Substitute specification-marked up
`
`055112-5004-US
`
`and ibrutinib, \Vere evaluated on human platelet-rnediated thrombosis by utilizing the in vivo
`
`human thrombus formation in the VWF HAI mice model, which has been previously described
`
`(Chen, et al., Nat Biotechnol. 2008, 26(1), 114-19). Purified human platelets were preincubated
`
`with various concentrations of the BTK inhibitors (0.1 µM, 0.5 µM, or I µM) or DMSO and then
`
`administered to VWF HAI mice, followed by laser-induced thrombus formation. The BTK
`
`inhibitor-treated human platelets were fluorescently labeled and infused continuously through a
`
`catheter inserted into the femoral artery. Their behavior in response to laser-induced vascular
`
`injury was monitored in real time using two-channel confocal intravital microscopy (Furie and
`
`The objective of this study was to evaluate in vivo thrombus formation in
`
`the presence of BTK inhibitors. In vivo testing of novel anti platelet agents requires informative
`
`biomarkers. By utilizing a genetic modified mouse von Willebrand factor (VWFR1326H) model
`
`that supports human but not mouse platelet-mediated thrombosis, we evaluated the effects of
`
`Formula (II), CC-292, and ibrutinib on thrombus formation. These results show that Formula
`
`(II) had no significant effect on human platelet-mediated thrombus formation while ibrutinib was
`
`able to limit this process, resulting in a reduction in maximal thrombus size by 61 % compared
`
`DBl/ 100334638.2
`
`102
`
`SANDOZ INC.
`
`IPR2023-00478
`
`Ex. 1004, p. 1182 of 1432
`
`

`

`Substitute specification-marked up
`
`055112-5004-US
`
`with control. CC-292 showed an effect similar to ibrutinib. These results, which show reduced
`
`thrombus formation for ibrutinib at physiologically relevant concentrations, may provide some
`
`mechanistic background for the Grade 2: 3 bleeding events ( eg, subdural hematoma,
`
`gastrointestinal bleeding, hematuria and postprocedural hemorrhage) that have been reported in
`
`:S 6% of patients treated with ibrutinib.
`
`GPVI platelet aggregation was measured for Formula (II) and ibrutinib.
`
`Blood was obtained from untreated humans, and platelets were purified from plasma-rich protein
`
`by centrifugation. Cells were resuspended to a final concentration of 350,000/µL in buffer
`
`containing 145 mmol/L NaCl, 10 mmol/L HEPES, 0.5 mmol/L Na2HPO4, 5 mmol/L KCl, 2
`
`mmol/L MgCh, 1 mmol/L CaCh, and 0.1 % glucose, at pH 7.4. Stock solutions of Convulxin
`
`(CVX) GPVI were prepared on the day of experimentation and added to platelet suspensions 5
`
`minutes (37 °C, 1200 rpm) before the induction of aggregation. Aggregation was assessed with
`
`a Chronolog Lumi-Aggregometer (model 540 VS; Chronolog, Havertown, PA) and permitted to
`
`proceed for 6 minutes after the addition of agonist. The results are reported as maximum percent
`
`change in light transmittance from baseline with platelet buffer used as a reference. The results
`are shown in FIG. 191:i
`
`In FIG. ;;1.9_12, the results of CVX-induced (250 ng/mL) human platelet
`
`aggregation results before and 15 minutes after administration of the BTK inhibitors to 6 healthy
`
`individuals are shown.
`
`The results depicted in FIG. -+-9-JsLand FIG. ;?,{c1-JLindicate that the BTK
`
`inhibitor ibrutinib significantly inhibits GPVI platelet aggregation, while the BTK inhibitor of
`
`Formula (II) does not, further illustrating the surprising benefits of the latter compound.
`
`Example 4 - Effects ofBTK Inhibition on Antibody-Dependent NK Cell Mediated Cytotoxicity
`
`Rituximab-combination chemotherapy is today's standard of care in
`
`CD20+ B-cell malignancies. Previous studies investigated and determined that ibrutinib
`
`antagonizes rituximab antibody-dependent cell mediated cytotoxicity (ADCC) mediated by NK
`
`cells. This may be due to ibrutinib's secondary irreversible binding to interleukin-2 inducible
`
`tyrosine kinase (ITK) which is required for FcR-stimulated NK cell function including calcium
`
`mobilization, granule release, and overall ADCC. H. E. Kohrt, et al., Blood 2014, 123, 1957-60.
`
`DBl/ 100334638.2
`
`103
`
`SANDOZ INC.
`
`IPR2023-00478
`
`Ex. 1004, p. 1183 of 1432
`
`

`

`Substitute specification-marked up
`
`055112-5004-US
`
`In this example, the effects of Formula (II) and ibrutinib on NK cell
`
`function were evaluated in primary NK cells from healthy volunteers and CLL patients. The
`
`activation ofNK cells co-cultured with antibody-coated target cells was strongly inhibited by
`
`ibrutinib. The secretion ofIFN-y was reduced by 48% (p = 0.018) and 72% (p = 0.002) in
`
`cultures treated with ibrutinib at 0.1 and 1.0 µM respectively and NK cell degranulation was
`
`significantly (p = 0.002) reduced, compared with control cultures. Formula (II) treatment at 1
`
`µM, a clinically relevant concentration, did not inhibit IFN-y or NK cell degranulation.
`
`Rituximab-mediated ADCC was evaluated in NK cells from healthy volunteers as well as assays
`
`ofNK cells from CLL patients targeting autologous CLL cells. In both cases, ADCC was not
`
`inhibited by Formula (II) treatment at 1 µM. In contrast, addition of ibrutinib to the ADCC
`
`assays strongly inhibited the rituximab-mediated cytotoxicity of target cells, and no increase over
`
`natural cytotoxicity was observed at any rituximab concentration. This result indicates that the
`
`combination of rituximab and Formula (II) provides an unexpected benefit in the treatment of
`
`CLL.
`
`F}~M,~-~-2,HM.s329]
`
`BTK is a non-receptor enzyme in the Tee kinase family that is expressed
`
`among cells of hematopoietic origin, including B cells, myeloid cells, mast cells and platelets,
`
`where it regulates multiple cellular processes including proliferation, differentiation, apoptosis,
`
`and cell migration. W. N. Khan, Immunol Res. 2001, 23, 147-56; A J. Mohamed, et al.,
`
`Immunol Rev. 2009, 228, 58-73; J.M. Bradshaw, Cell Signal. 2010, 22, 1175-84. Functional
`
`null mutations ofBTK in humans cause the inherited disease, X linked agammaglobulinemia,
`
`which is characterized by a lack of mature peripheral B cells. M. Vihinen, et al., Front Biosci.
`
`2000, 5, D917-28. Conversely, BTK activation is implicated in the pathogenesis of several B(cid:173)
`
`cell malignancies. S. E. Herman, etal., Blood 2011, 117, 6287-96; L. P. Kil, etal., Am. J Blood
`
`Res. 2013, 3, 71-83; Y. T. Tai, et al., Blood 2012, 120, 1877-87; J. J. Buggy, L. Elias, Int. Rev.
`
`Immunol. 2012, 31, 119-32 (Erratum in: Int. Rev. Immunol. 2012, 31, 428). In addition, BTK(cid:173)
`
`dependent activation of mast cells and other immunocytes in peritumoral inflammatory stroma
`
`has been shown to sustain the complex microenvironment needed for lymphoid and solid tumor
`
`maintenance. L. Soucek, et al., Neoplasia 2011, 13, 1093-100; S. Ponader, et al., Blood 2012,
`
`119, 1182-89; M. F. de Rooij, et al., Blood 2012, 119, 2590-94. Taken together, these findings
`
`have suggested that inhibition of BTK may offer an attractive strategy for treating B-cell
`
`neoplasms, other hematologic malignancies, and solid tumors.
`
`DBl/ 100334638.2
`
`104
`
`SANDOZ INC.
`
`IPR2023-00478
`
`Ex. 1004, p. 1184 of 1432
`
`

`

`Substitute specification-marked up
`
`055112-5004-US
`
`Ibrutinib (PCI-32765, IMBRUVICA), is a first-in-class therapeutic BTK
`
`inhibitor. This orally delivered, small-molecule drug is being developed by Pharmacyclics, Inc.
`
`for the therapy of B-cell malignancies. As described above, in patients with heavily pretreated
`
`indolent non-Hodgkin lymphoma (iNHL), mantle cell lymphoma (MCL), and CLL, ibrutinib
`
`showed substantial anti tumor activity, inducing durable regressions of lymphadenopathy and
`
`splenomegaly in the majority of patients. R.H. Advani, et al., J. Clin. Oncol. 31, 88-94 (2013);
`
`J.C. Byrd, et al., N Engl. J Med 2013, 369, 32-42; M. L. Wang, et al., N Engl. J Med 2013,
`
`369, 507-16. S. O'Brien, et al., Blood 2012, 119, 1182-89. The pattern of changes in CLL was
`
`notable. Inhibition ofBTK with ibrutinib caused rapid and substantial mobilization of malignant
`
`CLL cells from tissues sites into the peripheral blood, as described in J. A Woyach, et al., Blood
`
`2014, 123, 1810-17; this effect was consistent with decreased adherence of CLL to protective
`
`stromal cells. S. Ponader, et al., Blood 2012, 119, 1182-89; M. F. de Rooij, et al., Blood 2012,
`
`119, 2590-94. Ibrutinib has been generally well tolerated. At dose levels associated with total
`
`BTK occupancy, not dose-limiting toxicities were identified and subjects found the drug
`
`tolerable over periods extending to >2.5 years.
`
`H}~K~J,~WW33U
`
`Given the homology between BTK and interleukin-2 inducible tyrosine
`
`kinase (ITK), it has been recently confirmed that ibrutinib irreversibly binds ITK. J. A
`
`Dubovsky, et al., Blood 2013, 122, 2539-2549. ITK expression in Fe receptor (FcR)-stimulated
`
`NK cells leads to increased calcium mobilization, granule release, and cytotoxicity. D. Khurana,
`
`et al., J Immunol. 2007, 178, 3575-3582. As rituximab is a backbone of lymphoma therapy,
`
`with mechanisms of action including ADCC, as well as direct induction of apoptosis and
`
`complement-dependent cytotoxicity and FcR stimulation is requisite for ADCC, we investigated
`
`if ibrutinib or Formula (II) (lacking ITK inhibition) influenced rituximab' s anti-lymphoma
`
`activity in vitro by assessing NK cell IFN-y secretion, degranulation by CD107a mobilization,
`
`and cytotoxicity by chromium release using CD20+ cell lines and autologous patient samples
`
`with chronic lymphocytic leukemia (CLL ).
`
`Formula (II) is a more selective inhibitor than ibrutinib, as shown
`
`previously. Formula (II) is not a potent inhibitor of Itk kinase in contrast to ibrutinib (see Table
`
`1). Itk kinase is required for FcR-stimulated NK cell function including calcium mobilization,
`
`granule release, and overall ADCC. As anti-CD20 antibodies like rituximab are standard of care
`
`DBl/ 100334638.2
`
`105
`
`SANDOZ INC.
`
`IPR2023-00478
`
`Ex. 1004, p. 1185 of 1432
`
`

`

`Substitute specification-marked up
`
`055112-5004-US
`
`drugs, often as part of combination regimens, for the treatment of CD20+ B-cell malignancies,
`
`the potential of ibrutinib or Formula (II) to antagonize ADCC was evaluated in vitro. We
`
`hypothesized that Btk inhibitor, Formula (II) which does not have activity against Itk, may
`
`preserve NK cell function and therefore synergize rather than antagonize rituximab-mediated
`
`ADCC. Rituximab-dependent NK-cell mediated cytotoxicity was assessed using lymphoma cell
`
`lines as well as autologous CLL tumor cells.
`
`Cell culture conditions were as follows. Cell lines Raji and DHL-4 were
`
`maintained in RPMI 1630 supplemented with fetal bovine serum, L-glutamine, 2-
`
`mercaptoethanol and penicillin-streptomycin at 37 °Cina humidified incubator. The HER18
`
`cells were maintained in DEM supplemented with fetal bovine serum, penicillin-streptomycin
`
`and. Prior to assay, HER18 cells were harvested using trypsin-EDTA, washed with phosphate(cid:173)
`
`buffered saline (PBS) containing 5% serum and viable cells were counted. For culture of
`
`primary target cells, peripheral blood from CLL patients was subject to density centrifugation to
`
`obtain peripheral blood mononuclear cells (PBMC). Cell preparations were washed and then
`
`subject to positive selection of CD5+CD19+ CLL cells using magnetic beads (MACS, Miltenyi
`
`Biotech). Cell preparations were used fresh after selection. NK cells from CLL patients and
`
`healthy volunteers were enriched from peripheral blood collected in sodium citrate anti(cid:173)
`
`coagulant tubes and then subject to density centrifugation. Removal of non NK cells was
`
`performed using negative selection by MACS separation. Freshly isolated NK cells were
`
`washed three times, enumerated, and then used immediately for ADCC assays.
`
`Cytokine secretion was determined as follows. Rituximab and
`
`trastuzumab-dependent NK-cell mediated degranulation and cytokine release were assessed
`
`using lymphoma and HER2+ breast cancer cell lines (DHL-4 and HER18, respectively). Target
`
`cells were cultured in flat-bottom plates containing 10 µg/mL of rituximab (DHL-4) or
`
`trastuzumab (HER18) and test articles (0.1 or 1 µM ibrutinib, 1 µM Formula (II), or DMSO
`
`vehicle control). NK cells from healthy donors were enriched as described above and then added
`
`to the target cells and incubated for 4 hours at 37 °C. Triplicate cultures were performed on NK
`
`cells from donors. After incubation, supernatants were harvested, centrifuged briefly, and then
`
`analyzed for interferon-y using an enzyme-linked immunosorbent assay (ELISA) (R&D
`
`Systems, Minneapolis, MN, USA).
`
`DBl/ 100334638.2
`
`106
`
`SANDOZ INC.
`
`IPR2023-00478
`
`Ex. 1004, p. 1186 of 1432
`
`

`

`Substitute specification-marked up
`
`055112-5004-US
`
`Lytic granule release was determined as follows. NK cells from healthy
`
`donors were enriched and cultured in the presence of target cells, monoclonal antibodies and test
`
`articles as described above. After 4 hours, the cultures were harvested and cells were pelleted,
`
`washed, and then stained for flow cytometry evaluation. Degranulation was evaluated via by
`
`flow cytometery by externalization of CD107a, a protein normally present on the inner leaflet of
`
`lytic granules, and gating on NK cells (CD3-CD16+ lymphocytes). The percentage of CD107a
`
`positive NK cells was quantified by comparison with a negative control (isotype control,
`
`unstained cells/FMO). Control cultures (NK cells cultured without target cells, or NK, target cell
`
`co-cultures in the absence of appropriate monoclonal antibody) were also evaluated; all
`
`experiments were performed in triplicate.
`
`F}{\~~~°l"~m-~~H36]
`ADCC assays were performed as follows. Briefly, target cells (Raji or
`primary CLL) were labeled by incubation at 37 °C with 100 µCi 51Cr for 4 hours prior to co(cid:173)
`
`culture with NK cells. Cells were washed, enumerated, and then added in triplicate to prepared
`
`96-well plates containing treated NK cells at an effector:target (E:T) ratio of 25: 1. Rituximab
`
`(Genentech) was added to ADCC wells at concentrations of 0.1, 1.0 or 10 µg/mL and the assays
`
`were briefly mixed and then centrifuged to collect cells at the bottom of the wells. The effect of
`
`NK cell natural cytotoxicity was assessed in wells containing no rituximab. Cultures were
`
`incubated at 37 °C for 4 hours, and then centrifuged. Supernatants were harvested and 51Cr
`
`release was measured by liquid scintillation counting. All experiments were performed in
`
`triplicate.
`
`F~Z\}.4-~fm.~~H37]
`
`Ibrutinib-inhibited rituximab-induced NK cell cytokine secretion in a
`
`dose-dependent manner (0.1 and 1 µM) (FIG. ;?:11§.: 48% p = 0.018; 72% p = 0.002,
`
`respectively). At 1 µM, Formula (II) did not significantly inhibit cytokine secretion (FIG. ;;-1-Llt
`
`3.5%). Similarly, Formula (II) had no inhibitory effect on rituximab-stimulated NK cell
`
`degranulation (< 2%) while ibrutinib reduced degranulation by -50% (p = 0.24, FIG. ;?:21~'.).
`
`Formula (II) had no inhibitory effect while ibrutinib prevented trastuzumab-stimulated NK cell
`cytokine release and degranulation by -92% and -84% at 1 µM, respectively (FIG. ,~HJ_~ and
`FIG. ;;;;q): ***p = 0.004, **p = 0.002).
`
`{W,~J,H-Jrnitt.rnl
`cells was not inhibited by addition of Formula (II) at 1 µM, and increased cell lysis was observed
`
`In Raji cells samples, ex vivo NK cell activity against autologous tumor
`
`DBl/ 100334638.2
`
`107
`
`SANDOZ INC.
`
`IPR2023-00478
`
`Ex. 1004, p. 1187 of 1432
`
`

`

`Substitute specification-marked up
`
`055112-5004-US
`
`with increasing concentrations of rituximab at a constant E:T ratio (FIG. ;1J]Q). In primary CLL
`
`samples, ex vivo NK cell activity against autologous tumor cells was not inhibited by addition of
`
`Formula (II) at 1 µM, and increased cell lysis was observed with increasing concentrations of
`
`rituximab at a constant E:T ratio (FIG. ,;4_21). In contrast, addition of 1 µM ibrutinib completely
`
`inhibited ADCC, with less than 10% cell lysis at any rituximab concentration and no increase in
`
`cell lysis in the presence of rituximab, compared with cultures without rituximab. The difference
`
`between Formula (II) and ibrutinib was highly significant in this assay (p = 0.001). A plot
`
`highlighting the differences between Formula (II) and ibrutinib at 10 µMis shown in FIG. :;1~~_?}.
`
`In ADCC assays using healthy donor NK cells, antibody-dependent lysis
`
`of rituximab-coated Raji cells was not inhibited by addition of 1 µM Formula (II) (FIG. ;~~{t__;n_ ).
`
`In these experiments, addition of rituximab stimulated a 5- to 8-fold increase in cell lysis at 0.1
`
`and 1 µg/mL, compared with low (<20%) natural cytotoxicity in the absence ofrituximab. As
`
`previously reported, addition of 1 µM ibrutinib strongly inhibited the antibody-dependent lysis of
`
`target cells, with less than 20% cell lysis at all rituximab concentrations and no increase in
`
`ADCC with at higher rituximab concentrations. The difference between Formula (II) and
`
`ibrutinib was highly significant in this assay (p = 0.001).
`
`Ibrutinib is clinically effective as monotherapy and in combination with
`
`rituximab, despite inhibition of ADCC in vitro and in vivo murine models due to ibrutinib's
`
`secondary irreversible binding to ITK. Preclinically, the efficacy of therapeutics which do not
`
`inhibit NK cell function, including Formula (II), is superior to ibrutinib. Clinical investigation is
`
`needed to determine the impact of this finding on patients receiving rituximab as these results
`
`provide support for the unexpected property of Formula (II) as a better agent than ibrutinib to use
`
`in combination with antibodies that have ADCC as a mechanism of action.
`
`Example 5 - Effects ofBTK Inhibition on Generalized NK Cell Mediated Cytotoxicity
`
`F~t,~J,~A-UNH,fq
`Formula (II) on generalized NK killing (non-ADCC killing). The targets (K562 cells) do not
`
`An assay was performed to assess the effects of BTK inhibition using
`
`express MHC class I, so they do not inactivate NK cells. Target cells were grown to mid-log
`
`phase, and Sx 105 cells were labeled in 100 µL of assay medium (IMDM with 10% FCS and
`
`penicillin/streptomycin) with 100 µ Ci 51Cr for 1 hour at 37 °C. Cells were washed twice and
`
`DBl/ 100334638.2
`
`108
`
`SANDOZ INC.
`
`IPR2023-00478
`
`Ex. 1004, p. 1188 of 1432
`
`

`

`Substitute specification-marked up
`
`055112-5004-US
`
`resuspended in assay medium. A total of 5000 target cells/well was used in the assay. Effector
`
`cells were resuspended in assay medium, distributed on a V-bottom 96-well plate, and mixed
`
`with labeled target cells at 40: 1 E:T ratios. Maximum release was determined by incubating
`
`target cells in 1 % Triton X-100. For spontaneous release, targets were incubated without
`
`effectors in assay medium alone. After a 1 minute centrifugation at 1000 rpm, plates were
`incubated for 4 and 16 hours at 37 °C. Supernatant was harvested and 51Cr release was measured
`
`in a gamma counter. Percentage of specific release was calculated as ( experimental release(cid:173)
`
`spontaneous release)/(maximum release-spontaneous release) x 100. The results are shown in
`
`Example 6 - Effects ofBTK Inhibition on T Cells
`
`}1¥\":lo,tf]LtLbHJL
`
`An assay was performed to assess the effects of BTK inhibition using
`
`Formula (II) on T cells. Enriched CD4+ T cells are plated on 24-well culture dishes that have
`
`been precoated 2 hr with 250 µL anti-TCR~ (0.5 µg/mL) plus anti-CD28 (5 µg/mL) at 37 °C in
`
`PBS. The cells are then supplemented with media containing BTK inhibitors along with the
`
`skewing cytokines as indicated in the following. The Thl 7 and Treg cultures are grown for 4
`
`days before analysis. The cells are maintained for an additional 3 days with skewing cytokines
`
`(Thl 7; 20 ng/mL IL-6, 0.5 ng/mL TGF-~, 5 µg/mL IL-4, 5 µg/mL IFN-y and Treg; 0.5 ng/mL
`
`TGF-~, 5 µg/mL IL-4, 5 µg/mL IFN-y) and are supplemented with IL2 as a growth factor.
`
`The results are shown in FIG. 2-7-2d. and FIG. ;;i,g _ _L~., and further illustrate
`the surprising properties of Formula (II) in comparison to ibrutinib. Because of the lack of
`
`activity of Formula (II) on Itk and Txk, no adverse effects on Thl 7 and Treg development was
`
`observed. Since ibrutinib inhibits both Itk and Txk, a profound inhibition of Thl 7 cells and an
`
`increase in Treg development is observed, which is comparable to the murine Itk/Txk double
`
`knock-out cells which were used as a control.
`
`DBl/ 100334638.2
`
`109
`
`SANDOZ INC.
`
`IPR2023-00478
`
`Ex. 1004, p. 1189 of 1432
`
`

`

`Substitute specification-marked up
`
`055112-5004-US
`
`SEQUENCE LISTINGS
`
`<110> Acerta Pharma B.V.
`
`<120> METHODS OF TREATING CHRONIC LYMPHOCYTIC LEUKEMIA AND SMALL
`LYMPHOCYTIC LEUKEMIA USING A BTK INHIBITOR
`
`<130>
`
`055112-5004-WO
`
`<140> 62/000,000
`<141> 2015-01-15
`
`<150> 61/929,742
`<151> 2014-01-21
`
`<160>
`
`14
`
`<170> Patentin version 3.5
`
`1
`<210>
`451
`<211>
`<212> PRT
`<213> Artificial Sequence
`
`<220>
`<223> Heavy chain amino acid sequence of the anti-CD20 monoclonal
`antibody rituximab.
`
`<400>
`
`1
`
`Gln Val Gln Leu Gln Gln Pro Gly Ala Glu Leu Val Lys Pro Gly Ala
`1
`5
`10
`15
`
`Ser Val Lys Met Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr
`20
`25
`30
`
`Asn Met His Trp Val Lys Gln Thr Pro Gly Arg Gly Leu Glu Trp Ile
`35
`40
`45
`
`Gly Ala Ile Tyr Pro Gly Asn Gly Asp Thr Ser Tyr Asn Gln Lys Phe
`50
`55
`60
`
`Lys Gly Lys Ala Thr Leu Thr Ala Asp Lys Ser Ser Ser Thr Ala Tyr
`65
`70
`75
`80
`
`Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys
`85
`90
`95
`
`Ala Arg Ser Thr Tyr Tyr Gly Gly Asp Trp Tyr Phe Asn Val Trp Gly
`100
`105
`110
`
`DBl/ 100334638.2
`
`110
`
`SANDOZ INC.
`
`IPR2023-00478
`
`Ex. 1004, p. 1190 of 1432
`
`

`

`Substitute specification-marked up
`
`055112-5004-US
`
`Ala Gly Thr Thr Val Thr Val Ser Ala Ala Ser Thr Lys Gly Pro Ser
`115
`120
`125
`
`Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala
`130
`135
`140
`
`Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val
`145
`150
`155
`160
`
`Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala
`165
`170
`175
`
`Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val
`180
`185
`190
`
`Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His
`195
`200
`205
`
`Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys
`210
`215
`220
`
`Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly
`225
`230
`235
`240
`
`Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met
`245
`250
`255
`
`Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His
`260
`265
`270
`
`Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val
`275
`280
`285
`
`His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr
`290
`295
`300
`
`Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly
`305
`310
`315
`320
`
`Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile
`325
`330
`335
`
`DBl/ 100334638.2
`
`111
`
`SANDOZ INC.
`
`IPR2023-00478
`
`Ex. 1004, p. 1191 of 1432
`
`

`

`Substitute specification-marked up
`
`055112-5004-US
`
`Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val
`340
`345
`350
`
`Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser
`355
`360
`365
`
`Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu
`370
`375
`380
`
`Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro
`385
`390
`395
`400
`
`Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val
`405
`410
`415
`
`Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met
`420
`425
`430
`
`His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser
`435
`440
`445
`
`Pro Gly Lys
`450
`
`2
`<210>
`<211> 213
`<212> PRT
`<213> Artificial sequence
`
`<220>
`<223> Light chain amino acid sequence of the anti-CD20 monoclonal
`antibody rituximab.
`
`<400>
`
`2
`
`Gln Ile Val Leu Ser Gln Ser Pro Ala Ile Leu Ser Ala Ser Pro Gly
`1
`5
`10
`15
`
`Glu Lys Val Thr Met Thr Cys Arg Ala Ser Ser Ser Val Ser Tyr Ile
`20
`25
`30
`
`His Trp Phe Gln Gln Lys Pro Gly Ser Ser Pro Lys Pro Trp Ile Tyr
`35
`40
`45
`
`DBl/ 100334638.2
`
`112
`
`SANDOZ INC.
`
`IPR2023-00478
`
`Ex. 1004, p. 1192 of 1432
`
`

`

`Substitute specification-marked up
`
`055112-5004-US
`
`Ala Thr Ser Asn Leu Ala Ser Gly Val Pro Val Arg Phe Ser Gly Ser
`50
`55
`60
`
`Gly Ser Gly Thr Ser Tyr Ser Leu Thr Ile Ser Arg Val Glu Ala Glu
`65
`70
`75
`80
`
`Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Trp Thr Ser Asn Pro Pro Thr
`85
`90
`95
`
`Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg Thr Val Ala Ala Pro
`100
`105
`110
`
`Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly Thr
`115
`120
`125
`
`Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala Lys
`130
`135
`140
`
`Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln Glu
`145
`150
`155
`160
`
`Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser Ser
`165
`170
`175
`
`Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr Ala
`180
`185
`190
`
`Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser Phe
`195
`200
`205
`
`Asn Arg Gly Glu Cys
`210
`
`3
`<210>
`449
`<211>
`<212> PRT
`<213> Artificial sequence
`
`<220>
`<223> Heavy chain amino acid sequence of the anti-CD20 monoclonal
`antibody obinutuzumab.
`
`<400>
`
`3
`
`DBl/ 100334638.2
`
`113
`
`SANDOZ INC.
`
`IPR2023-00478
`
`Ex. 1004, p. 1193 of 1432
`
`

`

`Substitute specification-marked up
`
`055112-5004-US
`
`Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser
`1
`5
`10
`15
`
`Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Ala Phe Ser Tyr Ser
`20
`25
`30
`
`Trp Ile Asn Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met
`35
`40
`45
`
`Gly Arg Ile Phe Pro Gly Asp Gly Asp Thr Asp Tyr Asn Gly Lys Phe
`50
`55
`60
`
`Lys Gly Arg Val Thr Ile Thr Ala Asp Lys Ser Thr Ser Thr Ala Tyr
`65
`70
`75
`80
`
`Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys
`85
`90
`95
`
`Ala Arg Asn Val Phe Asp Gly Tyr Trp Leu Val Tyr Trp Gly Gln Gly
`100
`105
`110
`
`Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe
`115
`120
`125
`
`Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu
`130
`135
`140
`
`Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp
`145
`150
`155
`160
`
`Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu
`165
`170
`175
`
`Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser
`180
`185
`190
`
`Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro
`195
`200
`205
`
`Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys
`210
`215
`220
`
`DBl/ 100334638.2
`
`114
`
`SANDOZ INC.
`
`IPR2023-00478
`
`Ex. 1004, p. 1194 of 1432
`
`

`

`Substitute specification-marked up
`
`055112-5004-US
`
`Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro
`225
`230
`235
`240
`
`Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser
`245
`250
`255
`
`Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp
`260
`265
`270
`
`Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn
`275
`280
`285
`
`Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val
`290
`295
`300
`
`Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu
`305
`310
`315
`320
`
`Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys
`325
`330
`335
`
`Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr
`340
`345
`350
`
`Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr
`355
`360
`365
`
`Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu
`370
`375
`380
`
`Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu
`385
`390
`395
`400
`
`Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys
`405
`410
`415
`
`Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu
`420
`425
`430
`
`Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly
`435
`440
`445
`
`DBl/ 100334638.2
`
`115
`
`SANDOZ INC.
`
`IPR2023-00478
`
`Ex. 1004, p. 1195 of 1432
`
`

`

`Substitute specification-marked up
`
`055112-5004-US
`
`Lys
`
`4
`<210>
`<211> 219
`<212> PRT
`<213> Artificial sequence
`
`<220>
`<223> Light chain amino acid sequence of the anti-CD20 monoclonal
`antibody obinutuzumab.
`
`<400>
`
`4
`
`Asp Ile Val Met Thr Gln Thr Pro Leu Ser Leu Pro Val Thr Pro Gly
`1
`5
`10
`15
`
`Glu Pro Ala Ser Ile Ser Cys Arg Ser Ser Lys Ser Leu Leu His Ser
`20
`25
`30
`
`Asn Gly Ile Thr Tyr Leu Tyr Trp Tyr Leu Gln Lys Pro Gly Gln Ser
`35
`40
`45
`
`Pro Gln Leu Leu Ile Tyr Gln Met Ser Asn Leu Val Ser Gly Val Pro
`50
`55
`60
`
`Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile
`65
`70
`75
`80
`
`Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Ala Gln Asn
`85
`90
`95
`
`Leu Glu Leu Pro Tyr Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys
`100
`105
`110
`
`Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu
`115
`120
`125
`
`Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe
`130
`135
`140
`
`Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln
`145
`150
`155
`160
`
`Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser
`
`DBl/ 100334638.2
`
`116
`
`SANDOZ INC.
`
`IPR2023-00478
`
`Ex. 1004, p. 1196 of 1432
`
`

`

`Substitute specification-marked up
`
`055112-5004-US
`
`165
`
`170
`
`175
`
`Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu
`180
`185
`190
`
`Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser
`195
`200
`205
`
`Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys
`210
`215
`
`5
`<210>
`<211> 122
`<212> PRT
`<213> Artificial sequence
`
`<220>
`<223> Variable heavy chain amino acid sequence of the anti-CD20
`monoclonal antibody ofatumumab.
`
`<400>
`
`5
`
`Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Arg
`1
`5
`10
`15
`
`Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asn Asp Tyr
`20
`25
`30
`
`Ala Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
`35
`40
`45
`
`Ser Thr Ile Ser Trp Asn Ser Gly Ser Ile Gly Tyr Ala Asp Ser Val
`50
`55
`60
`
`Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Lys Ser Leu Tyr
`65
`70
`75
`80
`
`Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Leu Tyr Tyr Cys
`85
`90
`95
`
`Ala Lys Asp Ile Gln Tyr Gly Asn Tyr Tyr Tyr Gly Met Asp Val Trp
`100
`105
`110
`
`Gly Gln Gly Thr Thr Val Thr Val Ser Ser
`115
`120
`
`DBl/ 100334638.2
`
`117
`
`SANDOZ INC.
`
`IPR2023-00478
`
`Ex. 1004, p. 1197 of 1432
`
`

`

`Substitute specification-marked up
`
`055112-5004-US
`
`6
`<210>
`<211> 107
`<212> PRT
`<213> Artificial sequence
`
`<220>
`<223> Variable light chain amino acid sequence of the anti-CD20
`monoclonal antibody ofatumumab.
`
`<400>
`
`6
`
`Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly
`1
`5
`10
`15
`
`Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Ser Tyr
`20
`25
`30
`
`Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile
`35
`40
`45
`
`Tyr Asp Ala Ser Asn Arg Ala Thr Gly Ile Pro Ala Arg Phe Ser Gly
`50
`55
`60
`
`Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu Pro
`65
`70
`75
`80
`
`Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Arg Ser Asn Trp Pro Ile
`85
`90
`95
`
`Thr Phe Gly Gln Gly Thr Arg Leu Glu Ile Lys
`100
`105
`
`7
`<210>
`<211> 222
`<212> PRT
`<213> Artificial Sequence
`
`<220>
`<223> Fab fragment of heavy chain amino acid sequence of the anti-CD20
`monoclonal antibody ofatumumab.
`
`<400>
`
`7
`
`Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Arg
`1
`5
`10
`15
`
`DBl/ 100334638.2
`
`118
`
`SANDOZ INC.
`
`IPR2023-00478
`
`Ex. 1004, p. 1198 of 1432
`
`

`

`Substitute specification-marked up
`
`055112-5004-US
`
`Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asn Asp Tyr
`20
`25
`30
`
`Ala Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
`35
`40
`45
`
`Ser Thr Ile Ser Trp Asn Ser Gly Ser Ile Gly Tyr Ala Asp Ser Val
`50
`55
`60
`
`Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Lys Ser Leu Tyr
`65
`70
`75
`80
`
`Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Leu Tyr Tyr Cys
`85
`90
`95
`
`Ala Lys Asp Ile Gln Tyr Gly Asn Tyr Tyr Tyr Gly Met Asp Val Trp
`100
`105
`110
`
`Gly Gln Gly Thr Thr Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro
`115
`120
`125
`
`Ser Val Phe Pro Leu Ala Pro Gly Ser Ser Lys Ser Thr Ser Gly Thr
`130
`135
`140
`
`Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr
`145
`150
`155
`160
`
`Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro
`165
`170
`175
`
`Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr
`180
`185
`190
`
`Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn
`195
`200
`205
`
`His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro
`210
`215
`220
`
`8
`<210>
`<211> 211
`<212> PRT
`<213> Artificial Sequence
`
`DBl/ 100334638.2
`
`119
`
`SANDOZ INC.
`
`IPR2023-00478
`
`Ex. 1004, p. 1199 of 1432
`
`

`

`Substitute specification-marked up
`
`055112-5004-US
`
`<220>
`<223> Fab fragment of light chain amino acid sequence of the anti-CD20
`monoclonal antibody ofatumumab.
`
`<400>
`
`8
`
`Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly
`1
`5
`10
`15
`
`Glu Arg