NZ616433B2 - Bcma (cd269/tnfrsf17) -binding proteins - Google Patents

Bcma (cd269/tnfrsf17) -binding proteins Download PDF

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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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seq
antigen binding
binding protein
antibody
bcma
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NZ616433A
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NZ616433A (en
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Paul Algate
Stephanie Jane Clegg
Jennifer L Craigen
Paul Andrew Hamblin
Alan Peter Lewis
Patrick Mayes
Radha Shah Parmar
Trevor Anthony Kenneth Wattam
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Glaxo Group Limited
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Priority claimed from PCT/EP2012/059762 external-priority patent/WO2012163805A1/en
Publication of NZ616433A publication Critical patent/NZ616433A/en
Publication of NZ616433B2 publication Critical patent/NZ616433B2/en

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    • A—HUMAN NECESSITIES
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    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • A—HUMAN NECESSITIES
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    • A61K47/50—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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—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
    • A61K47/50—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
    • A61K47/51—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
    • A61K47/68—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
    • A61K47/6801—Drug-antibody or immunoglobulin conjugates defined by the pharmacologically or therapeutically active agent
    • A61K47/6803—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates
    • A61K47/6811—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates the drug being a protein or peptide, e.g. transferrin or bleomycin
    • A61K47/6817—Toxins
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—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
    • A61K47/50—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
    • A61K47/51—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
    • A61K47/68—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
    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A61P35/02—Antineoplastic agents specific for leukemia
    • A—HUMAN NECESSITIES
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    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • 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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    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • 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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • 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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    • C07—ORGANIC CHEMISTRY
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    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
    • C07K2317/33—Crossreactivity, e.g. for species or epitope, or lack of said crossreactivity
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    • C07—ORGANIC CHEMISTRY
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    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/40—Immunoglobulins specific features characterized by post-translational modification
    • C07K2317/41—Glycosylation, sialylation, or fucosylation
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    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C07K2317/565—Complementarity determining region [CDR]
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    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C07K2317/567—Framework region [FR]
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    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/72—Increased effector function due to an Fc-modification
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    • C07—ORGANIC CHEMISTRY
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    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/73—Inducing cell death, e.g. apoptosis, necrosis or inhibition of cell proliferation
    • C07K2317/732—Antibody-dependent cellular cytotoxicity [ADCC]
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    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/76—Antagonist effect on antigen, e.g. neutralization or inhibition of binding
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/77—Internalization into the cell
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    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
    • C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value

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 359% j Howieyy fit “a a W ?, M: , flflku{flaw1km(SI/MWj‘qu\fjfifw-H“WME ._ _ hfwwxmmm. 0X» H £3 .Nwfiw»%} Me :3 1% Me We a Mail?) a “j“ 3133951 806 (WMMAE) RM “6:: SSH-*1 269 Fj 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)

What we claim is:
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.
NZ616433A 2011-05-27 2012-05-24 Bcma (cd269/tnfrsf17) -binding proteins NZ616433B2 (en)

Applications Claiming Priority (5)

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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

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NZ616433B2 true NZ616433B2 (en) 2016-08-02

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