTACAACCAGAA
GTTCAAGGGCCGGGTGACCATCACCGCCGACAAGAGCACCAGCACCGCCTACATGGAACTGAG
CAGCCTCAGGAGCGAGGACACCGCTGTGTATTACTGCGCCAGGGGCGCCATCTACGACGGCTA
CGACGTGCTGGACAACTGGGGCCAGGGCACACTAGTGACCGTGTCCAGCGCCAGCACCAAGG
GCCCCAGCGTGTTCCCCCTGGCCCCCAGCAGCAAGAGCACCAGCGGCGGCACAGCCGCCCTG
GGCTGCCTGGTGAAGGACTACTTCCCCGAACCGGTGACCGTGTCCTGGAACAGCGGAGCCCTG
ACCAGCGGCGTGCACACCTTCCCCGCCGTGCTGCAGAGCAGCGGCCTGTACAGCCTGAGCAGC
GTGGTGACCGTGCCCAGCAGCAGCCTGGGCACCCAGACCTACATCTGTAACGTGAACCACAAG
CCCAGCAACACCAAGGTGGACAAGAAGGTGGAGCCCAAGAGCTGTGACAAGACCCACACCTGC
CCCCCCTGCCCTGCCCCCGAGCTGCTGGGAGGCCCCAGCGTGTTCCTGTTCCCCCCCAAGCCT
AAGGACACCCTGATGATCAGCAGAACCCCCGAGGTGACCTGTGTGGTGGTGGATGTGAGCCAC
GAGGACCCTGAGGTGAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCACAATGCCAAGACC
AAGCCCAGGGAGGAGCAGTACAACAGCACCTACCGGGTGGTGTCCGTGCTGACCGTGCTGCAC
CAGGATTGGCTGAACGGCAAGGAGTACAAGTGTAAGGTGTCCAACAAGGCCCTGCCTGCCCCTA
AAACCATCAGCAAGGCCAAGGGCCAGCCCAGAGAGCCCCAGGTGTACACCCTGCCCC
CTAGCAGAGATGAGCTGACCAAGAACCAGGTGTCCCTGACCTGCCTGGTGAAGGGCTTCTACCC
CATCGCCGTGGAGTGGGAGAGCAACGGCCAGCCCGAGAACAACTACAAGACCACCCC
CCCTGTGCTGGACAGCGATGGCAGCTTCTTCCTGTACAGCAAGCTGACCGTGGACAAGAGCAGA
TGGCAGCAGGGCAACGTGTTCAGCTGCTCCGTGATGCACGAGGCCCTGCACAATCACTACACC
CAGAAGAGCCTGAGCCTGTCCCCTGGCAAG
SEQ ID 59 — CA8 J8 Humanised heavy chain
QVQLVQSGAEVKKPGSSVKVSCKASGYTFTNYWMHWVRQAPGQGLEWMGATYRGHSDTYYNQK
FKGRVWTADKSTSTAYMELSSLRSEDTAVYYCTRGAWDGYDVUNMNGQGTLVTVSSASTKGPSVF
PLAPSSKSTSGGTAALGCLVKDYFPEPVTVSNNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSL
GTQTWCNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTUWSRTPEVTC
VVVDVSHEDPEVKHmNYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWlNGKEYKCKVSNK
ALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTT
PPVLDSDGSFFLYSKLTVDKSRWKMDGNVFSCSVMHEALHNHYTQKSLSLSPGK
SEQ ID 60 — CA8 J8 Humanised heavy chain (Polynucleotide)
CAGGTGCAGCTGGTCCAGAGCGGCGCCGAAGTGAAGAAGCCCGGCAGCTCCGTGAAAGTGAG
CTGCAAGGCCAGCGGCTACACCTTCACCAACTACTGGATGCACTGGGTGAGGCAGGCCCCCGG
ACAGGGCCTGGAGTGGATGGGCGCCACCTACAGGGGCCACAGCGACACCTACTACAACCAGAA
GTTCAAGGGCCGGGTGACCATCACCGCCGACAAGAGCACCAGCACCGCCTACATGGAACTGAG
CAGCCTCAGGAGCGAGGACACCGCTGTGTATTACTGCACCAGGGGCGCCATCTACGACGGCTA
GCTGGACAACTGGGGCCAGGGCACACTAGTGACCGTGTCCAGCGCCAGCACCAAGG
40 GCCCCAGCGTGTTCCCCCTGGCCCCCAGCAGCAAGAGCACCAGCGGCGGCACAGCCGCCCTG
WO 63805
GGCTGCCTGGTGAAGGACTACTTCCCCGAACCGGTGACCGTGTCCTGGAACAGCGGAGCCCTG
ACCAGCGGCGTGCACACCTTCCCCGCCGTGCTGCAGAGCAGCGGCCTGTACAGCCTGAGCAGC
GTGGTGACCGTGCCCAGCAGCAGCCTGGGCACCCAGACCTACATCTGTAACGTGAACCACAAG
CCCAGCAACACCAAGGTGGACAAGAAGGTGGAGCCCAAGAGCTGTGACAAGACCCACACCTGC
CCCCCCTGCCCTGCCCCCGAGCTGCTGGGAGGCCCCAGCGTGTTCCTGTTCCCCCCCAAGCCT
AAGGACACCCTGATGATCAGCAGAACCCCCGAGGTGACCTGTGTGGTGGTGGATGTGAGCCAC
GAGGACCCTGAGGTGAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCACAATGCCAAGACC
AAGCCCAGGGAGGAGCAGTACAACAGCACCTACCGGGTGGTGTCCGTGCTGACCGTGCTGCAC
TGGCTGAACGGCAAGGAGTACAAGTGTAAGGTGTCCAACAAGGCCCTGCCTGCCCCTA
TCGAGAAAACCATCAGCAAGGCCAAGGGCCAGCCCAGAGAGCCCCAGGTGTACACCCTGCCCC
CTAGCAGAGATGAGCTGACCAAGAACCAGGTGTCCCTGACCTGCCTGGTGAAGGGCTTCTACCC
CAGCGACATCGCCGTGGAGTGGGAGAGCAACGGCCAGCCCGAGAACAACTACAAGACCACCCC
CCCTGTGCTGGACAGCGATGGCAGCTTCTTCCTGTACAGCAAGCTGACCGTGGACAAGAGCAGA
TGGCAGCAGGGCAACGTGTTCAGCTGCTCCGTGATGCACGAGGCCCTGCACAATCACTACACC
AGCCTGAGCCTGTCCCCTGGCAAG
SEQ ID 61 — CA8 J9 sed heavy chain
QVQLVQSGAEVKKPGSSVKVSCKGSGYTFTNYMMMflNVRQAPGQGLEMHGATYRGHSDTYYNQKF
KGRATLTADTSTSTAYMELSSLRSEDTAVYYCTRGAIYDGYDVLDNWGQGTLVTVSSASTKGPSVFP
LAPSSKSTSGGTAALGCLVKDYFPEPVTVSVWVSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLG
TQTWCNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTUWSRTPEVTCV
VVDVSHEDPEVKHMNYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDMANGKEYKCKVSNKA
LPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTP
PVLDSDGSFFLYSKLTVDKSRWKDQGNVFSCSVMHEALHNHYTQKSLSLSPGK
SEQ ID 62 — CA8 J9 Humanised heavy chain (Polynucleotide)
CAGGTGCAGCTGGTCCAGAGCGGCGCCGAAGTGAAGAAGCCCGGCAGCTCCGTGAAAGTGAG
CTGCAAGGGCAGCGGCTACACCTTCACCAACTACTGGATGCACTGGGTGAGGCAGGCCCCCGG
ACAGGGCCTGGAGTGGATCGGCGCCACCTACAGGGGCCACAGCGACACCTACTACAACCAGAA
GTTCAAGGGCCGGGCGACCCTCACCGCCGACACGAGCACCAGCACCGCCTACATGGAACTGAG
CAGCCTCAGGAGCGAGGACACCGCTGTGTATTACTGCACCAGGGGCGCCATCTACGACGGCTA
CGACGTGCTGGACAACTGGGGCCAGGGCACACTAGTGACCGTGTCCAGCGCCAGCACCAAGG
GCCCCAGCGTGTTCCCCCTGGCCCCCAGCAGCAAGAGCACCAGCGGCGGCACAGCCGCCCTG
GGCTGCCTGGTGAAGGACTACTTCCCCGAACCGGTGACCGTGTCCTGGAACAGCGGAGCCCTG
ACCAGCGGCGTGCACACCTTCCCCGCCGTGCTGCAGAGCAGCGGCCTGTACAGCCTGAGCAGC
GTGGTGACCGTGCCCAGCAGCAGCCTGGGCACCCAGACCTACATCTGTAACGTGAACCACAAG
CCCAGCAACACCAAGGTGGACAAGAAGGTGGAGCCCAAGAGCTGTGACAAGACCCACACCTGC
CCCCCCTGCCCTGCCCCCGAGCTGCTGGGAGGCCCCAGCGTGTTCCTGTTCCCCCCCAAGCCT
AAGGACACCCTGATGATCAGCAGAACCCCCGAGGTGACCTGTGTGGTGGTGGATGTGAGCCAC
40 GAGGACCCTGAGGTGAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCACAATGCCAAGACC
AAGCCCAGGGAGGAGCAGTACAACAGCACCTACCGGGTGGTGTCCGTGCTGACCGTGCTGCAC
CAGGATTGGCTGAACGGCAAGGAGTACAAGTGTAAGGTGTCCAACAAGGCCCTGCCTGCCCCTA
TCGAGAAAACCATCAGCAAGGCCAAGGGCCAGCCCAGAGAGCCCCAGGTGTACACCCTGCCCC
CTAGCAGAGATGAGCTGACCAAGAACCAGGTGTCCCTGACCTGCCTGGTGAAGGGCTTCTACCC
CAGCGACATCGCCGTGGAGTGGGAGAGCAACGGCCAGCCCGAGAACAACTACAAGACCACCCC
CCCTGTGCTGGACAGCGATGGCAGCTTCTTCCTGTACAGCAAGCTGACCGTGGACAAGAGCAGA
TGGCAGCAGGGCAACGTGTTCAGCTGCTCCGTGATGCACGAGGCCCTGCACAATCACTACACC
CAGAAGAGCCTGAGCCTGTCCCCTGGCAAG
SEQ ID 63 — CA8 M0 Humanised light chain
D|QMTQSPSSLSASVGDRVTITCSASQDISNYLNWYQQKPGKAPKLLIYYTSNLHSGVPSRFSGSGS
GTDFTEHSSLQPEDFATYYCQQYRKLMNTFGQGTKLHKRTVAAPSVFFPPSDEQLKSGTASVVCLL
NNFYPREAKVCNVKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLS
SPVTKSFNRGEC
SEQ ID 64 — CA8 M0 Humanised light chain (Polynucleotide)
GACATCCAGATGACCCAGAGCCCTAGCTCACTGAGCGCCAGCGTGGGCGACAGGGTGACCATT
TCCGCCAGCCAGGACATCAGCAACTACCTGAACTGGTACCAGCAGAAGCCCGGCAAG
GCCCCCAAGCTGCTGATCTACTACACCTCCAACCTGCACTCCGGCGTGCCCAGCAGGTTCAGCG
GAAGCGGCAGCGGCACCGAITTCACCCTGACCATCTCCAGCCTGCAGCCCGAGGACTTCGCCA
CCTACTACTGCCAGCAGTACAGGAAGCTCCCCTGGACTTTCGGCCAGGGCACCAAACTGGAGAT
CAAGCGTACGGTGGCCGCCCCCAGCGTGTTCATCTTCCCCCCCAGCGATGAGCAGCTGAAGAG
CGGCACCGCCAGCGTGGTGTGTCTGCTGAACAACTTCTACCCCCGGGAGGCCAAGGTGCAGTG
GAAGGTGGACAATGCCCTGCAGAGCGGCAACAGCCAGGAGAGCGTGACCGAGCAGGACAGCA
AGGACTCCACCTACAGCCTGAGCAGCACCCTGACCCTGAGCAAGGCCGACTACGAGAAGCACA
AGGTGTACGCCTGTGAGGTGACCCACCAGGGCCTGTCCAGCCCCGTGACCAAGAGCTTCAACC
GGGGCGAGTGC
SEQ ID 65 — CA8 M1 sed light chain
D|QMTQSPSSLSASVGDRVTITCSASQDISNYLNWYQQKPGKAPKLLIYYTSNLHSGVPSRFSGSGS
HSSLQPEDFATYYCQQYRKLMNTFGQGTKLHKRTVAAPSVHFPPSDEQLKSGTASVVCLL
NNFYPREAKVCNVKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLS
SPVTKSFNRGEC
SEQ ID 66 — CA8 M1 Humanised light chain (Polynucleotide)
GACATCCAGATGACCCAGAGCCCTAGCTCACTGAGCGCCAGCGTGGGCGACAGGGTGACCATT
ACCTGCTCCGCCAGCCAGGACATCAGCAACTACCTGAACTGGTACCAGCAGAAGCCCGGCAAG
GCCCCCAAGCTGCTGATCTACTACACCTCCAACCTGCACTCCGGCGTGCCCAGCAGGTTCAGCG
GCAGCGGCACCGAITACACCCTGACCATCTCCAGCCTGCAGCCCGAGGACTTCGCCA
40 CCTACTACTGCCAGCAGTACAGGAAGCTCCCCTGGACTTTCGGCCAGGGCACCAAACTGGAGAT
WO 63805
CAAGCGTACGGTGGCCGCCCCCAGCGTGTTCATCTTCCCCCCCAGCGATGAGCAGCTGAAGAG
CGGCACCGCCAGCGTGGTGTGTCTGCTGAACAACTTCTACCCCCGGGAGGCCAAGGTGCAGTG
GAAGGTGGACAATGCCCTGCAGAGCGGCAACAGCCAGGAGAGCGTGACCGAGCAGGACAGCA
AGGACTCCACCTACAGCCTGAGCAGCACCCTGACCCTGAGCAAGGCCGACTACGAGAAGCACA
AGGTGTACGCCTGTGAGGTGACCCACCAGGGCCTGTCCAGCCCCGTGACCAAGAGCTTCAACC
GGGGCGAGTGC
SEQ ID 67 — CA8 M2 Humanised light chain
D|QLTQSPSSLSASVGDRVTITCSASQDISNYLNWYQQKPGKAPELVIYYTSNLHSGVPSRFSGSGSG
TDYTLTISSLQPEDFATYYCQQYRKLPWTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASWCLLN
NFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSS
PVTKSFNRGEC
SEQ ID 68 — CA8 M2 Humanised light chain (Polynucleotide)
GACATCCAGCTGACCCAGAGCCCTAGCTCACTGAGCGCCAGCGTGGGCGACAGGGTGACCATT
ACCTGCTCCGCCAGCCAGGACATCAGCAACTACCTGAACTGGTACCAGCAGAAGCCCGGCAAG
GCCCCCGAGCTGGTGATCTACTACACCTCCAACCTGCACTCCGGCGTGCCCAGCAGGTTCAGC
GGAAGCGGCAGCGGCACCGATTACACCCTGACCATCTCCAGCCTGCAGCCCGAGGACTTCGCC
ACCTACTACTGCCAGCAGTACAGGAAGCTCCCCTGGACTTTCGGCCAGGGCACCAAACTGGAGA
TCAAGCGTACGGTGGCCGCCCCCAGCGTGTTCATCTTCCCCCCCAGCGATGAGCAGCTGAAGA
GCGGCACCGCCAGCGTGGTGTGTCTGCTGAACAACTTCTACCCCCGGGAGGCCAAGGTGCAGT
TGGACAATGCCCTGCAGAGCGGCAACAGCCAGGAGAGCGTGACCGAGCAGGACAGC
AAGGACTCCACCTACAGCCTGAGCAGCACCCTGACCCTGAGCAAGGCCGACTACGAGAAGCAC
AAGGTGTACGCCTGTGAGGTGACCCACCAGGGCCTGTCCAGCCCCGTGACCAAGAGCTTCAAC
CGGGGCGAGTGC
SEQ ID 69 - 8GO3 mouse variable heavy
EVQLQQSGPELVKPGASVKISCKASGYTFTDYYMKWVKQSHGKSLEWIGEIYPNNGGITYNQKFKGK
ATLTVDKSSSTAYMELRSLTSEDSAVYYCANGYEFVYWGQGTLVTVSA
SEQ ID 70 - S307118G03 mouse variable heavy (DNA sequence)
GAGGTCCAGTTGCAACAATCTGGACCTGAGCTGGTGAAGCCTGGGGCTTCAGTGAAGATATCCT
GTAAGGCTTCTGGATACACATTCACTGACTACTACATGAAGTGGGTGAAGCAGAGCCATGGAAA
GAGCCTTGAGTGGATTGGAGAGATTTATCCTAATAATGGTGGTATTACCTACAACCAGAAGTTCA
AGGGCAAGGCCACATTGACTGTAGACAAGTCCTCCAGCACAGCCTACATGGAGCTCCGCAGCCT
GACATCTGAGGACTCTGCAGTCTATTACTGTGCAAATGGTTACGAGTTTGTTTACTGGGGCCAAG
GGACTCTGGTCACTGTCTCTGCA
SEQ ID 71 - S307118G03 mouse variable light
DIQMTQTASSLSASLGDRVTISCSASQGISNYLNWYQQKPDGTVKLLIYYTSSLHSGVPSRFSGSGSG
TDYSLTISNLEPEDIATYYCQQYSKLPWTF666TKLEIKR
SEQ ID 72 - S307118603 mouse variable light (DNA sequence)
6ATATCCA6AT6ACACA6ACT6CATCCTCCCTGTCTGCCTCTCTGG6A6ACA6A6TCACCATCA
6TT6CA6T6CAA6TCA666CATTA6CAATTATTTAAACT66TATCA6CA6AAACCA6AT66AACT
6TTAAACTCCT6ATCTATTACACATCAA6TTTACACTCA66A6TCCCATCAA66TTCA6T66CA6
T666TCT666ACA6ATTATTCTCTCACCATCA6CAACCT66AACCT6AA6ATATT6CCACTTACT
ATT6TCA6CA6TATA6TAA6CTTCC6T66AC6TTC66T66A66CACCAA6CT66AAATCAAAC6
G
SEQ ID 73 - S307118603 chimeric heavy chain
EVQLQQSGPELVKP6ASVK|SCKASGYTFTDYYMKWVKQSHGKSLEW|6E|YPNN66|TYNQKFK6K
KSSSTAYMELRSLTSEDSAVYYCANGYEFVYW6Q6TLVTVSAAKTTAPSVFPLAPSSKSTS
66TAAL6CLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSL6TQTY|CNVN
HKPSNTKVDKKVEPKSCDKTHTCPPCPAPELL66PSVFLFPPKPKDTLMISRTPEVTCVWDVSHED
PEVKFNWWD6VEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTIS
KAK6QPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSD|AVEWESN6QPENNYKTTPPVLDSDGSF
FLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSP6K
SEQ ID 74 - S307118603 chimeric heavy chain (DNA sequence)
6A66TCCA6TT6CAACAATCT66ACCT6A6CT66T6AA6CCT6666CTTCA6T6AA6ATATCCT
6TAA66CTTCT66ATACACATTCACT6ACTACTACAT6AA6T666T6AA6CA6A6CCAT66AAA
6A6CCTT6A6T66ATT66A6A6ATTTATCCTAATAAT66T66TATTACCTACAACCA6AA6TTCA
A66CCACATT6ACT6TA6ACAA6TCCTCCA6CACA6CCTACAT66A6CTCC6CA6CCT
6ACATCT6A66ACTCT6CA6TCTATTACT6T6CAAAT66TTAC6A6TTT6TTTACT6666CCAA6
T66TCACT6TCTCT6CA6CCAAAACAACA6CCCCCA6C6T6TTCCCCCT66CCCCCA6
CA6CAA6A6CACCA6C66C66CACA6CC6CCCT666CT6CCT66T6AA66ACTACTTCCCC6A
ACC66T6ACC6T6TCCT66AACA6C66A6CCCT6ACCA6C66C6T6CACACCTTCCCC6CC6T
6CT6CA6A6CA6C66CCT6TACA6CCT6A6CA6C6T66T6ACC6T6CCCA6CA6CA6CCT66
6CACCCA6ACCTACATCT6TAAC6T6AACCACAA6CCCA6CAACACCAA66T66ACAA6AA66T
66A6CCCAA6A6CT6T6ACAA6ACCCACACCT6CCCCCCCT6CCCT6CCCCC6A6CT6CT666
A66CCCCA6C6T6TTCCT6TTCCCCCCCAA6CCTAA66ACACCCT6AT6ATCA6CA6AACCCCC
6A66T6ACCT6T6T66T66T66AT6T6A6CCAC6A66ACCCT6A66T6AA6TTCAACT66TAC
6T66AC66C6T66A66T6CACAAT6CCAA6ACCAA6CCCA666A66A6CA6TACAACA6CACC
TACC666T66T6TCC6T6CT6ACC6T6CT6CACCA66ATT66CT6AAC66CAA66A6TACAA6
T6TAA66T6TCCAACAA66CCCT6CCT6CCCCTATC6A6AAAACCATCA6CAA66CCAA666CC
A6CCCA6A6A6CCCCA66T6TACACCCT6CCCCCTA6CA6A6AT6A6CT6ACCAA6AACCA66
T6TCCCT6ACCT6CCT66T6AA666CTTCTACCCCAGC6ACATC6CC6T66A6T666A6A6CA
40 AC66CCA6CCC6A6AACAACTACAA6ACCACCCCCCCT6T6CT66ACA6C6AT66CA6CTTCT
TCCTGTACAGCAAGCTGACCGTGGACAAGAGCAGATGGCAGCAGGGCAACGTGTTCAGCTGCT
CCGTGATGCACGAGGCCCTGCACAATCACTACACCCAGAAGAGCCTGAGCCTGTCCCCTGGCA
SEQ ID 75 - S307118603 chimeric light chain
D|QMTQTASSLSASL6DRVTISCSASQ6|SNYLNWYQQKPDGTVKLLIYYTSSLHSGVPSRFSGSGSG
TDYSLTISNLEPEDIATYYCQQYSKLPWTF666TKLELKRTVAAPSVFIFPPSDEQLKSGTASWCLLN
NFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSS
PVTKSFNRGEC
SEQ ID 76 - S307118603 chimeric light chain (DNA sequence)
CA6AT6ACACA6ACT6CATCCTCCCTGTCTGCCTCTCTGG6A6ACA6A6TCACCATCA
6TT6CA6T6CAA6TCA666CATTA6CAATTATTTAAACT66TATCA6CA6AAACCA6AT66AACT
6TTAAACTCCT6ATCTATTACACATCAA6TTTACACTCA66A6TCCCATCAA66TTCA6T66CA6
T666TCT666ACA6ATTATTCTCTCACCATCA6CAACCT66AACCT6AA6ATATT6CCACTTACT
ATT6TCA6CA6TATA6TAA6CTTCC6T66AC6TTC66T66A66CACCAA6CT66A6CT6AAAC6
TAC66T66CC6CCCCCA6C6T6TTCATCTTCCCCCCCA6C6AT6A6CA6CT6AA6A6C66CAC
C6CCA6C6T66T6T6TCT6CT6AACAACTTCTACCCCC666A66CCAA66T6CA6T66AA66T
66ACAAT6CCCT6CA6A6C66CAACA6CCA66A6A6C6T6ACC6A6CA66ACA6CAA66ACT
CCACCTACAGCCT6A6CA6CACCCT6ACCCT6A6CAA66CC6ACTAC6A6AA6CACAA66T6T
AC6CCT6T6A66T6ACCCACCA666CCT6TCCA6CCCC6T6ACCAA6A6CTTCAACC6666C6
A6T6C
SEQ ID 77 - S307118603 humanised H0 variable heavy
QVQLVQSGAEVKKPGSSVKVSCKASGGTFSDYYMKWVRQAP6Q6LEWM6E|YPNN66|TYNQKFK
6RVTITADKSTSTAYMELSSLRSEDTAVYYCARGYEFVYW6Q6TLVTVSS
SEQ ID 78 - S307118603 humanised H0 variable heavy (DNA sequence)
CA66T6CA6CT66T6CA6A6C66C6CC6AA6T6AA6AA6CCC66CTCCA6C6T6AA66T6A6
CT6CAA66CTA6C66C66CACCTTCA6C6ACTACTACAT6AA6T666T6A66CA66CCCCC66
ACT66A6T66AT666C6A6ATCTACCCCAACAAC66666CATCACCTACAACCA6AA
6TTCAA666CA666T6ACCATCACC6CC6ACAAAA6CACCA6CACC6CCTACAT66AACT6A6
CA6CCT6A66A6C6A66ACACC6CC6T6TACTACT6C6CCA6666CTAC6A6TTC6T6TATT6
666CCA666CACACTA6T6ACC6T6TCCA6C
SEQ ID 79 - 8603 humanised H1 variable heavy
QVQLVQSGAEVKKPGSSVKVSCKASGYTFTDYYMKWVRQAP6Q6LEWM6EIYPNN66ITYNQKFK
6RVTITADKSTSTAYMELSSLRSEDTAVYYCARGYEFVYW6Q6TLVTVSS
40 SEQ ID 80 - S307118603 humanised H1 le heavy (DNA sequence)
WO 63805 2012/059762
CAGGTGCAGCTGGTGCAGAGC66CGCCGAAGTGAAGAAGCCCGGCTCCAGCGTGAAGGTGAG
CTGCAAGGCTAGCG6CTACACCTTCACCGACTACTACATGAAGTGGGTGAGGCAGGCCCCCGG
CCAGGGACTGGAGTGGATGGGCGAGATCTACCCCAACAACGGGGGCATCACCTACAACCAGAA
GTTCAAGGGCAGGGTGACCATCACCGCCGACAAAAGCACCAGCACCGCCTACATGGAACTGAG
CA6CCTGAGGAGCGAGGACACCGCCGTGTACTACTGCGCCAGGGGCTACGAGTTCGTGTATTG
6GGCCAGGGCACACTAGTGACCGTGTCCAGC
SEQ ID 81 - S307118603 humanised H2 variable heavy
QVQLVQSGAEVKKPGSSVKVSCKASGYTFTDYYMKWVRQAP6Q6LEWM6EIYPNN66ITYNQKFK
6RVTITADKSTSTAYMELSSLRSEDTAVYYCAN6YEFVYW6Q6TLVTVSS
SEQ ID 82 - S307118603 sed H2 variable heavy (DNA sequence)
CA6CT66T6CA6A6C66C6CC6AA6T6AA6AA6CCC66CTCCA6C6T6AA66T6A6
CT6CAA66CTA6C66CTACACCTTCACC6ACTACTACAT6AA6T666T6A66CA66CCCCC66
CCA666ACT66A6T66AT666C6A6ATCTACCCCAACAAC66666CATCACCTACAACCA6AA
6TTCAA666CA666T6ACCATCACC6CC6ACAAAA6CACCA6CACC6CCTACAT66AACT6A6
CA6CCT6A66A6C6A66ACACC6CC6T6TACTACT6C6CCAAC66CTAC6A6TTC6T6TATT6
666CCA666CACACTA6T6ACC6T6TCCA6C
SEQ ID 83 - S307118603 humanised H3 variable heavy
QVQLVQSGAEVKKPGSSVKVSCKASGYTFTDYYMKWVRQAP6Q6LEW|6EIYPNN66ITYNQKFK6
RATLTVDKSTSTAYMELSSLRSEDTAVYYCAN6YEFVYW6Q6TLVTVSS
SEQ ID 84 - S307118603 humanised H3 variable heavy (DNA sequence)
CA66T6CA6CT66T6CA6A6C66C6CC6AA6T6AA6AA6CCC66CTCCA6C6T6AA66T6A6
CT6CAA66CTA6C66CTACACCTTCACC6ACTACTACAT6AA6T666T6A66CA66CCCCC66
CCA666ACT66A6T66ATA66C6A6ATCTACCCCAACAAC66666CATCACCTACAACCAGAA
6TTCAA666CA666C6ACCCTCACC6TC6ACAAAA6CACCA6CACC6CCTACAT66AACT6A6
CA6CCT6A66A6C6A66ACACC6CC6T6TACTACT6C6CCAAC66CTAC6A6TTC6T6TATT6
666CCA666CACACTA6T6ACC6T6TCCA6C
SEQ ID 85 - S307118603 humanised H4 variable heavy
QVQLVQSGAEVKKPGSSVKVSCKASGYTFTDYYMKWVRQAP6Q6LEWM6EIYPNN66ITYNQKFK
ADKSTSTAYMELSSLRSEDTAVYYCADGYEFVYW6Q6TLVTVSS
SEQ ID 86 - S307118603 humanised H4 variable heavy (DNA sequence)
CA66T6CA6CT66T6CA6A6C66C6CC6AA6T6AA6AA6CCC66CTCCA6C6T6AA66T6A6
CT6CAA66CTA6C66CTACACCTTCACC6ACTACTACAT6AA6T666T6A66CA66CCCCC66
CCA666ACT66A6T66AT666C6A6ATCTACCCCAACAAC66666CATCACCTACAACCA6AA
40 6TTCAA666CA666T6ACCATCACC6CC6ACAAAA6CACCA6CACC6CCTACAT66AACT6A6
2012/059762
CAGCCTGAGGAGCGAGGACACCGCCGTGTACTACTGCGCCGACGGCTACGAGTTCGTGTATTG
666CCAGGGCACACTAGTGACCGTGTCCAGC
SEQ ID 87 - 8603 humanised H5 variable heavy
QVQLVQSGAEVKKPGSSVKVSCKASGYTFTDYYMKWVRQAP6Q6LEW|6EIYPNN66ITYNQKFK6
RATLTVDKSTSTAYMELSSLRSEDTAVYYCAN6YEFDYW6Q6TLVTVSS
SEQ ID 88 - S307118603 humanised H5 variable heavy (DNA sequence)
CA66T6CA6CT66T6CA6A6C66C6CC6AA6T6AA6AA6CCC66CTCCA6C6T6AA66T6A6
CT6CAA66CTA6C66CTACACCTTCACC6ACTACTACAT6AA6T666T6A66CA66CCCCC66
ACT66A6T66ATA66C6A6ATCTACCCCAACAAC66666CATCACCTACAACCAGAA
6TTCAA666CA666C6ACCCTCACC6TC6ACAAAA6CACCA6CACC6CCTACAT66AACT6A6
6A66A6C6A66ACACC6CC6T6TACTACT6C6CCAAC66CTAC6A6TTC6ACTATT6
666CCA666CACACTA6T6ACC6T6TCCA6C
SEQ ID 89 - S307118603 humanised L0 variable light
D|QMTQSPSSLSASV6DRVTITCSASQGISNYLNWYQQKP6KAPKLLIYYTSSLHSGVPSRFSGSGS
T|SSLQPEDFATYYCQQYSKLPWTF6Q6TKLE|KR
SEQ ID 90 - S307118603 humanised L0 variable light (DNA sequence)
6ACATCCA6AT6ACCCA6A6CCCCTCAA6CCT6A6C6CCA6C6T666C6ACA666T6ACTATC
ACCT6CA6C6CCTCCCA666CATCA6CAACTACCT6AACT66TACCA6CA6AA6CCC66CAA6
6CCCCTAA6CT6CT6ATCTACTACACCA6CA6CCT6CACA6C66C6T6CCCA6CA66TTCTCC
66CA6C66CA6C66AACC6ACTTCACCCT6ACCATTA6CA6CCTCCA6CCC6A66ACTTC6CC
ACCTACTACTGCCAGCA6TACA6CAA6CT6CCCT66ACCTTC66CCA666CACCAAACT66A6
ATCAA6C6T
SEQ ID 91 - S307118603 humanised L1 variable light
D|QMTQSPSSLSASV6DRVTITCSASQGISNYLNWYQQKP6KAPKLLIYYTSSLHSGVPSRFSGSGS
6TDYTLTISSLQPEDFATYYCQQYSKLPWTF6Q6TKLE|KR
SEQ ID 92 - S307118603 humanised L1 variable light (DNA sequence)
6ACATCCA6AT6ACCCA6A6CCCCTCAA6CCT6A6C6CCA6C6T666C6ACA666T6ACTATC
ACCT6CA6C6CCTCCCA666CATCA6CAACTACCT6AACT66TACCA6CA6AA6CCC66CAA6
6CCCCTAA6CT6CT6ATCTACTACACCA6CA6CCT6CACA6C66C6T6CCCA6CA66TTCTCC
66CA6C66CA6C66AACC6ACTACACCCT6ACCATTA6CA6CCTCCA6CCC6A66ACTTC6CC
ACCTACTACTGCCAGCA6TACA6CAA6CT6CCCT66ACCTTC66CCA666CACCAAACT66A6
ATCAA6C6T
40 SEQ ID 93 - 8307118603 CDRH1
DYYMK
SEQ ID 94 - S307118G03 CDRH2
EIYPNNGGITYNQKFKG
SEQ ID 95 - S307118G03 CDRH3
GYEFVY
SEQ ID 96 - S307118G03 CDRL1
SASQGISNYLN
SEQ ID 97 - 8307118G03 CDRL2
YTSSLHS
SEQ ID 98 - S307118G03 CDRL3
QQYSKLPWT
SEQ ID 99 - S307118G03 sed H5 CDRH3
GYEFDY
SEQ ID 100 - 8307118603 humanised H0 heavy chain
QVQLVQSGAEVKKPGSSVKVSCKASGGTFSDYYMKWVRQAPGQGLEWMGEIYPNNGGITYNQKFK
GRVTITADKSTSTAYMELSSLRSEDTAVYYCARGYEFVYWGQGTLVTVSSASTKGPSVFPLAPSSKS
TSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICN
VNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCWVDVSH
EDPEVKFNWWDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAP|EK
TISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSD
GSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
SEQ ID 101 - S307118G03 humanised H0 heavy chain ucleotide)
CAGGTGCAGCTGGTGCAGAGCGGCGCCGAAGTGAAGAAGCCCGGCTCCAGCGTGAAGGTGAG
CTGCAAGGCTAGCGGCGGCACCTTCAGCGACTACTACATGAAGTGGGTGAGGCAGGCCCCCGG
CCAGGGACTGGAGTGGATGGGCGAGATCTACCCCAACAACGGGGGCATCACCTACAACCAGAA
GTTCAAGGGCAGGGTGACCATCACCGCCGACAAAAGCACCAGCACCGCCTACATGGAACTGAG
CAGCCTGAGGAGCGAGGACACCGCCGTGTACTACTGCGCCAGGGGCTACGAGTTCGTGTATTG
GGGCACACTAGTGACCGTGTCCAGCGCCAGCACCAAGGGCCCCAGCGTGTTCCCCCT
GGCCCCCAGCAGCAAGAGCACCAGCGGCGGCACAGCCGCCCTGGGCTGCCTGGTGAAGGACT
ACTTCCCCGAACCGGTGACCGTGTCCTGGAACAGCGGAGCCCTGACCAGCGGCGTGCACACCT
TCCCCGCCGTGCTGCAGAGCAGCGGCCTGTACAGCCTGAGCAGCGTGGTGACCGTGCCCAGCA
40 GCAGCCTGGGCACCCAGACCTACATCTGTAACGTGAACCACAAGCCCAGCAACACCAAGGTGG
ACAAGAAGGTGGAGCCCAAGAGCTGTGACAAGACCCACACCTGCCCCCCCTGCCCTGCCCCCG
AGCTGCTGGGAGGCCCCAGCGTGTTCCTGTTCCCCCCCAAGCCTAAGGACACCCTGATGATCA
GCAGAACCCCCGAGGTGACCTGTGTGGTGGTGGATGTGAGCCACGAGGACCCTGAGGTGAAGT
TCAACTGGTACGTGGACGGCGTGGAGGTGCACAATGCCAAGACCAAGCCCAGGGAGGAGCAGT
ACAACAGCACCTACCGGGTGGTGTCCGTGCTGACCGTGCTGCACCAGGATTGGCTGAACGGCA
AGGAGTACAAGTGTAAGGTGTCCAACAAGGCCCTGCCTGCCCCTATCGAGAAAACCATCAGCAA
GGCCAAGGGCCAGCCCAGAGAGCCCCAGGTGTACACCCTGCCCCCTAGCAGAGATGAGCTGAC
CAAGAACCAGGTGTCCCTGACCTGCCTGGTGAAGGGCTTCTACCCCAGCGACATCGCCGTGGA
GTGGGAGAGCAACGGCCAGCCCGAGAACAACTACAAGACCACCCCCCCTGTGCTGGACAGCGA
TGGCAGCTTCTTCCTGTACAGCAAGCTGACCGTGGACAAGAGCAGATGGCAGCAGGGCAACGT
GTTCAGCTGCTCCGTGATGCACGAGGCCCTGCACAATCACTACACCCAGAAGAGCCTGAGCCTG
TCCCCTGGCAAG
SEQ ID 102 - S307118G03 humanised H1 heavy chain
QVQLVQSGAEVKKPGSSVKVSCKASGYTFTDYYMKWVRQAPGQGLEWMGEIYPNNGGITYNQKFK
GRVTHADKSTSTAYMELSSLRSEDTAVYYCARGYEFVYMKRQGTLVTVSSASTKGPSVFPLAPSSKS
TSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICN
VNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLNHSRTPEVTCVVVDVSH
FNWWDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAP|EK
flSKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSD”MEWVESNGQPENNYKTTPPVLDSD
GSFFLYSKLTVDKSRWKMDGNVFSCSVMHEALHNHYTQKSLSLSPGK
SEQ ID 103 - 8G03 humanised H1 heavy chain (DNA sequence)
CAGGTGCAGCTGGTGCAGAGCGGCGCCGAAGTGAAGAAGCCCGGCTCCAGCGTGAAGGTGAG
CTGCAAGGCTAGCGGCTACACCTTCACCGACTACTACATGAAGTGGGTGAGGCAGGCCCCCGG
CCAGGGACTGGAGTGGATGGGCGAGATCTACCCCAACAACGGGGGCATCACCTACAACCAGAA
GTTCAAGGGCAGGGTGACCATCACCGCCGACAAAAGCACCAGCACCGCCTACATGGAACTGAG
CAGCCTGAGGAGCGAGGACACCGCCGTGTACTACTGCGCCAGGGGCTACGAGTTCGTGTATTG
GGGCCAGGGCACACTAGTGACCGTGTCCAGCGCCAGCACCAAGGGCCCCAGCGTGTTCCCCCT
CAGCAGCAAGAGCACCAGCGGCGGCACAGCCGCCCTGGGCTGCCTGGTGAAGGACT
ACTTCCCCGAACCGGTGACCGTGTCCTGGAACAGCGGAGCCCTGACCAGCGGCGTGCACACCT
TCCCCGCCGTGCTGCAGAGCAGCGGCCTGTACAGCCTGAGCAGCGTGGTGACCGTGCCCAGCA
GCAGCCTGGGCACCCAGACCTACATCTGTAACGTGAACCACAAGCCCAGCAACACCAAGGTGG
ACAAGAAGGTGGAGCCCAAGAGCTGTGACAAGACCCACACCTGCCCCCCCTGCCCTGCCCCCG
TGGGAGGCCCCAGCGTGTTCCTGTTCCCCCCCAAGCCTAAGGACACCCTGATGATCA
GCAGAACCCCCGAGGTGACCTGTGTGGTGGTGGATGTGAGCCACGAGGACCCTGAGGTGAAGT
TCAACTGGTACGTGGACGGCGTGGAGGTGCACAATGCCAAGACCAAGCCCAGGGAGGAGCAGT
ACAACAGCACCTACCGGGTGGTGTCCGTGCTGACCGTGCTGCACCAGGATTGGCTGAACGGCA
AGGAGTACAAGTGTAAGGTGTCCAACAAGGCCCTGCCTGCCCCTATCGAGAAAACCATCAGCAA
40 GGCCAAGGGCCAGCCCAGAGAGCCCCAGGTGTACACCCTGCCCCCTAGCAGAGATGAGCTGAC
2012/059762
CAAGAACCAGGTGTCCCTGACCTGCCTGGTGAAGGGCTTCTACCCCAGCGACATCGCCGTGGA
GTGGGAGAGCAACGGCCAGCCCGAGAACAACTACAAGACCACCCCCCCTGTGCTGGACAGCGA
TGGCAGCTTCTTCCTGTACAGCAAGCTGACCGTGGACAAGAGCAGATGGCAGCAGGGCAACGT
CTGCTCCGTGATGCACGAGGCCCTGCACAATCACTACACCCAGAAGAGCCTGAGCCTG
TCCCCTGGCAAG
SEQ ID 104 - S307118G03 humanised H2 heavy chain
QVQLVQSGAEVKKPGSSVKVSCKASGYTFTDYYMKWVRQAPGQGLEWMGEIYPNNGGITYNQKFK
GRVTHADKSTSTAYMELSSLRSEDTAVYYCANGYEFVYMKRQGTLVTVSSASTKGPSVFPLAPSSKS
TSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICN
VNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLNHSRTPEVTCVVVDVSH
EDPEVKFNWWDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAP|EK
flSKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSD”MEWVESNGQPENNYKTTPPVLDSD
SKLTVDKSRWKMDGNVFSCSVMHEALHNHYTQKSLSLSPGK
SEQ ID 105 - S307118G03 humanised H2 heavy chain (DNA sequence)
CAGGTGCAGCTGGTGCAGAGCGGCGCCGAAGTGAAGAAGCCCGGCTCCAGCGTGAAGGTGAG
CTGCAAGGCTAGCGGCTACACCTTCACCGACTACTACATGAAGTGGGTGAGGCAGGCCCCCGG
CCAGGGACTGGAGTGGATGGGCGAGATCTACCCCAACAACGGGGGCATCACCTACAACCAGAA
GTTCAAGGGCAGGGTGACCATCACCGCCGACAAAAGCACCAGCACCGCCTACATGGAACTGAG
CAGCCTGAGGAGCGAGGACACCGCCGTGTACTACTGCGCCAACGGCTACGAGTTCGTGTATTG
GGGCCAGGGCACACTAGTGACCGTGTCCAGCGCCAGCACCAAGGGCCCCAGCGTGTTCCCCCT
GGCCCCCAGCAGCAAGAGCACCAGCGGCGGCACAGCCGCCCTGGGCTGCCTGGTGAAGGACT
ACTTCCCCGAACCGGTGACCGTGTCCTGGAACAGCGGAGCCCTGACCAGCGGCGTGCACACCT
TCCCCGCCGTGCTGCAGAGCAGCGGCCTGTACAGCCTGAGCAGCGTGGTGACCGTGCCCAGCA
GCAGCCTGGGCACCCAGACCTACATCTGTAACGTGAACCACAAGCCCAGCAACACCAAGGTGG
ACAAGAAGGTGGAGCCCAAGAGCTGTGACAAGACCCACACCTGCCCCCCCTGCCCTGCCCCCG
AGCTGCTGGGAGGCCCCAGCGTGTTCCTGTTCCCCCCCAAGCCTAAGGACACCCTGATGATCA
GCAGAACCCCCGAGGTGACCTGTGTGGTGGTGGATGTGAGCCACGAGGACCCTGAGGTGAAGT
TCAACTGGTACGTGGACGGCGTGGAGGTGCACAATGCCAAGACCAAGCCCAGGGAGGAGCAGT
GCACCTACCGGGTGGTGTCCGTGCTGACCGTGCTGCACCAGGATTGGCTGAACGGCA
AGGAGTACAAGTGTAAGGTGTCCAACAAGGCCCTGCCTGCCCCTATCGAGAAAACCATCAGCAA
GGCCAAGGGCCAGCCCAGAGAGCCCCAGGTGTACACCCTGCCCCCTAGCAGAGATGAGCTGAC
CAAGAACCAGGTGTCCCTGACCTGCCTGGTGAAGGGCTTCTACCCCAGCGACATCGCCGTGGA
GTGGGAGAGCAACGGCCAGCCCGAGAACAACTACAAGACCACCCCCCCTGTGCTGGACAGCGA
TGGCAGCTTCTTCCTGTACAGCAAGCTGACCGTGGACAAGAGCAGATGGCAGCAGGGCAACGT
GTTCAGCTGCTCCGTGATGCACGAGGCCCTGCACAATCACTACACCCAGAAGAGCCTGAGCCTG
TCCCCTGGCAAG
40 SEQ ID 106 - 8307118603 humanised H3 heavy chain
QVQLVQSGAEVKKPGSSVKVSCKASGYTFTDYYMKWVRQAPGQGLEWIGEIYPNNGGITYNQKFKG
RATLTVDKSTSTAYMELSSLRSEDTAVYYCANGYEFVYWGQGTLVTVSSASTKGPSVFPLAPSSKST
SGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNV
NHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHE
DPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTI
SKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDG
SFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
SEQ ID 107 - S307118G03 humanised H3 heavy chain (DNA sequence)
CAGGTGCAGCTGGTGCAGAGCGGCGCCGAAGTGAAGAAGCCCGGCTCCAGCGTGAAGGTGAG
CTGCAAGGCTAGCGGCTACACCTTCACCGACTACTACATGAAGTGGGTGAGGCAGGCCCCCGG
CCAGGGACTGGAGTGGATAGGCGAGATCTACCCCAACAACGGGGGCATCACCTACAACCAGAA
GGGCAGGGCGACCCTCACCGTCGACAAAAGCACCAGCACCGCCTACATGGAACTGAG
CAGCCTGAGGAGCGAGGACACCGCCGTGTACTACTGCGCCAACGGCTACGAGTTCGTGTATTG
GGGCCAGGGCACACTAGTGACCGTGTCCAGCGCCAGCACCAAGGGCCCCAGCGTGTTCCCCCT
GGCCCCCAGCAGCAAGAGCACCAGCGGCGGCACAGCCGCCCTGGGCTGCCTGGTGAAGGACT
ACTTCCCCGAACCGGTGACCGTGTCCTGGAACAGCGGAGCCCTGACCAGCGGCGTGCACACCT
TCCCCGCCGTGCTGCAGAGCAGCGGCCTGTACAGCCTGAGCAGCGTGGTGACCGTGCCCAGCA
GCAGCCTGGGCACCCAGACCTACATCTGTAACGTGAACCACAAGCCCAGCAACACCAAGGTGG
ACAAGAAGGTGGAGCCCAAGAGCTGTGACAAGACCCACACCTGCCCCCCCTGCCCTGCCCCCG
AGCTGCTGGGAGGCCCCAGCGTGTTCCTGTTCCCCCCCAAGCCTAAGGACACCCTGATGATCA
GCAGAACCCCCGAGGTGACCTGTGTGGTGGTGGATGTGAGCCACGAGGACCCTGAGGTGAAGT
TCAACTGGTACGTGGACGGCGTGGAGGTGCACAATGCCAAGACCAAGCCCAGGGAGGAGCAGT
ACAACAGCACCTACCGGGTGGTGTCCGTGCTGACCGTGCTGCACCAGGATTGGCTGAACGGCA
AGGAGTACAAGTGTAAGGTGTCCAACAAGGCCCTGCCTGCCCCTATCGAGAAAACCATCAGCAA
GGCCAAGGGCCAGCCCAGAGAGCCCCAGGTGTACACCCTGCCCCCTAGCAGAGATGAGCTGAC
CCAGGTGTCCCTGACCTGCCTGGTGAAGGGCTTCTACCCCAGCGACATCGCCGTGGA
GTGGGAGAGCAACGGCCAGCCCGAGAACAACTACAAGACCACCCCCCCTGTGCTGGACAGCGA
TGGCAGCTTCTTCCTGTACAGCAAGCTGACCGTGGACAAGAGCAGATGGCAGCAGGGCAACGT
GTTCAGCTGCTCCGTGATGCACGAGGCCCTGCACAATCACTACACCCAGAAGAGCCTGAGCCTG
TCCCCTGGCAAG
SEQ ID 108 - S307118G03 humanised H4 heavy chain
QVQLVQSGAEVKKPGSSVKVSCKASGYTFTDYYMKWVRQAPGQGLEWMGEIYPNNGGITYNQKFK
GRVTITADKSTSTAYMELSSLRSEDTAVYYCADGYEFVYWGQGTLVTVSSASTKGPSVFPLAPSSKS
ALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICN
VNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCWVDVSH
EDPEVKFNWWDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAP|EK
GQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSD
40 GSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
SEQ ID 109 - S307118G03 humanised H4 heavy chain (DNA sequence)
CAGGTGCAGCTGGTGCAGAGCGGCGCCGAAGTGAAGAAGCCCGGCTCCAGCGTGAAGGTGAG
CTGCAAGGCTAGCGGCTACACCTTCACCGACTACTACATGAAGTGGGTGAGGCAGGCCCCCGG
CCAGGGACTGGAGTGGATGGGCGAGATCTACCCCAACAACGGGGGCATCACCTACAACCAGAA
GTTCAAGGGCAGGGTGACCATCACCGCCGACAAAAGCACCAGCACCGCCTACATGGAACTGAG
CAGCCTGAGGAGCGAGGACACCGCCGTGTACTACTGCGCCGACGGCTACGAGTTCGTGTATTG
GGGCCAGGGCACACTAGTGACCGTGTCCAGCGCCAGCACCAAGGGCCCCAGCGTGTTCCCCCT
GGCCCCCAGCAGCAAGAGCACCAGCGGCGGCACAGCCGCCCTGGGCTGCCTGGTGAAGGACT
ACTTCCCCGAACCGGTGACCGTGTCCTGGAACAGCGGAGCCCTGACCAGCGGCGTGCACACCT
TCCCCGCCGTGCTGCAGAGCAGCGGCCTGTACAGCCTGAGCAGCGTGGTGACCGTGCCCAGCA
GCAGCCTGGGCACCCAGACCTACATCTGTAACGTGAACCACAAGCCCAGCAACACCAAGGTGG
AGGTGGAGCCCAAGAGCTGTGACAAGACCCACACCTGCCCCCCCTGCCCTGCCCCCG
AGCTGCTGGGAGGCCCCAGCGTGTTCCTGTTCCCCCCCAAGCCTAAGGACACCCTGATGATCA
GCAGAACCCCCGAGGTGACCTGTGTGGTGGTGGATGTGAGCCACGAGGACCCTGAGGTGAAGT
TCAACTGGTACGTGGACGGCGTGGAGGTGCACAATGCCAAGACCAAGCCCAGGGAGGAGCAGT
ACAACAGCACCTACCGGGTGGTGTCCGTGCTGACCGTGCTGCACCAGGATTGGCTGAACGGCA
AGGAGTACAAGTGTAAGGTGTCCAACAAGGCCCTGCCTGCCCCTATCGAGAAAACCATCAGCAA
GGGCCAGCCCAGAGAGCCCCAGGTGTACACCCTGCCCCCTAGCAGAGATGAGCTGAC
CCAGGTGTCCCTGACCTGCCTGGTGAAGGGCTTCTACCCCAGCGACATCGCCGTGGA
GTGGGAGAGCAACGGCCAGCCCGAGAACAACTACAAGACCACCCCCCCTGTGCTGGACAGCGA
TGGCAGCTTCTTCCTGTACAGCAAGCTGACCGTGGACAAGAGCAGATGGCAGCAGGGCAACGT
GTTCAGCTGCTCCGTGATGCACGAGGCCCTGCACAATCACTACACCCAGAAGAGCCTGAGCCTG
TCCCCTGGCAAG
SEQ ID 110 - 8307118603 humanised H5 heavy chain
SGAEVKKPGSSVKVSCKASGYTFTDYYMKWVRQAPGQGLEWIGEIYPNNGGITYNQKFKG
RATLTVDKSTSTAYMELSSLRSEDTAVYYCANGYEFDYWKBQGTLVTVSSASTKGPSVFPLAPSSKST
SGGTAALGCLVKDYFPEPVTVSNNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTWCNV
NHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLNHSRTPEVTCVVVDVSHE
DPEVKFNWWDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTI
SKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDVMHBNESNGQPENNYKTTPPVLDSDG
SFFLYSKLTVDKSRWKMQGNVFSCSVMHEALHNHYTQKSLSLSPGK
SEQ ID 111 - S307118G03 humanised H5 heavy chain (DNA sequence)
CAGGTGCAGCTGGTGCAGAGCGGCGCCGAAGTGAAGAAGCCCGGCTCCAGCGTGAAGGTGAG
CTGCAAGGCTAGCGGCTACACCTTCACCGACTACTACATGAAGTGGGTGAGGCAGGCCCCCGG
CCAGGGACTGGAGTGGATAGGCGAGATCTACCCCAACAACGGGGGCATCACCTACAACCAGAA
GTTCAAGGGCAGGGCGACCCTCACCGTCGACAAAAGCACCAGCACCGCCTACATGGAACTGAG
40 CAGCCTGAGGAGCGAGGACACCGCCGTGTACTACTGCGCCAACGGCTACGAGTTCGACTATTG
GGGCCAGGGCACACTAGTGACCGTGTCCAGCGCCAGCACCAAGGGCCCCAGCGTGTTCCCCCT
GGCCCCCAGCAGCAAGAGCACCAGCGGCGGCACAGCCGCCCTGGGCTGCCTGGTGAAGGACT
ACTTCCCCGAACCGGTGACCGTGTCCTGGAACAGCGGAGCCCTGACCAGCGGCGTGCACACCT
TCCCCGCCGTGCTGCAGAGCAGCGGCCTGTACAGCCTGAGCAGCGTGGTGACCGTGCCCAGCA
GCAGCCTGGGCACCCAGACCTACATCTGTAACGTGAACCACAAGCCCAGCAACACCAAGGTGG
AGGTGGAGCCCAAGAGCTGTGACAAGACCCACACCTGCCCCCCCTGCCCTGCCCCCG
AGCTGCTGGGAGGCCCCAGCGTGTTCCTGTTCCCCCCCAAGCCTAAGGACACCCTGATGATCA
GCAGAACCCCCGAGGTGACCTGTGTGGTGGTGGATGTGAGCCACGAGGACCCTGAGGTGAAGT
TCAACTGGTACGTGGACGGCGTGGAGGTGCACAATGCCAAGACCAAGCCCAGGGAGGAGCAGT
ACAACAGCACCTACCGGGTGGTGTCCGTGCTGACCGTGCTGCACCAGGATTGGCTGAACGGCA
AGGAGTACAAGTGTAAGGTGTCCAACAAGGCCCTGCCTGCCCCTATCGAGAAAACCATCAGCAA
GGCCAAGGGCCAGCCCAGAGAGCCCCAGGTGTACACCCTGCCCCCTAGCAGAGATGAGCTGAC
CAAGAACCAGGTGTCCCTGACCTGCCTGGTGAAGGGCTTCTACCCCAGCGACATCGCCGTGGA
GTGGGAGAGCAACGGCCAGCCCGAGAACAACTACAAGACCACCCCCCCTGTGCTGGACAGCGA
TGGCAGCTTCTTCCTGTACAGCAAGCTGACCGTGGACAAGAGCAGATGGCAGCAGGGCAACGT
GTTCAGCTGCTCCGTGATGCACGAGGCCCTGCACAATCACTACACCCAGAAGAGCCTGAGCCTG
TCCCCTGGCAAG
SEQID112-S3OTH8G03humanbedLOHwficham
DK1MTQSPSSLSASVGDRVTWCSASQCHSNYUMNYQQKPGKAPKLUYYTSSLHSGVPSRFSGSGS
GTDFTEHSSLQPEDFATYYCQQYSKLMNTFGQGTKLHKRTVAAPSVHFPPSDEQLKSGTASVVCLL
NNFYPREAKVCNVKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLS
SPVTKSFNRGEC
SEQ ID 113 - S307118G03 humanised L0 light chain (DNA sequence)
GACATCCAGATGACCCAGAGCCCCTCAAGCCTGAGCGCCAGCGTGGGCGACAGGGTGACTATC
ACCTGCAGCGCCTCCCAGGGCATCAGCAACTACCTGAACTGGTACCAGCAGAAGCCCGGCAAG
GCCCCTAAGCTGCTGATCTACTACACCAGCAGCCTGCACAGCGGCGTGCCCAGCAGGTTCTCC
GGCAGCGGCAGCGGAACCGACTTCACCCTGACCATTAGCAGCCTCCAGCCCGAGGACTTCGCC
TACTGCCAGCAGTACAGCAAGCTGCCCTGGACCTTCGGCCAGGGCACCAAACTGGAG
ATCAAGCGTACGGTGGCCGCCCCCAGCGTGTTCATCTTCCCCCCCAGCGATGAGCAGCTGAAG
AGCGGCACCGCCAGCGTGGTGTGTCTGCTGAACAACTTCTACCCCCGGGAGGCCAAGGTGCAG
GTGGACAATGCCCTGCAGAGCGGCAACAGCCAGGAGAGCGTGACCGAGCAGGACAG
CAAGGACTCCACCTACAGCCTGAGCAGCACCCTGACCCTGAGCAAGGCCGACTACGAGAAGCA
GTACGCCTGTGAGGTGACCCACCAGGGCCTGTCCAGCCCCGTGACCAAGAGCTTCAA
CCGGGGCGAGTGC
SEQ ID 114 - S307118G03 humanised L1 light chain
DK1MTQSPSSLSASVGDRVTWCSASQCHSNYUMNYQQKPGKAPKLUYYTSSLHSGVPSRFSGSGS
40 GTDYTEHSSLQPEDFATYYCQQYSKLPMHFGQGTKLHKRTVAAPSVHFPPSDEQLKSGTASVVCLL
EAKVCNVKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLS
SPVTKSFNRGEC
SEQ ID 115 - S307118G03 humanised L1 light chain (DNA sequence)
GACATCCAGATGACCCAGAGCCCCTCAAGCCTGAGCGCCAGCGTGGGCGACAGGGTGACTATC
ACCTGCAGCGCCTCCCAGGGCATCAGCAACTACCTGAACTGGTACCAGCAGAAGCCCGGCAAG
GCCCCTAAGCTGCTGATCTACTACACCAGCAGCCTGCACAGCGGCGTGCCCAGCAGGTTCTCC
GGCAGCGGCAGCGGAACCGACTACACCCTGACCATTAGCAGCCTCCAGCCCGAGGACTTCGCC
ACCTACTACTGCCAGCAGTACAGCAAGCTGCCCTGGACCTTCGGCCAGGGCACCAAACTGGAG
ATCAAGCGTACGGTGGCCGCCCCCAGCGTGTTCATCTTCCCCCCCAGCGATGAGCAGCTGAAG
AGCGGCACCGCCAGCGTGGTGTGTCTGCTGAACAACTTCTACCCCCGGGAGGCCAAGGTGCAG
TGGAAGGTGGACAATGCCCTGCAGAGCGGCAACAGCCAGGAGAGCGTGACCGAGCAGGACAG
CAAGGACTCCACCTACAGCCTGAGCAGCACCCTGACCCTGAGCAAGGCCGACTACGAGAAGCA
CAAGGTGTACGCCTGTGAGGTGACCCACCAGGGCCTGTCCAGCCCCGTGACCAAGAGCTTCAA
CCGGGGCGAGTGC
SEQ ID 116 - S332121F02 murine variable heavy chain
EVQLQQSGPVLVKPGASVKMSCEASGYTFTDYYMNWVKQSHGKTLEWIGVINPYNGGTDYNQKFK
GKATLTVDKSSSTAYMELNSLTSEDSAVYYCARSVYDYPFDYWGQGTLVTVSS
SEQ ID 117 S332121F02 murine variable heavy chain (DNA sequence)
GAGGTGCAGCTGCAGCAGAGCGGCCCCGTGCTGGTGAAGCCTGGAGCCAGCGTGAAAATGAG
CTGCGAAGCCAGCGGCTACACCTTCACCGACTACTACATGAACTGGGTGAAGCAGAGCCACGG
CAAGACCCTGGAGTGGATCGGCGTGATCAACCCCTACAACGGGGGCACCGACTACAACCAGAA
GTTCAAGGGCAAGGCCACTCTGACCGTGGACAAGAGCTCCAGCACCGCCTACATGGAACTGAA
CAGCCTCACCTCTGAGGACAGCGCCGTCTATTACTGCGCCAGGAGCGTGTACGACTACCCCTTC
GACTACTGGGGCCAGGGCACACTAGTGACCGTGTCCAGC
SEQ ID 118 - 1F02 chimeric heavy chain
EVQLQQSGPVLVKPGASVKMSCEASGYTFTDYYMNWVKQSHGKTLEWIGVINPYNGGTDYNQKFK
GKATLTVDKSSSTAYMELNSLTSEDSAVYYCARSVYDYPFDYWGQGTLVTVSSASTKGPSVFPLAPS
SKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYI
CNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVWDV
VKFNWWDGVEVHNAKTKPREEQYNSTYRWSVLTVLHQDWLNGKEYKCKVSNKALPAPI
EKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLD
SDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
SEQ ID 119 - S332121F02 chimeric heavy chain (DNA sequence)
GAGGTGCAGCTGCAGCAGAGCGGCCCCGTGCTGGTGAAGCCTGGAGCCAGCGTGAAAATGAG
40 AGCCAGCGGCTACACCTTCACCGACTACTACATGAACTGGGTGAAGCAGAGCCACGG
CAAGACCCTGGAGTGGATCGGCGTGATCAACCCCTACAACGGGGGCACCGACTACAACCAGAA
GTTCAAGGGCAAGGCCACTCTGACCGTGGACAAGAGCTCCAGCACCGCCTACATGGAACTGAA
CACCTCTGAGGACAGCGCCGTCTATTACTGCGCCAGGAGCGTGTACGACTACCCCTTC
GACTACTGGGGCCAGGGCACACTAGTGACCGTGTCCAGCGCCAGCACCAAGGGCCCCAGCGT
GTTCCCCCTGGCCCCCAGCAGCAAGAGCACCAGCGGCGGCACAGCCGCCCTGGGCTGCCTGG
TGAAGGACTACTTCCCCGAACCGGTGACCGTGTCCTGGAACAGCGGAGCCCTGACCAGCGGCG
TGCACACCTTCCCCGCCGTGCTGCAGAGCAGCGGCCTGTACAGCCTGAGCAGCGTGGTGACCG
TGCCCAGCAGCAGCCTGGGCACCCAGACCTACATCTGTAACGTGAACCACAAGCCCAGCAACAC
CAAGGTGGACAAGAAGGTGGAGCCCAAGAGCTGTGACAAGACCCACACCTGCCCCCCCTGCCC
CGAGCTGCTGGGAGGCCCCAGCGTGTTCCTGTTCCCCCCCAAGCCTAAGGACACCCT
GATGATCAGCAGAACCCCCGAGGTGACCTGTGTGGTGGTGGATGTGAGCCACGAGGACCCTGA
GGTGAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCACAATGCCAAGACCAAGCCCAGGGA
GTACAACAGCACCTACCGGGTGGTGTCCGTGCTGACCGTGCTGCACCAGGATTGGCT
GAACGGCAAGGAGTACAAGTGTAAGGTGTCCAACAAGGCCCTGCCTGCCCCTATCGAGAAAACC
ATCAGCAAGGCCAAGGGCCAGCCCAGAGAGCCCCAGGTGTACACCCTGCCCCCTAGCAGAGAT
GAGCTGACCAAGAACCAGGTGTCCCTGACCTGCCTGGTGAAGGGCTTCTACCCCAGCGACATC
GCCGTGGAGTGGGAGAGCAACGGCCAGCCCGAGAACAACTACAAGACCACCCCCCCTGTGCTG
GACAGCGATGGCAGCTTCTTCCTGTACAGCAAGCTGACCGTGGACAAGAGCAGATGGCAGCAG
GGCAACGTGTTCAGCTGCTCCGTGATGCACGAGGCCCTGCACAATCACTACACCCAGAAGAGCC
TGAGCCTGTCCCCTGGCAAG
SEQ ID 120 - S332121F02 murine variable light chain
DIVLTQSPASLAVSLGQRATISCRASESVSIHGTHLMHWYQQKPGQPPKLLIYAASNLESGVPARFSG
SGSETDFTUWHPVEEEDAATYFCQQSEDPRTFGGGTKLHK
SEQ ID 121 - S332121F02 murine variable light chain (DNA sequence)
GACATCGTCCTGACCCAGAGCCCCGCCAGCCTGGCCGTGAGCCTGGGCCAGAGGGCCACAATC
AGCTGCAGGGCCTCTGAGTCCGTGAGCATCCACGGCACCCACCTGATGCACTGGTATCAGCAG
AAGCCCGGCCAGCCTCCCAAGCTGCTGATCTACGCCGCCAGCAACCTGGAGAGCGGCGTGCCC
GCTAGGTTCAGCGGAAGCGGCAGCGAGACCGACTTCACCCTGAACATCCACCCCGTGGAGGAG
GCCGCCACCTACTTCTGCCAGCAGAGCATCGAGGACCCCAGGACCTTCGGCGGGGGC
ACCAAGCTCGAGAITAAGCGT
SEQ ID 122 - S332121F02 chimeric light chain
MGWSCI|LFLVATATGVHSDIVLTQSPASLAVSLGQRATISCRASESVSIHGTHLMHWYQQKPGQPPK
LUYAASNLESGVPARFSGSGSETDFTUWHPVEEEDAATYFCQQSEDPRTFGGGTKLHKRTVAAPS
VHFPPSDEQLKSGTASVVCLLNNFYPREAKVCWVKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTL
SKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC
SEQ ID 123 - S332121F02 chimeric light chain (DNA sequence)
ATGGGCTGGTCCTGCATCATCCRFHTCTGGTGGCCACCGCCACCGGCGTGCACAGCGACATC
GTCCTGACCCAGAGCCCCGCCAGCCTGGCCGTGAGCCTGGGCCAGAGGGCCACAATCAGCTG
CAGGGCCTCTGAGTCCGTGAGCATCCACGGCACCCACCTGATGCACTGGTATCAGCAGAAGCC
CGGCCAGCCTCCCAAGCTGCTGATCTACGCCGCCAGCAACCTGGAGAGCGGCGTGCCCGCTAG
40 GTTCAGCGGAAGCGGCAGCGAGACCGACTTCACCCTGAACATCCACCCCGTGGAGGAGGAAGA
CACCTACTTCTGCCAGCAGAGCATCGAGGACCCCAGGACCTTCGGCGGGGGCACCAA
GCTCGAGAITAAGCGTACGGTGGCCGCCCCCAGCGTGTTCATCTTCCCCCCCAGCGATGAGCA
GCTGAAGAGCGGCACCGCCAGCGTGGTGTGTCTGCTGAACAACTTCTACCCCCGGGAGGCCAA
GGTGCAGTGGAAGGTGGACAATGCCCTGCAGAGCGGCAACAGCCAGGAGAGCGTGACCGAGC
AGGACAGCAAGGACTCCACCTACAGCCTGAGCAGCACCCTGACCCTGAGCAAGGCCGACTACG
AGAAGCACAAGGTGTACGCCTGTGAGGTGACCCACCAGGGCCTGTCCAGCCCCGTGACCAAGA
GCTTCAACCGGGGCGAGTGC
SEQ ID 124 - S322110D07 murine variable heavy chain
SGPELVKPGTSVKIPCKTSGYIFTDYSIDWVKQSHGKSLEWIGD|DPNYGDPIYNHKFKGKA
TLTVDRSSSTAYMELRSLTSEDTAVYFCARRATGTDWFAFWGQGTLVTVSS
SEQ ID 125 - S322110D07 murine variable heavy chain (DNA ce)
GAGGTGCAGCTGCAGCAGAGCGGCCCCGAGCTGGTGAAACCCGGCACCAGCGTGAAGATCCC
CTGCAAGACCTCTGGCTACATCTTCACCGACTACAGCATCGACTGGGTGAAGCAGAGCCACGGC
AAGTCTCTGGAGTGGATTGGGGACATCGACCCCAACTACGGCGACCCCATCTACAACCACAAGT
TCAAGGGCAAGGCCACCCTGACCGTGGACAGGAGCAGCAGCACCGCCTACATGGAACTCAGGA
GCCTGACCAGCGAGGACACCGCCGTGTATTTTTGCGCCAGGAGGGCCACCGGCACTGATTGGT
TCGCCTTCTGGGGCCAGGGCACACTAGTGACCGTGTCCAGC
SEQ ID 126 - S322110D07 chimeric heavy chain
EVQLQQSGPELVKPGTSVKIPCKTSGYIFTDYSIDWVKQSHGKSLEWIGD|DPNYGDPIYNHKFKGKA
TLTVDRSSSTAYMELRSLTSEDTAVYFCARRATGTDWFAFWGQGTLVTVSSASTKGPSVFPLAPSSK
AALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYIC
NVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCWVDVS
HEDPEVKFNWWDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIE
KTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDS
YSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
SEQ ID 127 - S322110D07 chimeric heavy chain (DNA sequence)
GAGGTGCAGCTGCAGCAGAGCGGCCCCGAGCTGGTGAAACCCGGCACCAGCGTGAAGATCCC
CTGCAAGACCTCTGGCTACATCTTCACCGACTACAGCATCGACTGGGTGAAGCAGAGCCACGGC
AAGTCTCTGGAGTGGATTGGGGACATCGACCCCAACTACGGCGACCCCATCTACAACCACAAGT
TCAAGGGCAAGGCCACCCTGACCGTGGACAGGAGCAGCAGCACCGCCTACATGGAACTCAGGA
GCCTGACCAGCGAGGACACCGCCGTGTATTTTTGCGCCAGGAGGGCCACCGGCACTGATTGGT
TCGCCTTCTGGGGCCAGGGCACACTAGTGACCGTGTCCAGCGCCAGCACCAAGGGCCCCAGCG
TGTTCCCCCTGGCCCCCAGCAGCAAGAGCACCAGCGGCGGCACAGCCGCCCTGGGCTGCCTG
GTGAAGGACTACTTCCCCGAACCGGTGACCGTGTCCTGGAACAGCGGAGCCCTGACCAGCGGC
ACCTTCCCCGCCGTGCTGCAGAGCAGCGGCCTGTACAGCCTGAGCAGCGTGGTGACC
GTGCCCAGCAGCAGCCTGGGCACCCAGACCTACATCTGTAACGTGAACCACAAGCCCAGCAAC
ACCAAGGTGGACAAGAAGGTGGAGCCCAAGAGCTGTGACAAGACCCACACCTGCCCCCCCTGC
CCTGCCCCCGAGCTGCTGGGAGGCCCCAGCGTGTTCCTGTTCCCCCCCAAGCCTAAGGACACC
CTGATGATCAGCAGAACCCCCGAGGTGACCTGTGTGGTGGTGGATGTGAGCCACGAGGACCCT
GAGGTGAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCACAATGCCAAGACCAAGCCCAGG
GAGGAGCAGTACAACAGCACCTACCGGGTGGTGTCCGTGCTGACCGTGCTGCACCAGGATTGG
CTGAACGGCAAGGAGTACAAGTGTAAGGTGTCCAACAAGGCCCTGCCTGCCCCTATCGAGAAAA
CCATCAGCAAGGCCAAGGGCCAGCCCAGAGAGCCCCAGGTGTACACCCTGCCCCCTAGCAGAG
40 ATGAGCTGACCAAGAACCAGGTGTCCCTGACCTGCCTGGTGAAGGGCTTCTACCCCAGCGACAT
CGCCGTGGAGTGGGAGAGCAACGGCCAGCCCGAGAACAACTACAAGACCACCCCCCCTGTGCT
GGACAGCGATGGCAGCTTCTTCCTGTACAGCAAGCTGACCGTGGACAAGAGCAGATGGCAGCA
GGGCAACGTGTTCAGCTGCTCCGTGATGCACGAGGCCCTGCACAATCACTACACCCAGAAGAG
CCTGAGCCTGTCCCCTGGCAAG
SEQ ID 128 - S322110D07 murine variable light chain
D|QMTQSPASLSVSVGETVTITCRASENIYNNLAWYQQKQGKSPQLLVYAATILADGVPSRFSGSGSG
TQYSLKINSLQSGDFGTYYCQHFWGTPLTFGAGTKLELKR
SEQ ID 129 - S322110D07 murine variable light chain (DNA sequence)
GACATCCAGATGACCCAGAGCCCCGCTAGCCTCAGCGTGTCCGTCGGCGAGACCGTGACCATC
ACCTGCAGGGCCAGCGAGAACATCTACAACAACCTGGCCTGGTATCAGCAGAAGCAGGGCAAA
AGCCCCCAGCTGCTGGTGTACGCCGCCACCATTCTGGCCGACGGCGTGCCCAGCAGGTTCTCT
GGAAGCGGCAGCGGCACCCAGTACAGCCTGAAGATCAACAGCCTGCAGAGCGGGGACTTCGG
CACCTACTACTGCCAGCACTTCTGGGGCACTCCCCTGACCTTCGGAGCCGGCACCAAGCTGGA
GCTGAAGCGT
SEQ ID 130 - S322110D07 chimeric light chain
D|QMTQSPASLSVSVGETVTITCRASENIYNNLAWYQQKQGKSPQLLVYAATILADGVPSRFSGSGSG
TQYSLKINSLQSGDFGTYYCQHFWGTPLTFGAGTKLELKRTVAAPSVFIFPPSDEQLKSGTASVVCLL
NNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLS
SPVTKSFNRGEC
SEQ ID 131 - 0D07 chimeric light chain (DNA ce)
GACATCCAGATGACCCAGAGCCCCGCTAGCCTCAGCGTGTCCGTCGGCGAGACCGTGACCATC
AGGGCCAGCGAGAACATCTACAACAACCTGGCCTGGTATCAGCAGAAGCAGGGCAAA
AGCCCCCAGCTGCTGGTGTACGCCGCCACCATTCTGGCCGACGGCGTGCCCAGCAGGTTCTCT
GGAAGCGGCAGCGGCACCCAGTACAGCCTGAAGATCAACAGCCTGCAGAGCGGGGACTTCGG
CACCTACTACTGCCAGCACTTCTGGGGCACTCCCCTGACCTTCGGAGCCGGCACCAAGCTGGA
GCTGAAGCGTACGGTGGCCGCCCCCAGCGTGTTCATCTTCCCCCCCAGCGATGAGCAGCTGAA
CACCGCCAGCGTGGTGTGTCTGCTGAACAACTTCTACCCCCGGGAGGCCAAGGTGCA
GTGGAAGGTGGACAATGCCCTGCAGAGCGGCAACAGCCAGGAGAGCGTGACCGAGCAGGACA
GCAAGGACTCCACCTACAGCCTGAGCAGCACCCTGACCCTGAGCAAGGCCGACTACGAGAAGC
ACAAGGTGTACGCCTGTGAGGTGACCCACCAGGGCCTGTCCAGCCCCGTGACCAAGAGCTTCA
GCGAGTGC
SEQ ID 132 — S332126E04 murine variable heavy chain
QVQLQQPGAELVKPGASVKLSCKASGYTFTNYWMHWVKQRPGQGLEWIGIIHPNSGSTNYNEKFKS
KATLTVDKSSSTAYMQLSSLTSEDSAVYYCARGIYDYPFAYWGQGTLVTVSS
SEQ ID 133 — S332126E04 murine variable heavy chain (DNA sequence)
CAGGTGCAGCTCCAGCAGCCCGGAGCCGAACTGGTGAAGCCCGGAGCCAGCGTCAAACTGTCC
TGCAAGGCCAGCGGCTACACCTTCACCAACTACTGGATGCACTGGGTGAAGCAGAGGCCCGGC
CAGGGCCTGGAGTGGATCGGCATCATCCACCCCAACAGCGGGAGCACCAACTACAACGAGAAG
TTCAAGAGCAAGGCCACCCTGACCGTGGACAAGAGCAGCAGCACTGCCTACATGCAGCTGAGC
AGCCTGACCAGCGAGGACAGCGCTGTGTACTACTGCGCCAGGGGCATCTACGACTACCCCTTC
GCCTATTGGGGCCAGGGCACACTAGTGACCGTGTCCAGC
SEQ ID 134 - S332126E04 Chimeric heavy chain
QVQLQQPGAELVKPGASVKLSCKASGYTFTNYWMHWVKQRPGQGLEWIGIIHPNSGSTNYNEKFKS
40 KATLTVDKSSSTAYMQLSSLTSEDSAVYYCARGIYDYPFAYWGQGTLVTVSSASTKGPSVFPLAPSS
KSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYI
CNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVWDV
SHEDPEVKFNWWDGVEVHNAKTKPREEQYNSTYRWSVLTVLHQDWLNGKEYKCKVSNKALPAPI
EKWSKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDVQHBNESNGQPENNYKTTPPVLD
LYSKLTVDKSRWKMDGNVFSCSVMHEALHNHYTQKSLSLSPGK
SEQ ID 135 - S332126E04 Chimeric heavy chain (DNA sequence)
CAGGTGCAGCTCCAGCAGCCCGGAGCCGAACTGGTGAAGCCCGGAGCCAGCGTCAAACTGTCC
TGCAAGGCCAGCGGCTACACCTTCACCAACTACTGGATGCACTGGGTGAAGCAGAGGCCCGGC
CAGGGCCTGGAGTGGATCGGCATCATCCACCCCAACAGCGGGAGCACCAACTACAACGAGAAG
TTCAAGAGCAAGGCCACCCTGACCGTGGACAAGAGCAGCAGCACTGCCTACATGCAGCTGAGC
AGCCTGACCAGCGAGGACAGCGCTGTGTACTACTGCGCCAGGGGCATCTACGACTACCCCTTC
GCCTATTGGGGCCAGGGCACACTAGTGACCGTGTCCAGCGCCAGCACCAAGGGCCCCAGCGTG
TTCCCCCTGGCCCCCAGCAGCAAGAGCACCAGCGGCGGCACAGCCGCCCTGGGCTGCCTGGT
GAAGGACTACTTCCCCGAACCGGTGACCGTGTCCTGGAACAGCGGAGCCCTGACCAGCGGCGT
GCACACCTTCCCCGCCGTGCTGCAGAGCAGCGGCCTGTACAGCCTGAGCAGCGTGGTGACCGT
CAGCAGCCTGGGCACCCAGACCTACATCTGTAACGTGAACCACAAGCCCAGCAACAC
CAAGGTGGACAAGAAGGTGGAGCCCAAGAGCTGTGACAAGACCCACACCTGCCCCCCCTGCCC
TGCCCCCGAGCTGCTGGGAGGCCCCAGCGTGTTCCTGTTCCCCCCCAAGCCTAAGGACACCCT
GATGATCAGCAGAACCCCCGAGGTGACCTGTGTGGTGGTGGATGTGAGCCACGAGGACCCTGA
GGTGAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCACAATGCCAAGACCAAGCCCAGGGA
GGAGCAGTACAACAGCACCTACCGGGTGGTGTCCGTGCTGACCGTGCTGCACCAGGATTGGCT
GAACGGCAAGGAGTACAAGTGTAAGGTGTCCAACAAGGCCCTGCCTGCCCCTATCGAGAAAACC
ATCAGCAAGGCCAAGGGCCAGCCCAGAGAGCCCCAGGTGTACACCCTGCCCCCTAGCAGAGAT
GAGCTGACCAAGAACCAGGTGTCCCTGACCTGCCTGGTGAAGGGCTTCTACCCCAGCGACATC
GCCGTGGAGTGGGAGAGCAACGGCCAGCCCGAGAACAACTACAAGACCACCCCCCCTGTGCTG
GACAGCGATGGCAGCTTCTTCCTGTACAGCAAGCTGACCGTGGACAAGAGCAGATGGCAGCAG
GGCAACGTGTTCAGCTGCTCCGTGATGCACGAGGCCCTGCACAATCACTACACCCAGAAGAGCC
TGAGCCTGTCCCCTGGCAAG
SEQ ID 136 — S332126E04 murine variable light chain
DIVLTQSPASLAVSLGQRATISCRASESVSIHGTHLMHWYQQKPGQPPKLLIYAASNLESGVPARFSG
SGSETDFTUWHPVEEEDAATYFCQQSEDPYTFGGGTKLHKR
SEQ ID 137 — S332126E04 murine le light chain (DNA sequence)
GACATCGTGCTGACCCAGTCTCCCGCTAGCCTGGCCGTGTCTCTGGGCCAGAGGGCCACAATC
AGCTGCAGGGCCAGCGAGAGCGTCAGCATTCACGGCACCCACCTGATGCACTGGTACCAGCAG
AAGCCCGGCCAGCCTCCCAAGCTCCTGATCTACGCCGCCAGCAACCTGGAAAGCGGAGTGCCC
GCCAGGTTCAGCGGCAGCGGCTCCGAGACCGACTTCACCCTGAACATCCACCCCGTGGAGGAG
GAGGACGCCGCCACCTACTTCTGCCAGCAGAGCATCGAGGACCCCTACACCTTCGGCGGCGGC
ACCAAGCTGGAGATCAAGCGTSEQID138-S33326E04ChhmncHgMChmn
SPASLAVSLGQRATISCRASESVSIHGTHLMHWYQQKPGQPPKLLIYAASNLESGVPARFSG
SGSETDFTUWHPVEEEDAATYFCQQSEDPYTFGGGTKLHKRTVAAPSVFFPPSDEQLKSGTASVV
CLLNNFYPREAKVCNVKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQ
GLSSPVTKSFNRGEC
40 SEQ ID 139 - S332126E04 Chimeric light chain (DNA sequence)
GTGCTGACCCAGTCTCCCGCTAGCCTGGCCGTGTCTCTGGGCCAGAGGGCCACAATC
AGCTGCAGGGCCAGCGAGAGCGTCAGCATTCACGGCACCCACCTGATGCACTGGTACCAGCAG
AAGCCCGGCCAGCCTCCCAAGCTCCTGATCTACGCCGCCAGCAACCTGGAAAGCGGAGTGCCC
GCCAGGTTCAGCGGCAGCGGCTCCGAGACCGACTTCACCCTGAACATCCACCCCGTGGAGGAG
GCCGCCACCTACTTCTGCCAGCAGAGCATCGAGGACCCCTACACCTTCGGCGGCGGC
ACCAAGCTGGAGATCAAGCGTACGGTGGCCGCCCCCAGCGTGTTCATCTTCCCCCCCAGCGAT
GAGCAGCTGAAGAGCGGCACCGCCAGCGTGGTGTGTCTGCTGAACAACTTCTACCCCCGGGAG
GCCAAGGTGCAGTGGAAGGTGGACAATGCCCTGCAGAGCGGCAACAGCCAGGAGAGCGTGAC
GGACAGCAAGGACTCCACCTACAGCCTGAGCAGCACCCTGACCCTGAGCAAGGCCGA
GAAGCACAAGGTGTACGCCTGTGAGGTGACCCACCAGGGCCTGTCCAGCCCCGTGAC
CAAGAGCTTCAACCGGGGCGAGTGC
SEQ ID 140 — S336105A07 murine variable heavy chain
EVKLLQSGGGLVQPGGSLKLSCAASGIDFSRYWMSWVRRAPGKGLEWIGEINPDRSTINYAPSLKDK
F||SRDNAKNTLYLQMSKVRSEDTALYYCAVFYYDYEGAMDYWGQGTSVTVSS
SEQ ID 141 — S336105A07 murine variable heavy chain (DNA sequence)
GAGGTGAAGCTTCTCCAGTCTGGAGGTGGCCTGGTGCAGCCTGGAGGATCCCTGAAACTCTCCT
GTGCAGCCTCAGGAATCGATTTTAGTAGATACTGGATGAGTTGGGTTCGGCGGGCTCCAGGGAA
AGGACTAGAATGGATTGGAGAAATTAATCCAGATAGGAGTACAATCAACTATGCACCATCTCTAA
AGGATAAATTCATCATCTCCAGAGACAACGCCAAAAATACGCTGTACCTGCAAATGAGCAAAGTG
AGATCTGAGGACACAGCCCTTTATTACTGTGCAGTTTTCTACTATGATTACGAGGGTGCTATGGA
CTACTGGGGTCAAGGAACCTCAGTCACCGTCTCCTCA
SEQ ID 142 - S336105A07 Chimeric heavy chain
EVKLLQSGGGLVQPGGSLKLSCAASGIDFSRYWMSWVRRAPGKGLEWIGEINPDRSTINYAPSLKDK
F||SRDNAKNTLYLQMSKVRSEDTALYYCAVFYYDYEGAMDYWGQGTSVTVSSAKTTAPSVFPLAPS
SKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYI
CNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVWDV
SHEDPEVKFNWWDGVEVHNAKTKPREEQYNSTYRWSVLTVLHQDWLNGKEYKCKVSNKALPAPI
EKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLD
SDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
SEQ ID 143 - 5A07 Chimeric heavy chain (DNA sequence)
GAGGTGAAGCTTCTCCAGTCTGGAGGTGGCCTGGTGCAGCCTGGAGGATCCCTGAAACTCTCCT
GTGCAGCCTCAGGAATCGATTTTAGTAGATACTGGATGAGTTGGGTTCGGCGGGCTCCAGGGAA
AGGACTAGAATGGATTGGAGAAATTAATCCAGATAGGAGTACAATCAACTATGCACCATCTCTAA
AGGATAAATTCATCATCTCCAGAGACAACGCCAAAAATACGCTGTACCTGCAAATGAGCAAAGTG
AGATCTGAGGACACAGCCCTTTATTACTGTGCAGTTTTCTACTATGATTACGAGGGTGCTATGGA
CTACTGGGGTCAAGGAACCTCAGTCACCGTCTCCTCAGCCAAAACAACAGCCCCCAGCGTGTTC
CCCCTGGCCCCCAGCAGCAAGAGCACCAGCGGCGGCACAGCCGCCCTGGGCTGCCTGGTGAA
GGACTACTTCCCCGAACCGGTGACCGTGTCCTGGAACAGCGGAGCCCTGACCAGCGGCGTGCA
CACCTTCCCCGCCGTGCTGCAGAGCAGCGGCCTGTACAGCCTGAGCAGCGTGGTGACCGTGCC
CAGCAGCAGCCTGGGCACCCAGACCTACATCTGTAACGTGAACCACAAGCCCAGCAACACCAAG
GTGGACAAGAAGGTGGAGCCCAAGAGCTGTGACAAGACCCACACCTGCCCCCCCTGCCCTGCC
CCCGAGCTGCTGGGAGGCCCCAGCGTGTTCCTGTTCCCCCCCAAGCCTAAGGACACCCTGATG
ATCAGCAGAACCCCCGAGGTGACCTGTGTGGTGGTGGATGTGAGCCACGAGGACCCTGAGGTG
40 AACTGGTACGTGGACGGCGTGGAGGTGCACAATGCCAAGACCAAGCCCAGGGAGGAG
CAGTACAACAGCACCTACCGGGTGGTGTCCGTGCTGACCGTGCTGCACCAGGATTGGCTGAAC
GGCAAGGAGTACAAGTGTAAGGTGTCCAACAAGGCCCTGCCTGCCCCTATCGAGAAAACCATCA
GCAAGGCCAAGGGCCAGCCCAGAGAGCCCCAGGTGTACACCCTGCCCCCTAGCAGAGATGAGC
TGACCAAGAACCAGGTGTCCCTGACCTGCCTGGTGAAGGGCTTCTACCCCAGCGACATCGCCGT
GGAGTGGGAGAGCAACGGCCAGCCCGAGAACAACTACAAGACCACCCCCCCTGTGCTGGACAG
CGATGGCAGCTTCTTCCTGTACAGCAAGCTGACCGTGGACAAGAGCAGATGGCAGCAGGGCAA
CAGCTGCTCCGTGATGCACGAGGCCCTGCACAATCACTACACCCAGAAGAGCCTGAGC
CTGTCCCCTGGCAAG
SEQ ID 144 — S336105A07 murine varaible light chain
DIVMTQSQKFMSTSVGDRVSVTCKASQNVDTNVAWYQQKPGQSPKALIYSASYRFSGVPDRFTGSG
SGTDFTLTISNVQSEDLAEYFCQQYNSFPFTFGSGTKLE|KR
SEQ ID 145 — S336105A07 murine variable light chain (DNA sequence)
GACATTGTGATGACCCAGTCTCAAAAATTCATGTCCACATCAGTAGGAGACAGGGTCAGCGTCAC
CTGCAAGGCCAGTCAGAATGTGGATACTAATGTAGCCTGGTATCAACAAAAACCAGGGCAATCTC
CTAAAGCACTGATTTACTCGGCATCCTACCGGTTCAGTGGAGTCCCTGATCGCTTCACAGGCAGT
GGGACAGATTTCACTCTCACCATCAGCAATGTGCAGTCTGAAGACTTGGCAGAGTATTT
CTGTCAGCAATATAACAGCTTTCCATTCACGTTCGGCTCGGGGACAAAGTTGGAAATAAAACGT
SEQ ID 146 - S336105A07 chimeric light chain
DIVMTQSQKFMSTSVGDRVSVTCKASQNVDTNVAWYQQKPGQSPKALIYSASYRFSGVPDRFTGSG
SGTDFTLTISNVQSEDLAEYFCQQYNSFPFTFGSGTKLE|KRTVAAPSVFIFPPSDEQLKSGTASVVCL
REAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGL
SSPVTKSFNRGEC
SEQ ID 147 - S336105A07 chimeric light chain (DNA sequence)
GACATTGTGATGACCCAGTCTCAAAAATTCATGTCCACATCAGTAGGAGACAGGGTCAGCGTCAC
CTGCAAGGCCAGTCAGAATGTGGATACTAATGTAGCCTGGTATCAACAAAAACCAGGGCAATCTC
CTAAAGCACTGATTTACTCGGCATCCTACCGGTTCAGTGGAGTCCCTGATCGCTTCACAGGCAGT
GGATCTGGGACAGATTTCACTCTCACCATCAGCAATGTGCAGTCTGAAGACTTGGCAGAGTATTT
CTGTCAGCAATATAACAGCTTTCCATTCACGTTCGGCTCGGGGACAAAGTTGGAAATAAAACGTA
CGGTGGCCGCCCCCAGCGTGTTCATCTTCCCCCCCAGCGATGAGCAGCTGAAGAGCGGCACCG
CCAGCGTGGTGTGTCTGCTGAACAACTTCTACCCCCGGGAGGCCAAGGTGCAGTGGAAGGTGG
ACAATGCCCTGCAGAGCGGCAACAGCCAGGAGAGCGTGACCGAGCAGGACAGCAAGGACTCCA
CCTACAGCCTGAGCAGCACCCTGACCCTGAGCAAGGCCGACTACGAGAAGCACAAGGTGTACG
CCTGTGAGGTGACCCACCAGGGCCTGTCCAGCCCCGTGACCAAGAGCTTCAACCGGGGCGAGT
GC
SEQ ID 148 — S335115G01 murine variable heavy chain
PVQLQQPGTELVRPGTSVKLSCKASGYTFTSYWMHWVKQRPGQGLEWIGVIDPSDSYTNYNQKFK
GKATLTVDTSSSTAYMQLSSLTSEDSAVYYCARQVFDYPMDYWGQGTSVTVSS
SEQID 149 — 5G01 murine variable heavy chain (DNA sequence)
CCGGTCCAACTGCAGCAGCCTGGGACTGAGCTGGTGAGGCCTGGGACTTCAGTGAAGTTGTCC
TGCAAGGCTTCTGGCTACACCTTCACCAGCTACTGGATGCACTGGGTAAAGCAGAGGCCTGGAC
AAGGCCTTGAGTGGATCGGAGTGATTGATCCTTCTGATAGTTATACTAACTACAATCAAAAGTTCA
AGGGCAAGGCCACATTGACTGTAGACACATCCTCCAGCACAGCCTACATGCAGCTCAGCAGCCT
GACATCTGAGGACTCTGCGGTCTATTACTGTGCAAGACAGGTGTTTGACTATCCTATGGACTACT
40 GGGGTCAAGGAACCTCAGTCACCGTCTCCTCA
SEQ ID 150 - S335115G01 Chimeric heavy chain
PVQLQQPGTELVRPGTSVKLSCKASGYTFTSHWMHWVKQRPGQGLEWIGVIDPSDSYTNYNQKFK
GKATLTVDTSSSTAYMQLSSLTSEDSAVYYCARQVFDYPMDYWGQGTLVTVSSASTKGPSVFPLAP
SSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSWTVPSSSLGTQT
YICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVD
VSHEDPEVKFNWWDGVEVHNAKTKPREEQYNSTYRWSVLTVLHQDWLNGKEYKCKVSNKALPAP
|EKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLD
SDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
SEQ ID 151 - S335115G01 Chimeric heavy chain (DNA sequence)
CCGGTCCAACTGCAGCAGCCTGGGACTGAGCTGGTGAGGCCTGGGACTTCAGTGAAGTTGTCC
GCTTCTGGCTACACCTTCACCAGCCACTGGATGCACTGGGTAAAGCAGAGGCCTGGAC
AAGGCCTTGAGTGGATCGGAGTGATTGATCCTTCTGATAGTTATACTAACTACAATCAAAAGTTCA
AGGGCAAGGCCACATTGACTGTAGACACATCCTCCAGCACAGCCTACATGCAGCTCAGCAGCCT
GACATCTGAGGACTCTGCGGTCTATTACTGTGCAAGACAGGTGTTTGACTATCCTATGGACTACT
GGGGTCAAGGAACACTAGTGACCGTGTCCAGCGCCAGCACCAAGGGCCCCAGCGTGTTCCCCC
TGGCCCCCAGCAGCAAGAGCACCAGCGGCGGCACAGCCGCCCTGGGCTGCCTGGTGAAGGAC
TACTTCCCCGAACCGGTGACCGTGTCCTGGAACAGCGGAGCCCTGACCAGCGGCGTGCACACC
TTCCCCGCCGTGCTGCAGAGCAGCGGCCTGTACAGCCTGAGCAGCGTGGTGACCGTGCCCAGC
AGCAGCCTGGGCACCCAGACCTACATCTGTAACGTGAACCACAAGCCCAGCAACACCAAGGTG
GACAAGAAGGTGGAGCCCAAGAGCTGTGACAAGACCCACACCTGCCCCCCCTGCCCTGCCCCC
GAGCTGCTGGGAGGCCCCAGCGTGTTCCTGTTCCCCCCCAAGCCTAAGGACACCCTGATGATC
AGCAGAACCCCCGAGGTGACCTGTGTGGTGGTGGATGTGAGCCACGAGGACCCTGAGGTGAAG
TTCAACTGGTACGTGGACGGCGTGGAGGTGCACAATGCCAAGACCAAGCCCAGGGAGGAGCAG
TACAACAGCACCTACCGGGTGGTGTCCGTGCTGACCGTGCTGCACCAGGATTGGCTGAACGGC
AAGGAGTACAAGTGTAAGGTGTCCAACAAGGCCCTGCCTGCCCCTATCGAGAAAACCATCAGCA
AGGCCAAGGGCCAGCCCAGAGAGCCCCAGGTGTACACCCTGCCCCCTAGCAGAGATGAGCTGA
CCAAGAACCAGGTGTCCCTGACCTGCCTGGTGAAGGGCTTCTACCCCAGCGACATCGCCGTGG
AGTGGGAGAGCAACGGCCAGCCCGAGAACAACTACAAGACCACCCCCCCTGTGCTGGACAGCG
ATGGCAGCTTCTTCCTGTACAGCAAGCTGACCGTGGACAAGAGCAGATGGCAGCAGGGCAACG
TGTTCAGCTGCTCCGTGATGCACGAGGCCCTGCACAATCACTACACCCAGAAGAGCCTGAGCCT
GTCCCCTGGCAAG
SEQ ID 152 — S335115G01 murine variable light chain
SPASLAVSLGQRATISCRASESVSIHGTHLMHWYQQKPGQPPKLLIYAASNLESGVPARFSG
SGSETDFTLN|HPVEEEDAATYFCQQSIEDPWTFGGGTKLEIKR
SEQ ID 153 — S335115G01 murine variable light chain (DNA sequence)
GACATTGTGCTGACCCAATCTCCAGCTTCTTTGGCTGTGTCTCTAGGGCAGAGGGCCACCATCT
CCTGCAGAGCCAGTGAAAGTGTCAGTATTCATGGTACTCATTTAATGCACTGGTACCAACAGAAA
CCAGGACAGCCACCCAAACTCCTCATCTATGCTGCATCCAACCTAGAATCTGGAGTCCCTGCCA
GTGGCAGTGGGTCTGAGACAGACTTCACCCTCAACATCCATCCTGTGGAGGAGGAGGA
TGCTGCAACCTATTTCTGTCAGCAAAGTATTGAGGATCCGTGGACGTTCGGTGGAGGCACCAAG
ATCAAACGT
SEQ ID 154 - 5G01 Chimeric light chain
DIVLTQSPASLAVSLGQRATISCRASESVSIHGTHLMHWYQQKPGQPPKLLIYAASNLESGVPARFSG
40 SGSETDFTLN|HPVEEEDAATYFCQQSIEDPWTFGGGTKLEINRTVAAPSVFIFPPSDEQLKSGTASVV
CLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQ
GLSSPVTKSFNRGEC
SEQ ID 155 - S335115G01 Chimeric light chain (DNA sequence)
GACATTGTGCTGACCCAATCTCCAGCTTCTTTGGCTGTGTCTCTAGGGCAGAGGGCCACCATCT
GAGCCAGTGAAAGTGTCAGTATTCATGGTACTCATTTAATGCACTGGTACCAACAGAAA
CCAGGACAGCCACCCAAACTCCTCATCTATGCTGCATCCAACCTAGAATCTGGAGTCCCTGCCA
GGTTCAGTGGCAGTGGGTCTGAGACAGACTTCACCCTCAACATCCATCCTGTGGAGGAGGAGGA
AACCTATTTCTGTCAGCAAAGTATTGAGGATCCGTGGACGTTCGGTGGAGGCACCAAG
CTGGAAATCAATCGTACGGTGGCCGCCCCCAGCGTGTTCATCTTCCCCCCCAGCGATGAGCAGC
TGAAGAGCGGCACCGCCAGCGTGGTGTGTCTGCTGAACAACTTCTACCCCCGGGAGGCCAAGG
TGCAGTGGAAGGTGGACAATGCCCTGCAGAGCGGCAACAGCCAGGAGAGCGTGACCGAGCAG
GACAGCAAGGACTCCACCTACAGCCTGAGCAGCACCCTGACCCTGAGCAAGGCCGACTACGAG
AAGCACAAGGTGTACGCCTGTGAGGTGACCCACCAGGGCCTGTCCAGCCCCGTGACCAAGAGC
TTCAACCGGGGCGAGTGC
SEQ ID 156 — S335122F05 murine variable heavy chain
QVQLQQSGAELVRPGASVTLSCKASGYTFTDYEMHWVKQTPVHGLEWIGAIDPETGGTAYNQKFKG
KAILTADKSSSTAYMELRSLTSEDSAVYYCTRSIYDYYFDYWGQGTTLTVSS
SEQ ID 157 — S335122F05 murine variable heavy chain (DNA sequence)
CAGGTTCAACTGCAGCAGTCTGGGGCTGAGCTGGTGAGGCCTGGGGCTTCAGTGACGCTGTCC
TGCAAGGCTTCGGGCTACACATTTACTGACTATGAAATGCACTGGGTGAAGCAGACACCTGTGC
TGGAATGGATTGGAGCTATTGATCCTGAAACTGGTGGTACTGCCTACAATCAGAAGTTC
AAGGGCAAGGCCATACTGACTGCAGACAAATCCTCCAGCACAGCCTACATGGAGCTCCGCAGCC
TGACATCTGAGGACTCTGCCGTCTATTACTGTACAAGATCGATTTATGATTACTACTTTGACTACT
GGGGCCAAGGCACCACTCTCACAGTCTCCTCA
SEQ ID 158 - S335122F05 Chimeric heavy chain
QVQLQQSGAELVRPGASVTLSCKASGYTFTDYEMHWVKQTPVHGLEWIGAIDPETGGTAYNQKFKG
KAILTADKSSSTAYMELRSLTSEDSAVYYCTRSIYDYYFDYWGQGTTLTVSSAKTTPPSVFPLAPSSK
STSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYIC
NVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCWVDVS
HEDPEVKFNWWDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIE
KTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDS
DGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
SEQ ID 159 - 2F05 Chimeric heavy chain (DNA sequence)
CAGGTTCAACTGCAGCAGTCTGGGGCTGAGCTGGTGAGGCCTGGGGCTTCAGTGACGCTGTCC
TGCAAGGCTTCGGGCTACACATTTACTGACTATGAAATGCACTGGGTGAAGCAGACACCTGTGC
ATGGCCTGGAATGGATTGGAGCTATTGATCCTGAAACTGGTGGTACTGCCTACAATCAGAAGTTC
AAGGGCAAGGCCATACTGACTGCAGACAAATCCTCCAGCACAGCCTACATGGAGCTCCGCAGCC
TGACATCTGAGGACTCTGCCGTCTATTACTGTACAAGATCGATTTATGATTACTACTTTGACTACT
GGGGCCAAGGCACCACTCTCACAGTCTCCTCAGCCAAAACGACACCCCCCAGCGTGTTCCCCCT
GGCCCCCAGCAGCAAGAGCACCAGCGGCGGCACAGCCGCCCTGGGCTGCCTGGTGAAGGACT
CCGAACCGGTGACCGTGTCCTGGAACAGCGGAGCCCTGACCAGCGGCGTGCACACCT
TCCCCGCCGTGCTGCAGAGCAGCGGCCTGTACAGCCTGAGCAGCGTGGTGACCGTGCCCAGCA
40 GCAGCCTGGGCACCCAGACCTACATCTGTAACGTGAACCACAAGCCCAGCAACACCAAGGTGG
ACAAGAAGGTGGAGCCCAAGAGCTGTGACAAGACCCACACCTGCCCCCCCTGCCCTGCCCCCG
AGCTGCTGGGAGGCCCCAGCGTGTTCCTGTTCCCCCCCAAGCCTAAGGACACCCTGATGATCA
GCAGAACCCCCGAGGTGACCTGTGTGGTGGTGGATGTGAGCCACGAGGACCCTGAGGTGAAGT
TCAACTGGTACGTGGACGGCGTGGAGGTGCACAATGCCAAGACCAAGCCCAGGGAGGAGCAGT
ACAACAGCACCTACCGGGTGGTGTCCGTGCTGACCGTGCTGCACCAGGATTGGCTGAACGGCA
AGGAGTACAAGTGTAAGGTGTCCAACAAGGCCCTGCCTGCCCCTATCGAGAAAACCATCAGCAA
GGCCAAGGGCCAGCCCAGAGAGCCCCAGGTGTACACCCTGCCCCCTAGCAGAGATGAGCTGAC
CAAGAACCAGGTGTCCCTGACCTGCCTGGTGAAGGGCTTCTACCCCAGCGACATCGCCGTGGA
GTGGGAGAGCAACGGCCAGCCCGAGAACAACTACAAGACCACCCCCCCTGTGCTGGACAGCGA
TGGCAGCTTCTTCCTGTACAGCAAGCTGACCGTGGACAAGAGCAGATGGCAGCAGGGCAACGT
GTTCAGCTGCTCCGTGATGCACGAGGCCCTGCACAATCACTACACCCAGAAGAGCCTGAGCCTG
TCCCCTGGCAAG
SEQ ID 160 — S335122F05 murine variable light chain
DIVLTQSPASLAVSLGQRATISCRASESVSIHGTHLMHWYQQKPGQPPKLLIYAASNLESGVPARFSG
FTLNIHPVEEEDGATYFCQQSIEYPRTFGGGTKLEINR
SEQ ID 161 — S335122F05 murine variable light chain (DNA sequence)
GACATTGTGCTGACCCAATCTCCAGCTTCTTTGGCTGTGTCTCTAGGGCAGAGGGCCACCATCT
CCTGCAGAGCCAGTGAAAGTGTCAGTATTCATGGTACTCATTTAATGCACTGGTACCAACAGAAA
CCAGGACAGCCACCCAAACTCCTCATCTATGCTGCATCCAACCTAGAATCTGGAGTCCCTGCCA
GGTTCAGTGGCGGTGGGTCTGAGACAGACTTCACCCTCAACATCCATCCTGTGGAGGAGGAGG
ATGGTGCAACCTATTTCTGTCAGCAAAGTATTGAGTATCCTCGGACGTTCGGTGGAGGCACCAA
GCTGGAAATCAATCGT
SEQ ID 162 - S335122F05 ic light chain
DIVLTQSPASLAVSLGQRATISCRASESVSIHGTHLMHWYQQKPGQPPKLLIYAASNLESGVPARFSG
GGSETDFTLNIHPVEEEDGATYFCQQSIEYPRTFGGGTKLEINRTVAAPSVFIFPPSDEQLKSGTASVV
CLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQ
GLSSPVTKSFNRGEC
SEQ ID 163 - S335122F05 Chimeric light chain (DNA sequence)
GACATTGTGCTGACCCAATCTCCAGCTTCTTTGGCTGTGTCTCTAGGGCAGAGGGCCACCATCT
CCTGCAGAGCCAGTGAAAGTGTCAGTATTCATGGTACTCATTTAATGCACTGGTACCAACAGAAA
CCAGGACAGCCACCCAAACTCCTCATCTATGCTGCATCCAACCTAGAATCTGGAGTCCCTGCCA
GGTTCAGTGGCGGTGGGTCTGAGACAGACTTCACCCTCAACATCCATCCTGTGGAGGAGGAGG
ATGGTGCAACCTATTTCTGTCAGCAAAGTATTGAGTATCCTCGGACGTTCGGTGGAGGCACCAA
AATCAATCGTACGGTGGCCGCCCCCAGCGTGTTCATCTTCCCCCCCAGCGATGAGCA
GCTGAAGAGCGGCACCGCCAGCGTGGTGTGTCTGCTGAACAACTTCTACCCCCGGGAGGCCAA
GGTGCAGTGGAAGGTGGACAATGCCCTGCAGAGCGGCAACAGCCAGGAGAGCGTGACCGAGC
AGGACAGCAAGGACTCCACCTACAGCCTGAGCAGCACCCTGACCCTGAGCAAGGCCGACTACG
AGAAGCACAAGGTGTACGCCTGTGAGGTGACCCACCAGGGCCTGTCCAGCCCCGTGACCAAGA
GCTTCAACCGGGGCGAGTGC
SEQ.|.D.NO: 164 - S332121F02 CDRH1
DYYNM
D.NO: 165 - S332121F02 CDRH2
VINPYNGGTDYNQKFG
40 SEQ.|.D.NO: 166 - 1F02 CDRH3
SVYDYPFDY
SEQ.|.D.NO: 167 - S332121F02 CDRL1
RASESVSIHGTHLMH
SEQ.|.D.NO: 168 - S332121F02 CDRL2
AASNLES
SEQ.|.D.NO: 169 - S332121F02 CDRL3
SEQ.|.D.NO: 170 - S322110D07 CDRH1
DYSID
SEQ.|.D.NO: 171 - S322110D07 CDRH2
DIDPNYGDPIYNHKFKG
SEQ.|.D.NO: 172 - S322110D07 CDRH3
RATGTDWFAF
D.NO: 173 - S322110D07CDRL1
RASENIYNNLA
SEQ.|.D.NO: 174 - S322110D07 CDRL2
SEQ.|.D.NO: 175 - S322110D07 CDRL3
QHFWGTPLT
SEQ.|.D.NO: 176 - S332126E04CDRH1
NYWMH
SEQ.|.D.NO: 177 - S332126E04 CDRH2
IIHPNSGSTNYNEKFKS
SEQ.|.D.NO: 178 - S332126E04 CDRH3
GIYDYPFAY
SEQ.|.D.NO: 179 - S332126E04 CDRL1
RASESVSIHGTHLMH
SEQ.|.D.NO: 180 - S332126E04 CDRL2
S
SEQ.|.D.NO: 181 - S332126E04 CDRL3
QQSIEDPYT
SEQ.|.D.NO: 182 - S336105A07 CDRH1
RYWMS
SEQ.|.D.NO: 183 - S336105A07 CDRH2
EINPDRSTINYAPSLKD
SEQ.|.D.NO: 184 - S336105A07 CDRH3
FYYDYEGAMDY
D.NO: 185 - S336105A07 CDRL1
KASQNVDTNVA
SEQ.|.D.NO: 186 - S336105A07 CDRL2
SASYRFS
SEQ.|.D.NO: 187 - S336105A07 CDRL3
QQYNSFPFT
40 D.NO: 188 - S335115G01CDRH1
SYWMH
SEQ.|.D.NO: 189 - S335115G01CDRH2
VIDPSDSYTNYNQKFKG
SEQ.|.D.NO: 190 - S335115G01CDRH3
QVFDYPMDY
SEQ.|.D.NO: 191 - S335115G01CDRL1
RASESVSIHGTHLMH
SEQ.|.D.NO: 192 - S335115G01CDRL2
AASNLES
SEQ.|.D.NO: 193 - S335115G01CDRL3
QQSIEDPWT
SEQ.|.D.NO: 194 - S335122F05 CDRH1
DYEMH
SEQ.|.D.NO: 195 - S335122F05 CDRH2
AIDPETGGTAYNQKFKG
SEQ.|.D.NO: 196 - S335122F05 CDRH3
SIYDYYFDY
SEQ.|.D.NO: 197 - 2F05 CDRL1
RASESVSIHGTHLMH
SEQ.|.D.NO: 198 - S335122F05 CDRL2
AASNLES
SEQ.|.D.NO: 199 - S335122F05 CDRL3
QQSIEYPRT
Claims (32)
1. An antigen binding protein which specifically binds to BCMA and which inhibits the binding of BAFF and/or APRIL to BCMA, wherein the antigen binding protein is capable of binding to FcγRIIIA or is capable of FcγRIIIA mediated effector function, and wherein the antigen binding n is capable of internalisation and wherein the antigen binding protein comprises CDRH3 of SEQ ID NO.3 or a variant of SEQ ID NO. 3, CDR H1 of SEQ. ID. NO: 1, CDRH2: SEQ. ID. NO: 2, CDRL1: SEQ. ID. NO: 4, CDRL2: SEQ. ID. NO: 5 and CDRL3: SEQ. ID. NO:
2. An antigen g protein according to claim 1 wherein the antigen binding protein has enhanced g to FcγRIIIA or has enhanced FcγRIIIA mediated effector function.
3. The antigen binding protein of claim 2 wherein the antigen binding fragment has enhanced ADCC effector function.
4. The antigen binding protein ing to any preceding claim wherein the n binding protein is defucosylated.
5. The antigen binding protein according to any preceding claim wherein the antigen binding fragment does not bind to Taci.
6. The antigen binding protein according to any preceding claim which comprises a heavy chain variable region encoded by any one of SEQ. ID. NO:23 or SEQ. ID. NO:27 or SEQ. ID. NO:29.
7. The antigen binding n ing to any preceding claim which comprises a light chain variable region encoded by any one of SEQ. ID. NO:31 or SEQ. ID. NO:33.
8. The antigen binding protein according to any one of claims 1-7 wherein the antigen binding protein comprises a heavy chain variable region encoded by SEQ. ID. NO:23 and a light chain variable region encoded by SEQ. ID. NO:31.
9. The antigen binding protein according to any preceding claim n the n binding protein ses a heavy chain encoded by SEQ. ID. NO:27 and a light chain encoded by SEQ. ID. NO:31.
10. The antigen binding protein according to any preceding claim n the antigen binding protein is a humanised onal antibody.
11. The antigen binding n according to claim 10 wherein the antibody is an IgG1 isotype.
12. The n binding protein of any ing claim wherein the antigen binding protein is a fragment which is a Fab, Fab’, F(ab’)2, Fv, diabody, triabody, tetrabody, miniantibody, minibody, isolated VH or ed VL.
13. The n binding protein according to any preceding claim wherein the antigen binding protein additionally binds non-human primate BCMA.
14. The antigen binding protein according to any one of the preceding claims and n the antigen binding protein binds BCMA with an affinity of stronger than 150pM.
15. An immunoconjugate sing the antigen binding protein according to any preceding claim and a cytotoxic agent.
16. The conjugate of claim 15 wherein the antigen binding n is linked to the cytotoxic agent via a linker.
17. The immunoconjugate of claim 15 or 16 wherein the cytotoxic agent is an auristatin or a dolostatin.
18. The immunoconjugate of any one of claims 15 to 17 wherein the cytotoxic agent is ed from MMAE and MMAF.
19. The immunoconjugate of any one of claims 15 to 18 wherein the cytotoxic agent is covalently bound to said antigen g protein.
20. The immunoconjugate of any one of claims 16 to 19 wherein said linker is a cleavable linker.
21. The immunoconjugate of any one of claims 16 to 19 wherein said linker is a non-cleavable linker
22. The immunoconjugate of any one of claims 16 to 21 wherein the linker is selected from 6- maleimidocaproyl (MC), maleimidopropanoyl (MP), valine-citrulline (val-cit), alaninephenylalanine (ala-phe), p-aminobenzyloxycarbonyl (PAB), N-Succinimidyl 4-(2- pyridylthio)pentanoate (SPP), N-succinimidyl 4-(N-maleimidomethyl)cyclohexane-1 carboxylate (SMCC), and N-Succinimidyl (4-iodo-acetyl) aminobenzoate (SIAB).
23. The immunoconjugate of any one of claims 16 to 22 wherein said immunoconjugate is engulfed by a tumor cell when contacted with a tumor cell.
24. A pharmaceutical composition comprising an antigen binding protein or immunoconjugate according to any preceding claim and a pharmaceutically acceptable carrier.
25. Use of an antigen binding protein ing to any one of claims 1 to 14, an immunoconjugate according to any one of claims 15 to 23 or a composition of claim 24, in the manufacture of a medicament for treating a human patient ted with a B cell lymphoma.
26. The use according to claim 25, wherein the B cell lymphoma is Multiple Myeloma (MM) or Chronic Lymphocytic mia (CLL).
27. The use of an antigen g protein according to any one of claims 1 to 14, an immunoconjugate according to any one of claims 15 to 23, or a composition of claim 24, in the manufacture of a medicament for treating a human patient afflicted with an inflammatory disorder or disease.
28. An antigen binding n ing to claim 1, substantially as herein described or ified.
29. An immunoconjugate according to claim 15, substantially as herein described or exemplified.
30. A pharmaceutical composition according to claim 24, substantially as herein described or exemplified.
31. A use according to claim 25, substantially as herein described or exemplified.
32. A use according to claim 27, substantially as herein described or exemplified.
Applications Claiming Priority (5)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US201161490732P | 2011-05-27 | 2011-05-27 | |
US61/490,732 | 2011-05-27 | ||
US201261647196P | 2012-05-15 | 2012-05-15 | |
US61/647,196 | 2012-05-15 | ||
PCT/EP2012/059762 WO2012163805A1 (en) | 2011-05-27 | 2012-05-24 | Bcma (cd269/tnfrsf17) -binding proteins |
Publications (2)
Publication Number | Publication Date |
---|---|
NZ616433A NZ616433A (en) | 2016-04-29 |
NZ616433B2 true NZ616433B2 (en) | 2016-08-02 |
Family
ID=
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